JP2003189883A - New ubiquitin-specific protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protease (USP) having a deubiquitinating activity, a (poly)peptide derived from the USP and a nucleotide encoding these, an antibody against the (poly)peptide, a physiologically active inhibitor, antagonist or activator of the new USP and a pharmaceutical composition utilizing these and to provide a method for producing the (poly)peptide by a genetic engineering technique and a method for identifying the inhibitor, the antagonist or the activator and a method for diagnosing diseases to which the new USP relates and to provide a kit. <P>SOLUTION: This polypeptide comprises an amino acid sequence having a specific sequence number and has a deubiquitinating activity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel protease having deubiquitinating activity (hereinafter sometimes referred to as USP) and a gene encoding the same. More specifically, a polypeptide or peptide having all or part of the amino acid sequence of the novel USP,
A polynucleotide encoding the polypeptide or peptide or a polynucleotide which is a complementary strand thereof, a recombinant vector containing the polynucleotide, a transformant containing the recombinant vector, an antibody against the polypeptide or peptide, the polypeptide Or a compound that interacts with the polynucleotide, a pharmaceutical composition containing one or more of these, a method for producing the polypeptide or peptide, a method for identifying a compound that interacts with the polypeptide or peptide or the polynucleotide, The present invention relates to a method for measuring the polypeptide or peptide or the polynucleotide, and a reagent kit used for the identifying method or the measuring method.

[0002]

Ubiquitin (hereinafter sometimes abbreviated as Ub) is a peptide chain consisting of 76 amino acid residues, and its amino acid sequence is highly conserved from yeast to human. The role of Ub in the body is various, and it includes carcinogenesis (Non-patent documents 1-4) and cell cycle (Non-patent documents 5-).
7), viral infection (Non-patent document 8), and neurodegenerative disease (Non-patent documents 9-11), etc. are involved in many biological reactions.

The most important function of Ub is to act as a signal in proteolysis in the 26S proteasome. Ub is isopeptide-bonded to a target protein by a series of ubiquitinating enzymes such as a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase (E3) to form a polyubiquitin chain. The polyubiquitin chain serves as a degradation signal and is recognized by the proteasome, whereby the ubiquitinated protein is degraded. As described above, the degradation of ubiquitinated proteins by the proteasome is performed by binding to the substrate protein and recognizing the polyubiquitin chain, and thus excessive polyubiquitin chain accumulation inhibits the degradation of the protein (Non-Patent Document 12). .

On the other hand, from the ubiquitinated protein to Ub
It has been reported that a deubiquitinating enzyme (DUB) that catalyzes the deubiquitinating reaction that dissociates the enzyme is present. DUB is
The structure is roughly classified into two families (Non-patent documents 13-15). One is Ubiquitin C-terminal hydrolase.
In most cases, the molecular weight is 20 kDa to 30 kDa, and the primary structure is conserved among different species. UCH is mainly Ub
Dissociates Ub from the low molecule using the low molecule bound through the C-terminus as a substrate. The other is ubiquitin-specific protease (Ubiquitinspecif).
ic protease) (USP, UBP or U)
CH type II), whose molecular weight is 40
It varies from kDa to 150 kDa, and there is little commonality of amino acid sequences among different species. USP has a cysteine (Cys) domain (Cys bo) as its active domain.
x), histidine (His) domain (His bo
x) and aspartic acid (Asp) domains,
It is a cysteine protease having a cysteine residue existing in the Cys domain as an active site. USP is Ub
Dissociates Ub from the polymer using the polymer bound via the C-terminus as a substrate.

The function of this USP in the living body can be roughly divided into three. One of them is the function of generating Ub from precursor Ub such as ribosome protein-fused ubiquitin and peptide-bonded polyubiquitin chain. Ub-CEP52, which is one of USPs, is involved in this. The second is a function of dissociating Ub from a ubiquitinated protein having an isopeptide bond, which suppresses protein degradation by suppressing ubiquitination of the protein. Three
The claw has a function of disassembling the isopeptide-bound polyubiquitin chain after being decomposed by the proteasome, and isopeptidase T known as USP5, for example.
Has this function (Non-Patent Document 16).

[0006] One of the functions of the ubiquitin system in which deubiquitinase (DUB) is involved is the removal of abnormal proteins produced in vivo and the quantitative regulation by the degradation of transcription factors, signal transduction factors, etc. ( Non-Patent Document 17), D
UB dysfunction causes abnormalities in this system. recent years,
Numerous reports have suggested a relationship between abnormalities in the ubiquitin system and carcinogenesis and neurodegenerative diseases (Non-Patent Document 1).
7-19). For example, aggregates such as tau and α-synuclein in Alzheimer's disease and Parkinson's disease are recognized by anti-ubiquitin antibodies (Non-patent Document 17). UCH-L1 is highly expressed in nerves and neuroendocrine cells (Non-Patent Document 20), and recently, in a family with autosomal dominant juvenile Parkinson's disease, UCH-L1.
A missense mutation in one gene was discovered (Non-Patent Document 1
0). Further, the gene responsible for the recessive inherited neurodegenerative disease model mouse (gad mousse) is UCH-L1.
It has become clear that this is caused by the deletion of
1), the involvement of deubiquitinase in neurodegenerative diseases is being clarified. Neuronal damage to oxidative stress and the ubiquitin system are also closely related. The ubiquitin gene is expressed in the hippocampus, cerebellum, and dentate gyrus in normal nervous tissue, but oxidative stress increases the expression of the ubiquitin gene in the hippocampus and cerebellar granule cell layer (Non-Patent Document 21). In addition, in an in vitro experimental system, it has been confirmed that ubiquitinated proteins accumulate in nerve cells due to oxidative stress (Non-Patent Document 22). Given that one of the causes of neurodegenerative diseases is neuronal cell death due to the accumulation of abnormal proteins, the abnormal proteins produced by oxidative stress are promptly removed by the ubiquitin system also activated by oxidative stress to prevent cell death. It is thought to be out. further,
A link between abnormal ubiquitin system and muscular atrophy has been reported (Non-Patent Document 23).

[0007] USP is also involved in the maintenance of chromosome structure, and it is known that deubiquitination of ubiquitinated histones is important for chromosome aggregation (Non-patent Document 24). However, little was known about the deubiquitinating enzyme. Recently Ub-
It was reported that M (USP16) deubiquitinates histone H2A (Non-patent Document 25). Ub-M is H
Histone proteins other than 2A are also considered to be deubiquitinated.

There are various kinds of USPs in the living body, and 16 kinds of USPs have been reported in yeast (Non-patent document 17) and 27 kinds in humans (Non-patent document 26). Each of these USPs seems to have different substrate selectivity and a function of discriminating the binding mode of the polyubiquitin chain.
Many structural features of the USP family have long unique sequences at the N- or C-termini other than the active sites Cys box and His box, which are substrate-selective and intracellular localization to individual USPs. It is presumed that the function and the function specificity are given (Non-patent document 27). Therefore, it is necessary to discover and utilize a large number of new USPs in order to clarify diseases caused by abnormal USPs, such as carcinogenesis and neurodegenerative diseases, and to prevent, treat and diagnose them. .

[0009]

[Non-Patent Document 1] "Febs Letters (FEBS L
etters) ", 1997, Volume 420, p. 25
−

[Non-Patent Document 2] “Oncogene (Oncogen)
e) ", 1993, volume 8, p. 2307-

[Non-Patent Document 3] "Oncogene
e) ”, 1995, Volume 10, p. 2179-

[Non-Patent Document 4] "Nature", 1
993, Volume 366, p. 313-

[Non-Patent Document 5] “Annual Review of Biochemistry (Annual Review of
Biochemistry) ", 1998, 67th.
Vol., P. 425-

[Non-Patent Document 6] "Proceeding of the National Academy of Sciences of the
United States of America (Proce
edings Of The National Ac
ademy OfSciences Of The U
nitted States of American
a) ", 1996, Vol. 93, p. 3275-

[Non-Patent Document 7] "Journal of Biological Chemistry"
ical Chemistry) ", 1997, second
72, p. 51-

[Non-Patent Document 8] "Embo Journal (EMBO J
), 1997, Vol. 16, p. 151
9-

[Non-Patent Document 9] "Trends In Neurosciences (Trends In Neuroscience)
s) ", 1998, Volume 21, p. 516-

[Non-Patent Document 10] "Nature",
1998, Volume 395, p. 451-452

[Non-Patent Document 11] "Nature Genetics (N
Nature Genetics) ", 1999, second
Volume 3, p. 47-51

[Non-Patent Document 12] "Embo Journal (EMBO
Journal) ", 1997, Volume 16, p. 482
6-

[Non-Patent Document 13] "Biochemical and Biophysical Research Communications (Bioc
chemical And Biophysical R
essearch Communications) ",
1999, Vol. 266, p. 633-

[Non-Patent Document 14] “Fasebu Journal (FASE
B Journal) ", 1997, Vol. 11, p.
1245-

[Non-Patent Document 15] "Critical Reviews in Biological and Molecular Biology (Critical Reviews In Bi
chemistry And Molecular
Biology) ", 1998, vol. 33, p. 33
7-

[Non-Patent Document 16] "Biochemistry (Bioch
), 1995, Vol. 34, p. 14
535-

[Non-Patent Document 17] Toshiaki Suzuki, Hideki Shimura, Nobutaka Hattori,
"Ubiquitin and neurodegenerative diseases", "Experimental medicine", 200
Year 0, Volume 18, p. 1478-1482

[Non-Patent Document 18] Toshiaki Suzuki, "Various Actions of Deubiquitinating Enzymes", "Experimental Medicine", 2001, Volume 19,
p. 193-

[Non-patent Document 19] Tadashi Anan, Mitsuyoshi Nakao, "Molecular Mechanism of Ubiquitin Disease", "Protein / Nucleic Acid / Enzyme", 1999,
Volume 44, p. 776-

[Non-Patent Document 20] "Biochemical Society
Transactions (Biochemical So
ciency Transactions) ", 1992
Year, Volume 20, p. 631-637

[Non-Patent Document 21] "Neurochemical Research (N
eurochemical Research) ", 1
997, Vol. 22, p. 345-350

[Non-Patent Document 22] "Molecular Biology Report (Molecular Biology Report)
orts) ”, 1997, Vol. 24, p. 35-38

[Non-patent document 23] "Current opinion in clinical nutrition and metabolic
Care (Current Opinion InClin
ical Nutrition And Metabo
lic care) ", 2001, Volume 4, p. 18
3-190

[Non-Patent Document 24] "BioEsses (BioEss
ays) ", 1992, Volume 14, p. 9-

[Non-Patent Document 25] "Proceeding of the National Academy of Sciences of the"
United States of America (Proce
edings Of The National Ac
ademy Of Sciences Of The
United States of American
a) ”, 1999, Vol. 96, p. 2828-

[Non-Patent Document 26] “Hugo Gene Nome Committee (Hugo Gene Nome)
nculture Committee) ”, <http
p: // www. gene. ucl. ac. uk / no
mencture>

[Non-Patent Document 27] "Journal of Biological Chemistry"
physical Chemistry) ", 2001, vol. 276, p. 20357-20363

[0010]

One of the problems to be solved by the present invention is to find a novel USP and enable the control of the USP in a living body. More specifically, it is to provide a USP having novel properties, and accordingly, a novel USP-derived polypeptide or peptide, a polynucleotide encoding them, and an antibody against the polypeptide or peptide. Is to provide. Furthermore, it is to identify an inhibitor, an antagonist, a promoter or the like of the expression of novel USP and its physiological activity, and to provide the identified compound. Another object is to provide a pharmaceutical composition using the above-mentioned polypeptide or peptide, the above-mentioned polynucleotide, the above-mentioned antibody, and the above-mentioned compound, and a method for measuring the above-mentioned polypeptide or peptide or the above-mentioned polynucleotide. Furthermore, it is to provide a method for producing a novel USP-derived polypeptide or peptide by a genetic engineering technique using the above-mentioned polynucleotide.

[0011]

Means for Solving the Problems The present invention provides (1) a polypeptide having deubiquitinating activity selected from the following groups; SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 in the sequence listing, A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, a polypeptide containing said polypeptide, and said polypeptide and at least about 70% amino acid sequence A polypeptide having homology and deubiquitinating activity, and a polypeptide having a mutation such as deletion, substitution, addition, or insertion of 1 to several amino acids in the amino acid sequence and having deubiquitinating activity Peptide, (2) a polypeptide selected from the following group, which is a ubiquitin (Ub) -bound glutathione S-transfectant: A polypeptide having an activity of dissociating Ub from an enzyme; described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 in the sequence listing. A polypeptide consisting of an amino acid sequence, a polypeptide containing the above polypeptide, a polypeptide having at least about 70% amino acid sequence homology with the above polypeptide and having deubiquitinating activity, and the above amino acid sequence A polypeptide having a mutation such as deletion, substitution, addition, or insertion of 1 to several amino acids and having deubiquitinating activity, (3) encoding the polypeptide of (1) or (2) above Or a complementary strand thereof, (4) deubiquitinating activity, and / or ubiquitin (Ub) Encoding a polypeptide having an activity to dissociate Ub from glutathione S- transferase, the sequence in the sequence listing No. 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a complementary strand thereof, (5) the above (3) or the above (4)
(6) A polynucleotide which hybridizes with the polynucleotide or a complementary strand thereof under stringent conditions, (6) A recombinant vector containing the polynucleotide according to any one of (3) to (5) above, (7) Recombination The recombinant vector according to (6) above, wherein the vector is an expression recombinant vector, (8) a transformant obtained by introducing the recombinant vector according to (6) or (7) above, (9) above (1) or The method for producing a polypeptide according to (2) above, which comprises culturing a transformant into which the recombinant vector according to (7) is introduced, or the recombinant vector according to (6) or (7) above. (10) an antibody that immunologically recognizes the polypeptide of (1) or (2) above, and (11) the antibody of (10) above Therefore, an antibody that suppresses deubiquitination activity, (12) a compound that interacts with the polypeptide of (1) or (2) above to inhibit or activate its physiological activity, and / or (3) ) To the compound of any one of the above (5), which inhibits or promotes the expression thereof, which comprises the polypeptide of (1) or (2) above, (3) above. Of the polynucleotide of any of (5), the recombinant vector of (6) or (7), the transformant of (8), and the antibody of (10) or (11) above. A method comprising using at least one of the above, (13) a compound which interacts with the polypeptide according to (1) or (2) above to inhibit or promote its physiological activity, and Alternatively, there is provided a method for identifying a compound that interacts with the polynucleotide according to any one of (3) to (5) above to inhibit or promote its expression, the interaction between the compound and the polypeptide or the polynucleotide. Under conditions that allow
By contacting the polypeptide or the polynucleotide with a compound, and then detecting the presence or absence or change of a signal caused by the interaction between the compound and the polypeptide or the polynucleotide, the compound Or a method for determining whether to inhibit or promote the physiological activity of the polypeptide or the expression of the polynucleotide by interacting with the polynucleotide, (14) the polypeptide according to (1) or (2) above, A method for identifying a compound that interacts with and inhibits or promotes its physiological activity, and / or a compound that interacts with the polynucleotide according to any one of (3) to (5) above to inhibit or promote its expression And contacting the transformant of the above (8) with a compound,
Using a system using a signal and / or marker capable of detecting the presence or absence of the expression or physiological activity of the polypeptide of (1) or (2) above, the presence or absence or change of this signal and / or marker is used. Is detected to determine whether the compound promotes or inhibits the expression or physiological activity of the polypeptide of (1) or (2), (15) the above (12) to (14) (16) a compound identified by any of the methods, (16) a compound which interacts with the polypeptide of (1) or (2) above to inhibit or activate deubiquitination activity, or (3) above To (17) a compound which interacts with any of the polynucleotides of the above (5) to inhibit or promote the expression thereof, (17) the above (1) or the above 2) polypeptide, or a polynucleotide of (5) from the (3), a recombinant vector of the (6) or (7), wherein (8)
(10) or the antibody of (11), and at least one of the compounds of (15) or (16). 18) The above (1) or (2)
The polypeptide of any of (3) to (5) above, the recombinant vector of (6) or (7) above, the transformant of (8) above, (10) above.
Or an agent for preventing and / or treating a neurodegenerative disease, which comprises the antibody of (11) above and at least one of the compounds of (15) or (16) above. ) The preventive and / or therapeutic agent for the neurodegenerative disease according to (18), wherein the neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease, (20) the polypeptide according to (1) or (2) above, 3) to the polynucleotide of any one of (5) above, the recombinant vector of (6) or (7) above, the transformant of (8) above, the antibody of (10) or (11) above, and (15) or (1)
6) A preventive agent and / or therapeutic agent for muscular atrophy, which comprises at least one of the compounds of 6), (21) the polypeptide of (1) or (2) above, 3) A method for quantitatively or qualitatively measuring the polynucleotide according to any one of 5) to 5),
2) From (12) to (14), and (2
A reagent kit for use in any one of 1),
The polypeptide of (1) or (2) above, the polynucleotide of any of (3) to (5) above,
It comprises the recombinant vector of (6) or (7), the transformant of (8), and a reagent kit containing at least one or more of the antibody of (10) or (11).

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION (New USP) Human USP provided in the present invention was selected as a gene having a protease motif from a human brain-derived long chain cDNA library of Kazusa DNA Research Institute or a GenBank human cDNA library. Eight kinds of genes, KIAA1
097, AK024318, KIAA1003, KIA
A1372, KIAA1453, KIAA1063, K
It is a protein encoded by IAA0190 and KIAA0891, respectively. These eight kinds of USPs were obtained by expressing in E. coli into which an expression plasmid incorporating each of the above genes was introduced. These USPs are known USPs
Similarly, it possesses a Cys domain and a His domain in its amino acid sequence. Each of the above eight types of USPs exhibits deubiquitinating activity by acting on the artificial substrate in an in vitro system for reacting the USP with the artificial substrate or when expressed with the artificial substrate in a gene co-expression system, Ub was dissociated. The above genes have already been disclosed in publicly known databases, but the enzymatic activity of these genes has not been confirmed. For the first time in the present invention, it was possible to obtain a protein having physiological activity by expressing these genes and confirming their enzymatic activity.

KIAA1097 is a USP having a molecular weight of 103 kDa, which is composed of 911 amino acid residues encoded by a gene having a base length of 4271 bp, and has a Cys box from 155th Gly to 170th Glu as an active motif. Had a His box from the 626th Tyr to the 643rd Tyr.
The amino acid sequence of KIAA1097 and the nucleotide sequence of its gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the sequence listing, respectively.
It was shown to. BLAST of amino acids estimated from ORF
In the homology search by search, KI described later
60% agreement with AA1003. KIAA1097 is
Ub-M-GST, Ub-R-GST and Ub-I-
In the co-expression system with GST, it acted on these substrates and exhibited deubiquitinating enzyme activity, but showed no enzyme activity against Ub-P-GST. Ub-M-GST is
It is an artificial substrate in which glutathione-S transferase (GST) is bound to the C terminus of Ub. Ub-R-G
ST, Ub-I-GST and Ub-P-GST are U
It is an artificial substrate in which GST was bound to the C-terminus of b via arginine (R), isoleucine (I), and proline (P), respectively. On the other hand, in the in vitro system, multi-ubiquitin chain (Multi Ubchain)
s), Ub-CEP52 and Ub-M-GST did not show deubiquitinating enzyme activity. In addition, K
It was found that the active center of IAA1097 is a cysteine in the Cys box (a cysteine at the 163rd amino acid sequence of KIAA1097: ¹⁶³ Cys).

KIAA1097 is highly expressed in the cerebellum, and subsequently highly expressed in the liver and ovary. Also, KIAA
TATA of AL138790 which is 1097 gene
About 400 bp upstream of the box, the DNA binding site consensus sequence of the androgen receptor (“Molecular Endocrinolo”).
gy) ", 1992, Volume 6, p. 2229-223
Half of 5) (GGTACA) was present. SAGE
According to the map information, the androgen analog compound R1881 (D
UPONT) stimulation of KIAA1097 mRNA
Induction of <http: // www. nc
bi. nlm. nih. gov / SAGE / SAGEt
ag. cgi? tag = ATGGCTTTGT>, KI
The involvement of the androgen receptor in the gene expression of AA1097 was suggested. On the other hand, it has been confirmed that the resistance to oxidative stress in rat cerebellar granule cells is enhanced by androgen treatment (“European Journal of Neuroscience”).
Journal Of Neuroscience) ",
1999, Volume 11, p. 1285-1291). These results are shown by androgen KIAA1097.
Expression is induced, which activates the ubiquitin system and protects cells from oxidative stress. In addition, KIAA1097 was predicted to be distributed in the nucleus in the cell, and USP1
It had the same Zn-finger domain as 6. These facts suggested a relationship between KIAA1097 and chromosome condensation.

Furthermore, the inventors of the present application have conducted KIAA1097.
Was found to bind to X11L (X11β, Mint2). That is, XII was determined by yeast two-hybrid screening using two types of baits of KIAA1097 (the 70th to 169th amino acids and the 70th to 369th amino acids).
Overlapping plays of the 284th to 554th amino acids and the 243rd to 415th amino acids of L (full length 749 amino acids) were selected. X11L is a protein that binds to Munc18-1 localized in synaptic vesicles (Journal of Biological Chemistry).
), 1997, vol. 272, p. 31
459-31464), and amyloid precursor protein (AP) which is one of the genes responsible for early-onset familial Alzheimer's disease.
(P) was identified as a protein that binds to the cytoplasmic domain (Journal of Biological Chemistry).
), 1999, vol. 274, p. Two
243-2254), an adapter protein specifically expressed in the central nervous system. Munc18-1 binding region at the 185th to 279th amino acids, the 368th to
PID which is the APP binding region at the 555th amino acid
P at the 568th to 654th amino acids in the domain
PDZ domain 2 exists in DZ domain 1 and amino acids 659 to 734. X11L is Mun
It is considered to be involved in exocytosis of synaptic vesicles by interacting with c18-1 (Journal
Of Biological Chemistry (Journal
of Biological Chemistr
y) ", 1997, vol. 272, p. 31459-3
1464). It is also known to regulate the metabolism of APP that undergoes membrane protein transport (“European Journal of Neuroscience”).
Journal of Neuroscience
e) ", 1999, Vol. 11, p. 1988-199
7). A Zn finger domain exists in the 70th to 169th amino acids, which is the common region of the two baits of KIAA1097 involved in the binding of X11L.
Since this part is considered to play an important role in deubiquitinating enzyme activity, X11L may have a function of regulating this enzyme activity in the brain. Also,
The play fragment of X11L is P, which is the APP binding domain.
Since it partially overlaps with the ID domain, KI
By interacting with X11L by AA1097, X
It may also regulate 11L regulation of APP metabolism. However, the PID domain of X11L is known to recognize the NPXY motif, but this motif does not exist in the binding domain on the KIAA1097 side.

AK024318 has a base length of 3176 bp.
Is a USP consisting of 355 amino acid residues encoded by the gene for Gl and has the 25th Gl as an active motif.
Cys box from y to 40th Gln, 28th
His b from the 5th Tyr to the 303rd Tyr
had ox. The amino acid sequence of AK024318 and the nucleotide sequence of its gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4 of the sequence listing, respectively. AK024318 is U
b-M-GST, Ub-R-GST and Ub-IG
In the co-expression system with ST, it acted on these substrates and exhibited deubiquitinating enzyme activity, but showed no enzyme activity against Ub-P-GST. On the other hand, in the in vitro system, multi-ubiquitin chain and Ub-CEP52
On the other hand, it showed no deubiquitinase activity. The active center of AK024318 was found to be cysteine in the Cys box (the 33rd cysteine in the amino acid sequence of AK024318: ³³ Cys).
AK024318 was strongly expressed in the heart, brain, placenta, skeletal muscle, kidney, pancreas, and lung by Northern blotting, particularly in the brain and placenta. The size of the gene detected in each organ was slightly different, suggesting the presence of splicing variants.

KIAA1003 has a base length of 4252 bp.
Is a USP having a molecular weight of about 105 kDa and consisting of 913 amino acid residues encoded by the gene for γ, and has an active motif of 146th Gly to 161st Gln.
Cys box from the 626th Tyr to 64th
It had a His box in the third Tyr. KIA
The amino acid sequence of A1003 and the base sequence of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6 of the sequence listing, respectively. KIAA1003 is Ub-M-GST, Ub-R
In the co-expression system with -GST and Ub-I-GST, it acted on these substrates and showed deubiquitinating enzyme activity, but showed no enzyme activity against Ub-P-GST. On the other hand, in the in vitro system, Ub-MG
It showed no deubiquitinase activity to ST.
The active center of KIAA1003 is cysteine in Cys box (amino acid sequence No. 15 of KIAA1003).
Fourth cysteine: ¹⁵⁴ Cys). KIAA
1003 was expressed in E. coli and was obtained as a soluble protein. Although KIAA1003 is expressed in various tissues, its expression in brain and ovary is slightly higher than that in other tissues.

KIAA1372 has a base length of 3771 bp.
Is a USP consisting of 749 amino acid residues with a molecular weight of about 86.8 kDa, which is encoded by the gene for γ.
Cys box for n, 5th to 576th Tyr
It had a His box at the 94th Tyr. KI
The amino acid sequence of AA1372 and the nucleotide sequence of its gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8 of the sequence listing, respectively. KIAA1372 is Ub-in vitro.
M-GST, Ub-R-GST and Ub-I-GST
And showed deubiquitinating enzyme activity on Ub-P
-No enzymatic activity was shown against GST. USP showing deubiquitinating enzyme activity in vitro
It is considered that the recombinant KIAA1372 tends to take an active form. In addition, KIAA137
The active center of 2 is cysteine in the Cys box (KIA
The cysteine at position 181 of the amino acid sequence of A1372:
¹⁸¹ Cys). Moreover, the expression of KIAA1372 is recognized in various tissues.

KIAA1453 has a base length of 5879 bp.
Is a USP consisting of 1121 amino acid residues encoded by the gene of, and the Cys box from the 123rd Gly to the 138th Gln as an active motif,
Hi from the 365th Tyr to the 383rd Tyr
s box. The amino acid sequence of KIAA1453 and the nucleotide sequence of its gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10 of the sequence listing, respectively. KIAA145
3 is Ub-M-GST, Ub-R-GST, Ub-I
In the co-expression system with -GST and Ub-P-GST, it acted on any of these substrates and showed deubiquitinating enzyme activity. On the other hand, in the in vitro system, Ub
-No deubiquitinating enzyme activity was shown against M-GST. The active center of KIAA1453 is Cys.
Cysteine in the box (cysteine 131st in the 131st amino acid sequence of KIAA1453: ¹³¹ Cys).
Although KIAA1453 is expressed in various tissues, its expression in testis is slightly higher than that of other tissues. In addition, the intracellular expression was found to be particularly higher in the nucleus than in the cytoplasm. Also, KIAA1453 is U
It has a homology of 33.8% with mouse DUB-2 reported to be SP (Non-patent Document 6). DUB
-2 is a protein whose expression is induced by interleukin-2 stimulation in T cells, and is considered to be involved in the regulation of the ubiquitination state of cell growth regulators (Non-Patent Document 6). That is, it was expected that the cell growth regulator was quantitatively regulated by the ubiquitin system. Moreover, since DUB-1 which shows high homology with DUB-2 and similarly has USP activity is induced by interleukin-3 (Non-patent Document 6), USP specifically induced by each cytokine is It is considered to be present, suggesting that KIAA1453 may be one of them.

KIAA1063 has a base length of 5223 bp.
Is a USP consisting of 514 amino acid residues encoded by the gene for G, and the 166th G as an active motif.
Cys box from ly to 181st Gln, His from 452nd Tyr to 469th Tyr
had a box. The amino acid sequence of KIAA1063 and the nucleotide sequence of its gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12 of the sequence listing, respectively. KIAA106
3 is Ub-M-GST, Ub-R-GST, and U
In the co-expression system with b-I-GST, it acted on any of these substrates and showed deubiquitinating enzyme activity.
It showed no enzyme activity against b-P-GST. On the other hand, the in vitro system showed no deubiquitinase activity against Ub-M-GST. In addition, K
The active center of IAA1063 is a cysteine in the Cys box (cysteine at the 174th amino acid sequence of KIAA1063: ¹⁷⁴ Cys). Also, KIAA1
Expression of 063 is recognized in various tissues.

KIAA0190 has a base length of 3280 bp.
It consists of 803 amino acid residues encoded by the gene
USP with a molecular weight of about 88 kDa
Then from the 421st Gly to the 436th Gln
Cys box from the 737th Tyr to the 755th
The second Tyr had a His box. KIAA
The amino acid sequence of 0190 and the nucleotide sequence of its gene
Shown in SEQ ID NO: 13 and SEQ ID NO: 14 of the sequence listing, respectively.
did. KIAA0190 is an in vitro and co-expression system
In both, Ub-M-GST, Ub-R-GST
And acts on Ub-I-GST to deubiquitinate the enzyme
, But showed enzymatic activity against Ub-P-GST.
Not shown. Deubiquitinating enzyme in vitro
There are few USPs showing activity, and the recombinant KI
AA0190 is considered to be easy to take an active form
It The active center of KIAA0190 is Cys bo.
Cysteine in x (amino acid sequence of KIAA0190
The 429th cysteine: ⁴²⁹Cys). Well
Moreover, KIAA0190 is highly expressed in skeletal muscle and testis.
Is shown. KIAA0190 is mouse US
P10 (P52479) and amino acids with a phase of about 80% or more
Having the same sex (Kazusa DNA Research Institute, HUGE Dat
a base <http: // www. kazusa.
or. jp / huge / gfpage>). Mouse US
For P10, currently only nucleotide sequence information has been published.
The function of is unknown. In addition, KIAA0190 is Hum
an Gene Nomenclature Data
Named and registered as USP10 on base
However, there are still no reports showing actual deubiquitinating enzyme activity.
However, the enzyme activity of the present invention was confirmed for the first time by the present invention.
It has been approved.

KIAA0891 has a base length of 4401 bp.
Is a USP consisting of 1318 amino acid residues encoded by the gene of the above and having a molecular weight of about 146 kDa, and has 498th Gly to 513th Gl as an active motif.
The nth had a Cys box, and the 1149th Tyr to the 1166th Tyr had a His box.
The amino acid sequence of KIAA0891 and the nucleotide sequence of its gene are shown in SEQ ID NO: 15 and SEQ ID NO: 16 of the sequence listing, respectively. KIAA0891 is Ub-M-GST, Ub-R-GST, Ub-IG in vitro.
It acted on both ST and Ub-P-GST substrates and showed deubiquitinating enzyme activity. In vitro, few USPs showed deubiquitinase activity, and it is considered that the recombinant KIAA0891 easily takes an active form. Also, in general, Ub-P-GS
It is considered that T is not cleaved by yeast USP and human USP shows a cleavage activity. Since KIAA0891 acted on Ub-P-GST and showed an enzymatic activity although it was weak, KIAA0891 was a substrate selection characteristic for humans similar to USP4 and USP15 known as USP showing Ub-P-GST activity. It is considered to have sex.
The active center of KIAA0891 is cysteine in Cysbox (amino acid sequence No. 506 of KIAA0891).
Th cysteine: ⁵⁰⁶ Cys). KIAA0
Although 891 is expressed in most tissues, its expression level is generally low (Kazusa DNA Research Institute, HUGE).
Data base, <http: // www. ka
zusa. or. jp / huge / gfpage / KI
AA0891>). In addition, toxicity to E. coli, which is considered to be due to overexpression of KIAA0891, was observed.
From these facts, KIAA0891 is an inducible USP that acts specifically during inflammation and biological invasion, in addition to controlling proteolysis on a daily basis, and its overexpression disturbs intracellular homeostasis and causes apoptosis. Can cause.

(Polypeptide or Peptide) The polypeptide according to the present invention is a gene product of any of the above eight kinds of USP genes, and is a polypeptide obtained by expressing the gene in cells such as Escherichia coli. . Here, the polypeptide means a long-chain peptide such as a protein among any peptides containing two or more amino acids bound to each other by a peptide bond or a modified peptide bond, and it means oligopeptide and Short peptides, also called oligomers, are simply called peptides. In the present specification, amino acids may be represented by three letters or one letter.

The polypeptide according to the present invention comprises the amino acids described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15. It can be a polypeptide consisting of a sequence. The polypeptide according to the present invention has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. It may be a polypeptide containing a polypeptide consisting of

The polypeptide according to the present invention is described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 in the sequence listing. 70% or more, preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more homologous to the polypeptide of the above and having deubiquitinating activity. It may be one. The deubiquitinating activity is, for example, as shown in Examples described later, an artificial substrate such as human ubiquitin having CST bound to GST, or ubiquitin bound to GST via arginine, isoleucine, or proline. Whether or not ubiquitin can be dissociated from the substrate using
It can be measured by detecting ST by a known method such as an immunoblotting method using an anti-GST antibody. Techniques for determining the homology of amino acid sequences are known per se, and for example, a method of directly determining the amino acid sequence, a method of determining the base sequence of cDNA, and then deducing the amino acid sequence encoded thereby, and the like are possible. Note that homologous gene products of animal species other than human are naturally included in the scope of the present invention.

Further, based on the thus-identified polypeptide, the deubiquitinating activity is used as an index to determine one or more, for example, 1 to 100, preferably 1 to 30, more preferably 1 to 20
There is also provided a polypeptide comprising an amino acid sequence having a mutation such as deletion, substitution, addition, or insertion of one, more preferably one to ten, and particularly preferably one to several amino acids. Deletion, substitution, addition, or means of insertion is known per se, for example, site-directed mutagenesis method, gene homologous recombination method, primer extension method or polymerase chain amplification method (PCR) alone or in appropriate combination, For example, Sambrook et al., "Molecular Cloning Laboratory Manual", 2nd
Edition, cold_spring_harbor_laboratory_
Press, 1989, edited by Masamitsu Muramatsu, "Lab Manual Genetic Engineering", Maruzen Co., Ltd., 1988, edited by Ehrlich, "PCR Technology",
"Principles and Applications of DNA Amplification", Stockton Press, 1
It can be carried out according to the method described in the publication such as 989 or by modifying those methods. For example, Ulmer technology ("Science (Science)
e) ", 1983, Volume 219, p. 666-) can be used.

From the viewpoint of not changing the basic properties (physical properties, physiological activity, immunological activity, etc.) of the polypeptide upon introduction of the mutation as described above, for example, a cognate amino acid (polar amino acid, non-amino acid, non-amino acid) Mutual substitution between polar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned. Furthermore, these peptides can be modified to the extent that they do not cause a significant change in function, such as modification of the constituent amino group or carboxyl group. According to the present invention, it is possible to provide a polypeptide having an activity equivalent to the physiological activity possessed by the above eight kinds of USPs, for example, a deubiquitinating activity, but in addition to these, a polypeptide having a modified activity intensity or substrate specificity is also available. Can be provided.

The present invention also comprises the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 in the sequence listing. The polypeptide or the peptide having a partial sequence of the polypeptide consisting of the amino acid sequence described in the following polypeptide is included. The minimum unit of the polypeptide or peptide having the partial sequence is 5 or more amino acids, preferably 8 or more amino acids,
More preferably, it consists of 12 or more, and even more preferably 15 or more consecutive amino acids. For example, the present invention also provides a polypeptide or peptide comprising a minimum activity unit (region or domain) of physiological activity possessed by any of the above eight types of USPs. The polypeptide or peptide having the above-mentioned partial sequence is a reagent used for identifying a substance that regulates the activity of any of the above-mentioned 8 types of USPs or the physiological activity equivalent to the USP, for example, the activity of the above-mentioned polypeptide having deubiquitin activity. Is useful as Further, in the case of a peptide that can be immunologically recognized, for example, an epitope peptide, as will be described later, alone or as a carrier (for example, as an antigen for producing an antibody specific to any of the above eight types of USPs, Keyhole limpate hemocyanin or ovalbumin) can be used in combination.

The scope of the present invention also includes proteins to which different kinds of proteins or substances such as carriers are bound as described above. For example, in order to facilitate the detection or purification of the polypeptide or the like according to the present invention, or to add another function, a protein or peptide of another species such as alkaline phosphatase, β is added to its N-terminal side or C-terminal side. -Galactosidase, immunoglobulin Fc fragment such as IgG, or FLAG-tag can also be added directly or indirectly through a linker peptide or the like using a known method such as a genetic engineering method.

(Polynucleotide) The present invention provides the above-mentioned polypeptide or a polynucleotide encoding the above-mentioned peptide and a complementary strand thereof. For example, the polynucleotide according to the present invention has SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 in the sequence listing.
It may be a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 13, or SEQ ID NO: 15, or its complementary strand. Preferably, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 in the sequence listing,
A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a complementary strand thereof.

The present invention further provides the above polynucleotide or its complementary strand, preferably SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 in the Sequence Listing. Alternatively, the present invention provides a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 or a polynucleotide that hybridizes to the corresponding region of the complementary strand thereof under stringent conditions. The hybridization conditions can be based on, for example, Sambrook et al., "Molecular Cloning Laboratory Manual", Second Edition, Cold_Spring_Harbor_Laboratory_Press, 1989.
These polynucleotides are the polynucleotide of interest,
Preferably SEQ ID NO: 2, SEQ ID NO: 4 of the sequence listing,
Sequence number 6, sequence number 8, sequence number 10, sequence number 1
2, SEQ ID NO: 14, or SEQ ID NO: 16, or any complementary sequence so long as it hybridizes to its complementary strand. For example, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 1 in the sequence listing
0, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16
The homology to the polynucleotide consisting of the nucleotide sequence described in 1 above or its complementary strand is at least about 70% or more, preferably about 80% or more, more preferably about 90%.
Above, more preferably about 95% or more. In addition, the polynucleotide according to the present invention has 10 consecutive nucleotides corresponding to the designated nucleotide sequence region of the above-mentioned polynucleotide.
Polynucleotides or oligonucleotides consisting of sequences of 1 or more nucleotides, preferably 15 or more, more preferably 20 or more, or their complementary strands are included.

These polynucleotides provide genetic information useful for the production of the polypeptide of the present invention, or can be used as reagents or standard products for nucleic acids. For example, a nucleic acid encoding any of the above eight types of USPs, for example, the gene or mR thereof.
It can be used as a probe or primer for the detection of NA, or as an antisense oligonucleotide for regulating gene expression. In that sense, the polynucleotide and the oligonucleotide according to the present invention include not only the translated region but also those corresponding to the untranslated region. Here, the selection of a polynucleotide encoding any of the above eight kinds of USPs or a polypeptide having the same physiological activity as the USP can be performed by, for example, confirming the expressed protein using a known protein expression system. Done,
The physiological activity, for example, deubiquitination activity can be used as an index. As a known protein expression system, for example, a cell-free protein expression system utilizing the technique of a ribosome system derived from embryos or rabbit reticulocytes (“Nature (N
Aure), 1957, Volume 179, p. 160-
161) can be illustrated.

(Recombinant Vector) A recombinant vector can be obtained by incorporating the above-mentioned polynucleotide into an appropriate vector DNA. The vector DNA used is appropriately selected depending on the type of host and the purpose of use. Vector D
The NA may be one obtained by extracting a naturally occurring one, or may be one in which a part of DNA other than the part necessary for growth is partially deleted. For example, vectors derived from chromosomes, episomes and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, such as baculovirus, papovavirus, SV.
40, vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors combining them, such as plasmids and vectors derived from genetic elements of bacteriophage, such as cosmids and phagemids. Etc. Moreover, an expression vector, a cloning vector, or the like can be used depending on the purpose.

The recombinant vector comprises, as constituent elements, a gene sequence of interest and a gene sequence carrying information relating to replication and control, for example, a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer, etc., and these are known per se. It is created by combining the methods. As a method for incorporating the polynucleotide of the present invention into the vector DNA, a method known per se can be applied.
For example, a method is used in which an appropriate restriction enzyme is selected and treated to cut the DNA at a specific site, which is then mixed with DNA similarly used as a vector and religated with ligase. Alternatively, the desired recombinant vector can also be obtained by ligating an appropriate linker to the polynucleotide of interest and inserting this into the multi-cloning site of the vector suitable for the purpose.

(Transformant) A vector DNA having the above-mentioned polynucleotide incorporated therein is introduced into a host known per se, such as Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells, etc., by a method known per se. A transformant is obtained. In the case of introducing a gene, a more preferable system is the method of integrating into the chromosome if the stability of the gene is taken into consideration. For convenience, an autonomous replication system utilizing an extranuclear gene can be used. Introduction of vector DNA into a host cell is standard, for example, described in Sambrook et al., "Molecular Cloning Laboratory Manual", 2nd edition, Cold_Spring_Harbor_Laboratory_Press, 1989. It can be performed by a method. Specifically, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading (scrape).
loading, ballistic introduction (ballis)
tic induction) and infection.

Further, by using an expression vector as the vector DNA to be introduced into the host, the polypeptide or peptide according to the present invention can be provided. The transformant introduced with the expression vector DNA incorporating the above-mentioned polynucleotide is cultivated by selecting the optimum conditions for culturing conditions of each host known per se. The culturing may be performed using the action of the polypeptide or peptide of the present invention expressed by the transformant, for example, at least the deubiquitinating activity or the amount of the polypeptide or peptide produced in or outside the host as an index. However, subculture or batch culture may be carried out using the amount of transformant in the medium as an index.

(Recovery of USP and its Derivatives) The polypeptide or peptide according to the present invention can be recovered from the medium in which the above transformant is cultured, for example, by using at least deubiquitination activity as an index, molecular sieve, ion column chromatography. It can be purified and recovered by a combination of chromatography, affinity chromatography and the like, or by fractionating means such as ammonium sulfate and alcohol based on the difference in solubility. Preferably, a method is used in which a polyclonal antibody or a monoclonal antibody specific to these is prepared based on the information of the amino acid sequence of the peptide to be recovered, and the antibody is specifically adsorbed and recovered using the antibody.

(Antibody) An antibody is prepared by using the above-mentioned polypeptide or the above-mentioned peptide as an antigen. The antigen may be the above-mentioned polypeptide or the above-mentioned peptide or a fragment thereof, and at least 8, preferably at least 10
, More preferably at least 12 and even more preferably 15 or more amino acids. To generate an antibody specific to any of the above eight types of USP, U
It is preferable to use a region consisting of an amino acid sequence unique to any of the above eight types of USPs, other than the conserved region between SP families. The amino acid sequence of this region is not necessarily homologous to those described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 in the sequence listing. It is not necessary to be present, and an exposed site on the outside of the protein on the three-dimensional structure is preferable, and even if the amino acid sequence of the exposed site is discontinuous on the primary structure, it may be a continuous amino acid sequence for the exposed site. The antibody is not particularly limited as long as it immunologically binds or recognizes a peptide or polypeptide consisting of any of the above eight types of USPs and their derivatives. The presence or absence of this binding or recognition is determined by a known antigen-antibody binding reaction.

In order to produce the antibody, a method for producing an antibody known per se can be used. For example, an antibody can be obtained by administering the above-mentioned antigen, alone or in combination with a carrier, to an animal in the presence or absence of an adjuvant to induce immunity such as humoral response and / or cellular response. The carrier is not particularly limited as long as it does not cause a harmful effect on the host, and examples thereof include cellulose, polymerized amino acids, albumin and the like. As the animal to be immunized, mouse, rat, rabbit, goat, horse, etc. are preferably used.

The polyclonal antibody is obtained from the serum of the animal immunized as described above by a method known per se for antibody recovery. As a preferable means, it is obtained by an immunoaffinity chromatography method.

In order to produce a monoclonal antibody,
Antibody-producing cells (for example, spleen or lymph node-derived lymphocytes) are collected from the animal to which the above-mentioned immunization means has been applied, and permanently proliferating cells known per se (for example, P3-X63-Ag).
8 strains such as myeloma strain). For example, an antibody-producing cell and a permanently proliferating cell are fused by a method known per se to prepare a hybridoma, which is then cloned, and a hybridoma producing an antibody that specifically recognizes the above-mentioned polypeptide or the above-mentioned peptide is selected. The antibody is recovered from the culture medium of the hybridoma.

The thus obtained polyclonal antibody or monoclonal antibody which recognizes and binds to the above-mentioned polypeptide or peptide can be used as an antibody for purification of the above-mentioned polypeptide or peptide, a reagent, a labeling marker or the like. For example, among the polyclonal or monoclonal antibodies that recognize and bind to the above-mentioned polypeptide or peptide, any USP according to the present invention can be directly used.
An antibody that binds to and suppresses its deubiquitinating activity is
It is useful for the prevention and / or treatment of various diseases caused by abnormal USP, such as neurodegenerative diseases and muscular atrophy.

(Screening) The polypeptide or peptide of the present invention, the polynucleotide of the present invention and its complementary chain, a vector incorporating the polynucleotide and its complementary chain, a transformant obtained by introducing the vector, or A protein synthesis system using these, and an antibody that immunologically recognizes the polypeptide or the peptide, can be used alone or in combination of two or more thereof to inhibit the activity or enhancer of the activity of the polypeptide or peptide, and / or Provided is a method effective for identifying an expression inhibitor or expression promoter of a polynucleotide. The method can be carried out using a drug screening system known per se. According to the identification method of the present invention, selection of antagonists by drug design based on the three-dimensional structure of the peptide or polypeptide, selection of expression regulator at the gene level using a protein synthesis system,
It is possible to select an antibody recognizing substance using an antibody.

For example, using the polypeptide or peptide according to the present invention, conditions that allow the interaction between the test compound and these polypeptides or peptides are selected, and under the conditions, these polypeptides or peptides are selected. By contacting the compound with the compound and detecting the presence, absence, or change of a signal generated by the interaction, the USP of any of the above eight types or the U
It is possible to identify a compound that promotes or inhibits the activity of a polypeptide having a physiological activity similar to SP, for example, the deubiquitinating activity.

Further, the conditions that allow the interaction between the polynucleotide of the present invention and the test compound are selected,
A compound that binds to these polynucleotides can be identified by contacting these polynucleotides with the compound under such conditions and detecting the presence, absence, or change in the signal caused by the interaction. From the obtained compound, a compound that inhibits the expression of any of these polynucleotides, for example, any of the above eight kinds of USPs can be identified by the following method using a transformant.

Furthermore, the transformant of the present invention is used to bring a test compound or the above-identified compound into contact with it under appropriate conditions to determine whether or not any of the USPs of the present invention is expressed or altered. To detect the USP
It is possible to identify compounds that inhibit or promote the expression of. The detection of the presence or absence or the change of the expression of the USP is conveniently carried out, for example, by using the enzymatic activity of the expressed USP, for example, the deubiquitination effect as an index, for example, the artificial substrate Ub-RG.
It can be carried out by measuring GST generated by decomposition of ST. Further, in order to detect the presence or absence or the change of expression of any of the above eight types of USP, a system known per se using a signal or marker for detection is introduced, and the presence or absence of this signal or marker or Changes may be detected. Here, the signal is
The substance itself can be directly detected by its physical or chemical properties, and the marker can be indirectly detected by using its physical or biological properties as an index. Known signals such as luciferase or green fluorescent protein (GFP) as a signal, a reporter gene such as a chloramphenicol acetyltransferase (CAT) gene, or a detection tag such as a 6 × His tag are used as markers. Available. A vector incorporating these signals or markers may be prepared, and the vector may be introduced into a host cell to prepare a transformant. Further, methods for detecting these signals or markers are well known to those skilled in the art.

Specifically, for example, an experimental system for measuring the deubiquitinating activity of the USPs by co-expressing any of the above 8 types of USPs with a substrate, for example, in Escherichia coli according to the Examples described later. In the above, by adding a test compound here, a compound that inhibits or promotes the expression or physiological activity of the USP can be identified. This experimental system explains one of the identification methods, and the identification method of the compound according to the present invention is not limited to this.

(Compound) The compound identified by the above method is an inhibitor, antagonist, or the like of any of the above eight kinds of USPs or a polypeptide having physiological activity similar to the USP, for example, deubiquitination activity. Alternatively, it can be used as a candidate compound for an accelerator. It can also be used as a candidate compound for an inhibitor, an antagonist, or an accelerator for the expression of any of the above eight kinds of USPs at the gene level or a polypeptide having the same physiological activity as the USPs. These candidate compounds are various diseases caused by expression or physiological activity of any of the above eight kinds of USPs and polypeptides having the same physiological activity as the USPs, for example, increase, decrease or deletion of deubiquitination activity. The preventive effect and / or the therapeutic effect of the physical symptoms can be expected. As will be described later, since there is a possible relationship between USP and neurodegenerative diseases and muscular atrophy, agents for preventing these diseases and / or
Or it can be used as a therapeutic agent.

(Pharmaceutical Composition) The candidate compounds thus selected can be prepared as a pharmaceutical composition by further selection in consideration of the balance between biological utility and toxicity. In addition, the new US according to the present invention, which is one of the above eight types
A polypeptide or peptide comprising P and a derivative thereof, a polynucleotide according to the present invention or a complementary strand thereof,
A vector containing the polynucleotide or its complementary strand,
And an antibody that immunologically recognizes a polypeptide or peptide consisting of any of the above eight types of USPs and their derivatives, is itself a USP of any of the above eight types.
And expression of a polypeptide having physiological activity similar to that of the USP and increase in physiological activity, for example, deubiquitination activity,
It can be used for prevention and / or treatment of various pathological symptoms caused by reduction or deletion. That is, the present invention, by using these alone or in combination,
There is provided a pharmaceutical composition containing at least one of these. Upon formulation, a formulation means suitable for each object such as a polypeptide, peptide, protein, polynucleotide, antibody, etc. known per se may be introduced.

One of the methods for treating abnormal symptoms caused by the decrease or deletion of any expression of USP and its physiological activity according to the present invention is to use the USP itself or the same physiological activity as the USP. A method comprising administering a therapeutically effective amount of a compound (promoter) that activates a polypeptide having the same or a gene encoding the USP, together with a pharmaceutically acceptable carrier, thereby improving abnormal symptoms. Is mentioned. Alternatively, gene therapy may be used to produce the USP within cells in the subject. Gene therapy using the above polynucleotide,
Although known methods can be used, for example, a replication-defective retrovirus vector incorporating the polynucleotide of the present invention as described above may be prepared and used for gene therapy. In addition, for example, by using a DNA or RNA encoding a protein, for example, by using a retrovirus plasmid vector, ex vivo
It is also possible to treat the cells from the subject in o) and then introduce the cells into the subject.

When the expression of any of the USPs according to the present invention and its physiological activity are excessive, an effective amount of the above inhibitor compound is administered to a subject together with a pharmaceutically acceptable carrier to inhibit the activity of the USP, It can also improve abnormal symptoms. Alternatively, the expression block method may be used to inhibit the expression of the gene encoding the endogenous USP. The expression of the gene can be inhibited using the antisense sequence of the gene produced intracellularly or administered separately. These oligonucleotides can be obtained by designing and synthesizing the above-mentioned polynucleotide according to the present invention. The oligonucleotide can be administered per se or the relevant oligomer can be expressed in vivo.

Dysfunction such as increase, decrease or deletion of USP expression and activity causes abnormality of ubiquitin system associated with USP and eventually causes pathological symptoms. For example, it has been suggested that a USP abnormality is associated with carcinogenesis or a neurodegenerative disease (Non-Patent Document 17). USP is also involved in maintenance of chromosomal structure, and it is known that deubiquitination of ubiquitinated histones is important for chromosome aggregation (Non-patent Document 24), and USP family 1
It has been reported that USP16, which is one of them, deubiquitinates H2A (Non-patent Document 25). Further, many protein aggregates observed in Alzheimer's disease and Parkinson's disease react with anti-ubiquitin antibody (Non-patent Document 17), which suggests a relationship between neurodegenerative disease and USP dysfunction. Expression of USP according to the present invention in various tissues has been confirmed, and particularly KIAA1097 and AK024318 have strong expression in the brain. In addition, in cells, KIAA1097, KIAA1003,
KIAA1372, KIAA1063, and KIAA
In each of 0190, about 50% to about 65% of the expression level is K
IAA089 and AK024318 are each about 26
% And about 39% were found in the nucleus. Further KIA
About 87% of the expression amount of A1453 was found in the nucleus.
Therefore, the present invention is to elucidate the biological functions involved in USP,
For example, elucidation of carcinogenic processes, elucidation of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and elucidation of muscular atrophy, development of their preventive agents and / or therapeutic agents, and assay methods used as their diagnostic means. It is very useful in enabling development.

As described above, the inventor of the present application is KIAA1
It was found that 097 binds to X11L in the yeast two-hybrid system as shown in Example 1. Therefore,
It is considered that this binding has high specificity and can be sufficiently reproduced in vivo. This bond plays a central role in the fusion of synaptic vesicles and presynaptic membranes.
It is an important factor in unc18-1 complex formation.
By inhibiting this binding, abnormalities in synaptic transmission may be ameliorated to normal, and therefore inhibitors of this binding are
Can be a therapeutic agent. Furthermore, because X11L is a protein that binds to the cytoplasmic domain of the amyloid precursor protein and is an adapter protein that is specifically expressed in the central nervous system, it inhibits the production of X11L and mutation of the part involved in binding. Can inhibit the formation of amyloid precursor protein complex towards Alzheimer's disease.

The administration form may be systemic administration or local administration. One preferred aspect of systemic administration is injection,
Examples include intravenous injection. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another mode of administration is oral administration, provided that the enteric or capsule formulation is successfully formulated. In addition, transmucosal or transdermal administration with penetrants such as bile salts, fusidic acids, or other surfactants can be used.
For topical administration, forms such as salves, pasta and gel can be used.

The dose range required is a polypeptide or peptide comprising USP according to the present invention and its derivatives,
Polynucleotides encoding them or a complementary chain thereof, a vector containing the polynucleotide or a complementary chain thereof, an antibody that immunologically recognizes a polypeptide or peptide consisting of novel USP and its derivative, the above compound, and the above pharmaceutical composition The efficacy of the product, the route of administration, the nature of the formulation, the nature of the subject's symptoms, and the judgment of the attending physician. Specifically, an appropriate dose is, for example, a subject's body weight of 1 k.
The range is 0.1 to 100 μg per gram. However, these dosage variations can be made using routine routine experimentation for optimization well known in the art.

For formulation, for example, peptides, proteins, oligonucleotides, polynucleotides, antibodies,
A known formulation means may be introduced according to the physical properties of each target such as a compound. Specifically, for example, a method for formulating powders, pills, tablets, capsule formulations, aqueous solution formulations, ethanol solution formulations, liposome formulations, fat emulsions, inclusion bodies such as cyclodextrins, etc. can be used.

Powders, pills, capsules and tablets can be used as excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol. , A hydroxypropyl cellulose, a binder such as gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like. Solid pharmaceutical carriers are used to manufacture tablets and capsules.

The suspension can be produced using water, sugars such as sucrose, sorbitol and fructose, glycols such as PEG and oils.

Injectable solutions can be prepared using carriers that consist of salt solutions, glucose solutions, or a mixture of saline and glucose solutions.

To form liposomes, for example, a solution prepared by dissolving the substance in a solvent (such as ethanol) is added to a solution prepared by dissolving a phospholipid in an organic solvent (such as chloroform), and then the solvent is distilled off. It can be carried out by adding a buffer solution, shaking, sonicating and centrifuging, and then filtering and collecting the supernatant.

Fat emulsification is carried out by, for example, the substance, oil component (soybean oil, sesame oil, vegetable oil such as olive oil, MCT).
Etc.), an emulsifier (phospholipid, etc.) and the like, and after heating to form a solution, add the required amount of water and emulsify using a homogenizer (homogenizer, such as high-pressure jet type or ultrasonic type).
It can be performed by homogenizing. It is also possible to freeze-dry this. When emulsifying into a fat, an emulsification aid may be added, and examples of the emulsification aid include glycerin and sugars (eg glucose, sorbitol, fructose, etc.).
Is exemplified.

For inclusion of cyclodextrin, for example, a solution prepared by dissolving cyclodextrin in water or the like is added to a solution prepared by dissolving the substance in a solvent (ethanol or the like), then cooled and the deposited precipitate is filtered. Can be sterilized and dried. At this time, the cyclodextrin used is
Cyclodextrins (α, β, γ type) having different void diameters may be appropriately selected according to the size of the substance.

(Determination Method and Reagent for Diagnosis) A polypeptide or peptide comprising any USP according to the present invention and its derivative, a polynucleotide according to the present invention and its complementary chain, and the USP and its derivative. An antibody that immunologically recognizes a polypeptide consisting of or is a quantitative marker for any USP according to the present invention and a polypeptide derived therefrom, or a polynucleotide encoding these, as a diagnostic marker or reagent. Or qualitatively.
The present invention also provides a reagent kit comprising one or more containers filled with one or more of these. It should be noted that upon formulation, a formulation means suitable for each known polypeptide or peptide, protein, polynucleotide, antibody or the like may be introduced. According to the above-mentioned measuring method, it becomes possible to diagnose various pathological symptoms caused by the increase, decrease or deletion of the expression or activity of any of the above 8 kinds of USPs.

A diagnostic means for a disease caused by abnormal expression or physiological activity of a peptide or polypeptide comprising any of the USPs and its derivatives according to the present invention is, for example, an interaction with a nucleic acid encoding the USP. And / or reactivity to determine the abundance of the corresponding nucleic acid and / or to determine the biodistribution in the individual for the USP, and / or the presence of the USP, in a sample derived from the individual. This is done by determining the abundance. Specifically, any of the above eight types of USPs is assayed as a diagnostic marker. Assays that can be used to detect or determine the abundance of the USP of interest in a sample are well known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, Western Blot analysis and ELISA assays. Examples of the method for detecting and quantifying the polynucleotide encoding USP according to the present invention include amplification, PCR, reverse transcription polymerase amplification reaction (RT-PCR), RNase protection, Northern blotting, and other hybridization methods. It can be used to measure at the RNA level.

As a sample to be measured, cells derived from an individual,
For example, blood, urine, saliva, spinal fluid, tissue biopsy or autopsy material and the like can be mentioned. The nucleic acid to be measured can be obtained from each of the above samples by a nucleic acid preparation method known per se. Nucleic acids may use genomic DNA directly for detection, or may be enzymatically amplified by using PCR or other amplification methods prior to analysis. RNA or cDNA
A may be used as well. Deletions and insertions can be detected by a change in the size of the amplification product in comparison to the normal genotype. The above USP labeled with amplified DNA
Point mutations can be identified by hybridizing to DNA encoding

By detecting a mutation, a decrease or an increase in the USP according to the present invention and the DNA encoding the USP by the above-mentioned measurement, a disease caused by the abnormality of the USP, such as cancer or neurodegenerative disease, such as Diagnosis of Alzheimer's disease, Parkinson's disease, etc. becomes possible.

[0067]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 (Isolation / Identification of KIAA1097) KIA
A1097 cDNA is obtained by bioinformatics from the human long-chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
1097 is ORF predictive analysis software Gene Mark
Analysis by Analysis suggested that the N-terminal may be deleted (Kazusa DNA Research Institute, HUGE).
Data base, <http: // www. kaz
usa. or. jp / huge / gfpage / KIA
A1097>). Therefore, an attempt was made to further extend the N-terminal by an EST search using BLAST search, but no translation initiation site (Met) could be confirmed in the upstream base sequence. In addition, the cDNA of KIAA1097 is genomic AL1.
Found to match 38790, KIAA1097
AL13879 corresponding to about 300 bp upstream of cDNA
It was confirmed that the TATA box (TATAAT) was present at the 0 position. Since there is no Met codon between the AL138790 base sequences from the KIAA1097 cDNA to the TATA box, pBluescript II SK (+) was obtained from Kazusa DNA Research Institute.
It was revealed that the KIAA1097 cDNA obtained as a plasmid having the SalI-NotI site inserted thereinto was full-length. Therefore, the 210th to the 212th ATG (s) in the nucleotide sequence published in GenBank (
²¹⁰ ATG ^{21 2)} was used as a translation initiation site, following
²⁹⁴³ TAA Area up to ²⁹⁴⁵ ORF (2.7K
b).

KIAA1097 has an estimated amino acid number of 91
1, thought to be USP with a molecular weight of 103 kDa, and Cys box ¹⁵⁵ GLKNIGNTC as an active motif.
YMNAALQ ¹⁷⁰ (SEQ ID NO: 17) (G- [LIV
MF [-x (1,3)-[AGC [-[NASM [-x
-C- [FYW [-[LIVMFC [-[NST [-
[SACV [-x [LIVMS [-Q) and His bo
x ⁶²⁶ YDLLSVICHHGTASSGHY
⁶⁴³ (SEQ ID NO: 18) (YxL- [SAG [-
[LIVMFT [-x (2) -Hx-G-x (4
5) -G-H-Y). A homology search by BLAST search for the amino acid deduced from the ORF is considered to be one of the human USPs, KI.
60% agreement with AA1003. Also, KIAA109
About 400 bp upstream of the TATA box of AL138790, which is a 7-gene, a DNA binding site consensus sequence of the androgen receptor (“Molecular Endocrinology”).
gy) ", 1992, Volume 6, p. 2229-223
Half of 56) (GGTACA) was present. SAG
According to Emap information, in the prostate cancer-derived cell line LNCa cells, the androgen analog compound R1881 (DU
PONA) stimulated KIAA1097 mRNA
Induction has been recognized ("Serial Analysis of Gene Expression Tag to Gene Mapping (Serial Analysis of
Gene Expression Tag to G
ene Mapping (SAGEmap) ", <ht
tp: // www. ncbi. nlm. nih. gov
/ SAGE / SAGEtag. cgi? tag = ATG
It was suggested that the androgen receptor is involved in the gene expression of GCTTTGT>) and KIAA1097.

KIAA obtained from Kazusa DNA Research Institute
In order to confirm that the 1097 clone was present in the actual human brain mRNA, the ORF region of KIAA1097 was amplified by RT-PCR, part of the sequence was performed, and the nucleotide sequence was compared with the published data. Human brain pol
y (A) ⁺ RNA (CLONTECH) to TaKa
CDNA was prepared using the Ra RNA PCR-kit (AMV.2.1), and a template (te) for gene confirmation was prepared.
plate). O of KIAA1097 cDNA
About 500-80 for bp201-bp2974 including RF
Divide into 4 regions of 0 bp (No. 1 to No. 4), design primers for each region (Table 1), and perform PCR (Klen T
aq-1, Advantage cDNA polymerase
Mix, CLONTECH). The resulting PCR product was cloned into pCR2.1
Vector (Original TA Cloning
Kit, Invitrogen), and the nucleotide sequence was confirmed by sequence. The sequence is a universal primer (M-
13 reverse primer, T7 promoter primer, M13 forward primer).
The sequence reaction is a Thermo Sequence Cy5.5 Terminator Sequencing Kit (Thermos
equipment Cy5.5 Terminator
Sequencing kit) (Amersham
Pharmacia biotech) and Big Dye Terminator Cycle Sequencing FS
Ready Reaction Kit (BigDye Te
rminator Cycle Sequencing
FS Ready Reaction Kit (Am
ersham pharmacia biotech)
Was carried out.

[0070]

[Table 1]

Ge of the nucleotide sequence obtained by the sequence
The homology processing with the nBank information is performed by the genetic information processing software Genetyx-WIN Version.
3 (Software Development Co., Ltd.).

PCR fragment No. 2 to No. About 4,
The base sequence in the sequenced range was identical to that published in GenBank. However, PCR fragment No.
In 1, the 837th T is the human cDNA adjusted this time
Has been converted to C, which is different from the public information. Such base substitution of T837C does not affect Cys box and His box, but causes substitution of 210th Y to H in the amino acid sequence (Y210H). The base sequence of KIAA1097 is NEDO hu
man cDNA sequencing process
It is also registered in GenBank as AK022864 from ct. In AK022864, the 837th nucleotide sequence is C and the amino acid is H in the same manner as the sequence result.
Is coded. Therefore, in the present invention, human cDNA
It was also suggested that the base substitution confirmed by the sequence of A may be single nucleotide polymorphism (SNPs).

KIAA1097 is HUGE Database <http: // w of Kazusa DNA Research Institute
ww. kazusa. or. jp / huge / gfpa
According to ge / KIAA1097>, RT-PCR results show that the expression is high in the cerebellum, followed by high expression in the liver and ovary. In addition, a database of 3 terminal fragments of cDNA, BODY MAP <http: // bodyma
p. ims. u-tokyo. ac. jp> also shows the expression of KIAA1097 in the cerebellum. In addition, about 400 bp upstream of the TATA box of AL138790, which is a KIAA1097 gene, half of the DNA binding site consensus sequence of the androgen receptor (G
GTACA) was present. In the SAGEmap information, induction of KIAA1097 mRNA was observed in the prostate cancer-derived cell line LNCaP cells upon stimulation with the androgen analog compound R1881 (DUPONT), suggesting the involvement of the androgen receptor in KIAA1097 gene expression. On the other hand, it has been confirmed that resistance to oxidative stress in rat cerebellar granule cells is enhanced by androgen treatment (“European Journal of Neuroscience (Eur
open journal of neurosci
ence) ", 1999, Volume 11, p. 1285-
1291). These results are
This indicates that the expression of IAA1097 is induced to activate the ubiquitin system and protect the cells from oxidative stress. In addition, KIAA1097 is an intracellular protein localization prediction program PSORT <http: // ps
ort. nibb. ac. When analyzed by jp>, the KIAA1097 protein was predicted to be distributed in the nucleus. In addition, KIAA1097 has US in its sequence.
Same Zn-finger domain as P16 ( ³⁰ CQD
CKVQGPNLWACLENRCSYVGCGESQ
VDHSTIHSQETKHYLTVNLTTLRVW
CYAC ⁸⁶ ) (SEQ ID NO: 27). These facts suggested a relationship between KIAA1097 and chromosome condensation.

(Expression of KIAA1097) KIAA10
Gateway cloning technology (GAT
EWAY Cloning Technology)
(LIFE TECHNOLOGY). That is, KIAA1 obtained from Kazusa DNA Research Institute
Using 097 cDNA as a template, the primer Pr-DO
NR1097 (+) 5'-GGGACAAGTTTTGT
ACAAAAAAGCAGGCTACAAAATGTC
AGCTTTTCGA-3 '(SEQ ID NO: 28) and P
r-DONR1097 (-) 5'-GGGGACCAC
TTTTGCAAGAAAAGCTGGGTCTCAT
PCR of the ORF region was performed using TAGAACTCTCTACATCC-3 ′ (SEQ ID NO: 29) (HF-2 PCR).
Kit, CLONTECH) and the resulting PCR product is BP clonase enzyme (BP CL
It was introduced into an entry vector (Entry Vector) by recombination reaction with ONASE Enzyme (pENTRY1097). pENTRY1097
E. coli DH5α competent cells (LIFE TEC
HNOLOGY) and the plasmid was extracted (GFX ^™ Micro Plasmid Pr).
ep Kit, Amersham pharmacia
biotech), perform the same sequence as above.
It was confirmed that the RF base sequence was correctly inserted. The ORF region extends from pENTRY1097 to the N-terminal 6
× pD expressed as a histidine (His) fusion protein
EST17 vector T7 promoter) and LR Clonase Enzyme (LR CLONASE Enzym).
Expression vector pD introduced by recombination reaction according to e)
EST17 / KIAA1097 was constructed. E. coli BL21-SI competent cell (LIFE TEC
HNOLOGY) to obtain recombinant E. coli.

The KIAA1097-introduced E. coli was cultured in ampicillin-containing LBON medium (NaCl-free LB medium) at 37 ° C. in the late logarithmic growth phase (OD ₆₀₀ =
The culture was continued until it reached about 1.0). Then E. coli BL
To induce T7 RNA polymerase in 21-SI, NaCl was added to a final concentration of 0.3 M, and the mixture was further cultured for 4 hours. The cells were collected by centrifugation, and 1/10 volume of the culture solution was 50 mM Tris / 50 mM.
After suspending with NaCl / 1 mM EDTA / 1 mM DTT aqueous solution and crushing the cells by ultrasonic treatment (10 seconds × 5),
Centrifugation in the presence of 1% Triron X-100 (150
00 rpm × 30 minutes). The obtained supernatant was used as a soluble fraction and the precipitate was used as an insoluble fraction. Add an equivalent amount of 2 × SDS sample buffer to each of the obtained fractions,
After heating at 100 ° C. for 5 minutes, electrophoresis was performed on 8% SDS-polyacrylamide gel. Protein was detected by Coomassie Brilliant Blue (CBB) staining (Rapid)
Stain CBB Kit, Nacalai Tesque) and Western blot. After Western blotting, proteins were analyzed by PVDF membrane (Polyscr).
ee NEF-1000, Daiichi Kagaku) and then anti-H
The His fusion protein was detected with an is antibody (Penta-His antibody, QIAGEN). The above-mentioned electrophoresis and analysis are performed using Gene Rapid (Amersham pha).
rmciabiotech) and ABI PRIs
M 310 (Amersham pharmacia
biotech).

As a result, a protein corresponding to the estimated molecular weight of 103 kDa was expressed in a NaCl-dependent manner. Most of the expressed protein was found in the insoluble fraction, and the distribution in the soluble fraction was small (Fig. 1). It is known that when USP is expressed in Escherichia coli, most of them are insoluble, and a similar tendency was observed in this time USP. In addition, a protein having a molecular weight of about ½ (66 kDa) was observed, suggesting that the synthesized protein may be degraded in the cells.

(Preparation of Escherichia coli crude extract) N prepared above
Crude extracts were made from aCl-induced and non-induced KIAA1097 recombinant E. coli. First, a TED buffer extract was prepared. The bacterial cells were mixed with TED buffer (50 mM Tris (pH 7.5), 1 mM) in 1/15 volume of the culture solution.
After suspending in EDTA, 5 mM DTT), the cells were disrupted by sonication (5 seconds x 3) on ice and centrifuged (1500).
The supernatant obtained at 0 rpm × 10 minutes, 4 ° C.) was used as a TED buffer extract. Next, a TN buffer extract was prepared. The microbial cells were mixed with 2/15 volumes of TN buffer (50 mM Tris (pH 7.4), 150 mM N).
aCl, 1 mM DTT, 0.25 mg / mL lysozyme), suspended on ice for 10 minutes, disrupted by ultrasonic waves (5 seconds × 3), and centrifuged (1500).
The supernatant obtained at rpm × 5 minutes, 4 ° C.) was used as a TN buffer extract. Each extract was stored at -80 ° C until use.

For use as a positive control, USP15, which is one of the USP family and confirmed to have deubiquitinating activity, was expressed in Escherichia coli, and a crude extract was prepared in the same manner as above. Therefore, KI that codes USP15
The AA0529 cDNA clone was obtained from Kazusa DNA Research Institute and expressed in E. coli BL21-SI as an N-terminal 6 × His fusion protein using the gateway cloning technology in the same manner as KIAA1097. The KIAA0529 cDNA clone from Kazusa DNA Research Institute lacked the N-terminal, so
r) et al. (“Genomics (Genomics
s) ", 1999, Vol. 59, p. 264-), Me
t-Ala-Glu was designed in a PCR primer, and these 3 residues were fused to the existing N-terminus to prepare a complete ORF, and the protein was expressed. Recombinant E. coli is KIAA1
TN buffer crude extract was made after being induced by NaCl as in 097.

(In Vitro Deubiquitinating Enzyme Activity) As a substrate used for studying deubiquitinating enzyme activity, multiubiquitin chain (Multi ubiq) is used.
Uichin chain) and Ub-CEP52 were used. The multi-ubiquitin chain is a mixture of isopeptide-bonded 2-4 tetramers of Ub, and Ub-CEP52 is a peptide of 52 residues of ribosomal protein bound to the C-terminus of Ub. These are Affinity res
It was purchased from earl Products. Multi-ubiquitin chain 2 μL (4 μg) or Ub-CEP52
8 to 10 μL (protein mass 86 μg) of the E. coli TED buffer extract prepared above was added to 2 μL (2 μg) and adjusted to 20 μL with TED buffer, and then 37 ° C.
Incubate overnight. As a positive control, isopeptidase T (Affinity) was added to the multi-ubiquitin chain.
research products) to a final concentration of 0.1
μM, Ub-CEP52 contains ubiquitin C-terminal hydrolase rabbit (Ubiquitin C-term)
internal hydrorabbit (CA)
LBIOCHEM) was added at a final concentration of 0.1 μM. 2x sample buffer (2% SDS / 50 mM containing reaction solution and 2-ME digested overnight
Tris (pH 6.8) / 30% glycerol /
0.01% BPB) was mixed in equal amounts, boiled for 2 minutes, and electrophoresed on a 15% polyacrylamide gel. After transfer P
VDF membrane (Polyscreen NEF-1000, Daiichi Pure Chemicals) was added to TBS-T (10 mM Th) containing 3% BSA.
is (pH 7.5) / 150 mM NaCl / 0.05%
After blocking with Tween 20) for 1 hour at room temperature, an anti-Ub antibody (rabbit antiserum) (Biogenes) diluted 1500 times with 3% BSA / TBS-T was reacted at room temperature for 1 hour. Then 100% PVDF membrane
Pre-treatment with shaking for 15 seconds in methanol and 2 minutes in H ₂ O, then 100mA by semi-dry method from the electrophoresed gel.
It was transferred for 1 hour. After the reaction, the PVDF membrane was washed with TBS-T three times for 10 minutes, and the secondary antibody (HRP-labeled anti-rabbit I
gG) diluted 2000 times with 10% skim milk / TBS-T was reacted at room temperature for 1 hour, and then with TBS-T for 1 hour.
Washing for 0 minutes was performed 3 times. After that, the Eb kit and Hyperfilm were used to detect Ub released from each substrate.

Furthermore, as a substrate, C of mammalian-derived Ub is used.
At the end is a cystoma japonicum (Schistoma)
Ub- to which GST derived from
It examined using M-GST. First, Ub-M-GST
Expression plasmid pTV118N / Ub-GST was added to pTV
Constructed using 118N (TaKaRa), E. coli JM
109 competent cells were transformed. Ub-M
-GST-introduced E. coli in the presence of 10 mM isopropyl 1-thio-β-D-galactoside (IPTG) 3
After culturing at 7 ° C. for 4 hours, a TN buffer extract was prepared from the cells and used as a substrate for in vitro reaction. Ub-M
-15 μL of KIAA1097 TN buffer extract (protein mass 90 μg) was added to 10 μL of GST extract, and the mixture was incubated at 37 ° C. overnight. KIAA0529 was added to 1 μL of the Ub-M-GST extract as a positive control.
14 μL of TN buffer extract (15 μg protein amount)
A sample added with was prepared. After reaction, 15% SDS
-After electrophoresis on a polyacrylamide gel to transfer the protein to the PVDF membrane, the anti-GST antibody (Amersham)
Pharmacia biotech) Ub-MG
GST released from ST was detected.

In the in vitro system, the KIAA1097 expressed protein did not show deubiquitinating enzyme activity against multi-ubiquitin chains, Ub-CEP52 and Ub-M-GST. UC used as a positive control
Hrabbit, isopeptidase T and KIA
Since A0529 showed enzyme activity with each substrate, it was revealed that there was no problem in the test system.

(Deubiquitinating Enzyme Activity in Co-expression System with Substrate) One of the reasons why the deubiquitinating activity of KIAA1097 is not detected in vitro is that the expressed protein is extremely unstable.
Attempts were made to detect the activity in a co-expression system used in many papers as a method for detecting the USP activity (“Achieves of Biochemistry and Biophysics”).
And Biophysics) ", 2000, volume 379, p. 198). This co-expression system shows compatibility with the ori of the pBR322 system contained in the enzyme expression plasmid.
By using a plasmid for expressing a substrate (Ub-GST) having an ori of 15A, the enzyme and the substrate are co-expressed in the same microbial cell, and the enzyme reaction occurs immediately after protein expression, so the enzyme is inactivated. It is considered useful when it is easy to do. As a substrate, GST to which Ub is bound via methionine (M), arginine (R), proline (P), or isoleucine (I), Ub
-M-GST, Ub-R-GST, Ub-P-GST,
Alternatively, Ub-I-GST was used. These are collectively called Ub-X-GST. In addition, this co-expression system may be referred to as in vivo.

To prepare Ub-M-GST, Ub-M-
After the Ub-M-GST coding region was amplified by PCR using the GST expression plasmid pTV118N / Ub-GST as a template, pDEST14 Ub-M-GST was prepared using the gateway cloning technology. The region containing the T7 promoter and the Ub-M-GST coding region was excised from pDEST14 Ub-M-GST by SphI and HindIII treatment, and p
PACYC184 (New with ori derived from 15A)
SphI-Hin from England, Biolabs)
It was inserted at the dIII site to prepare a Ub-M-GST expression plasmid pACUb-GST for coexpression. Next, using this pACUb-GST as a template, a site-directed mutagenesis kit (Site-Dire).
cted Mutagenesis Kit) (STRA
UTAG and GST binding by Arg
(Ub-R-GST), Pro (Ub-P-GST), and Ile (Ub-I-GST) were prepared into plasmids, and the plasmid pACUb- for co-expression was prepared.
R-GST, pACUb-P-GST and pACUb
-I-GST. Escherichia coli BL21-SI containing these plasmids was made into competent cells by the calcium chloride method, and the expression vector pDEST17 / KIAA was prepared.
1097, pDEST17 / KIAA0529 and p
DEST17 / Luciferas was added for transformation. In addition, luciferase
Was used as a negative control. pDEST17 / Lucif
eras is a pGL3-Luc vector (Promega).
6xHistidine fusion protein using Gateway Cloning Technology as a template. Colonies were selected with ampicillin (50 μg / mL) and chloramphenicol (34 μg / mL) to obtain a co-expressing strain carrying both plasmids.

It was confirmed by Western blot whether the KIAA1097 protein was expressed in the co-expression system. That is, the bacterial cells were diluted with 1/10 volume of PBS.
After suspending in (-), the cells were disrupted by ultrasonic treatment (10 seconds x 3). The disrupted cell suspension was electrophoresed on a 10% SDS-polyacrylamide gel to transfer the protein to a PVDF membrane, and the His fusion protein was detected with an anti-His antibody. As a result, the amount of KIAA1097 protein detected in the co-expression system that did not induce NaCl was very small compared with the case of single expression. However, in KIAA0529 and luciferase, many proteins were expressed even under non-induction,
A difference was observed in the amount of expressed protein from KIAA1097.

After culturing each co-expressing strain in LBON medium containing ampicillin and chloramphenicol at 37 ° C. overnight, cells were collected by centrifugation. 1 of the culture medium
After suspending in / 10 volume of PBS (-), ultrasonic treatment (10
The cells were disrupted for 3 seconds for 3 seconds. Bacteria disruption solution is 15% S
GST released from each Ub-fused GST by Western blotting after electrophoresis on DS-polyacrylamide gel
Was detected. The result is shown in FIG. Ub-M-GS
In the co-expression system with T, Ub-R-GST and Ub-I-GST, GST was released from the fused GST by Western blotting, and it was shown that the KIAA1097 expressed protein has deubiquitinating activity. On the other hand, Ub-P-GST showed no release of GST, and K
IAA1097 was found to be difficult to cleave the Ub-P bond. KIAA0529 showed deubiquitinating enzyme activity in vivo as well as in vitro. KIAA
Since 0529 cleaved the Ub-P bond, KIAA
1097 was shown to have a different substrate selectivity than KIAA0529. Further, free GST was not observed in co-expression with luciferase, and it was revealed that there is no deubiquitinase activity due to the E. coli endogenous protein.

(Confirmation of enzyme active center) KIAA1097
The enzyme active center of E. coli was confirmed in a co-expression system with a substrate using the KIAA1097 mutant. First, ¹⁵⁵ GLKNIGNTCYMNAA of Cys box of KIAA1097
A mutant Mut1097 ^C163S in which ¹⁶³ C in LQ ¹⁷⁰ (SEQ ID NO: 17) was converted to S was prepared by a site-directed mutagenesis kit.
That is, using pENTRY1097 as a template, the primer 5'-GGAAATACATTAGTACATGAA was prepared.
TGCAGCTTTGCAGGCTC-3 '(SEQ ID NO: 30) and 5'-GAGCCTGCAAAGCTGC
ATTCATGTAACTAGATTATTCC-3 '
(SEQ ID NO: 31) was used to Pfu turbo DNA polymerase (Pfu turboDNA polymer).
race). Then D
The template plasmid was digested with pnI and the reaction mixture was used for E. coli DH.
5α competent cells were added for transformation. A plasmid was extracted from E. coli and sequenced to confirm that the nucleotide sequence containing the mutation was inserted correctly. The ORF region containing the mutation was introduced into the pDEST17 vector by recombination reaction with LR clonase enzyme,
Expression vector pDEST17 / KIAA1097
^C163S was constructed. This was transformed into the competent cell prepared above and selected with ampicillin and chloramphenicol to obtain recombinant Escherichia coli. C16
The activity disappeared in the 3S mutant, and this activity was detected in the Cys box.
It was revealed to be due to ¹⁶³ Cys in the inside (Fig. 2).

(Identification of protein to which KIAA1097 binds) Based on the amino acid sequence of KIAA1097, a primer for constructing a bait used in the yeast two-hybrid (Y2H) screening was designed and synthesized. A bait fragment was amplified using the above-synthesized primers with a commercial cDNA library derived from 9 types of human organs / tissues as a template for cloning. Using homologous recombination of Mat α type yeast PNY200, GAL4 D
The bait fragment was inserted into a yeast expression vector (pGBT.Q, selectable marker: trp1) that is expressed in a form in which a bait protein is fused to the C-terminus of the NA-binding domain. Colony pc
The bait insertion region in the plasmid was amplified using R, the chain length was confirmed by agarose gel electrophoresis, and the nucleotide sequence was confirmed by direct sequencing.

By assaying whether or not the transcription activity of GAL4 was present in the bait protein, bait having an autoactivating effect was excluded. That is, the bait recombinant yeast was mated with the Matα-type yeast BK100 containing an empty vector, and it was examined whether or not the diploid yeast grows on the selective medium plate. When significant growth was detected, this bait was judged to be a self-activator and was not used for screening.

Next, an expression vector (pGA that induces a prey protein at the C-terminal side of the GAL4 transcriptional activation domain).
D. PN2, selectable marker: leu2), cDNA with 5,000,000 or more independent clones derived from various organs
C of Mat α-type yeast BK100 obtained by fusing the fragments
The DNA library and the above bait yeast were mixed in approximately equimolar numbers for each cell number capable of covering an independent clone of the library, and joined by the filter mating method. This zygote yeast is auxotrophic for histidine and adenine, while Gal1p-HIS3 and Gal1p-HIS3
Since the 2p-ADE2 two-hybrid promoter / reporter gene system is integrated, a protein that complements the histidine-adenine requirement appears only through the interaction of the bait protein and the prey protein in the library, and histidine Yeast colonize on agar lacking adenine (and tryptophan, leucine). For the emerged colonies (positive colonies), the DNA sequences of the bait and prey inserts were
It was examined by CR direct sequencing.

The above-mentioned positive colonies were subjected to small-scale liquid culture to obtain D
NA was extracted. Escherichia coli KC8 was transformed with this DNA by electroporation, and selected on tryptophan-deficient and leucine-deficient plates to obtain recombinants containing bait plasmid and prey plasmid, respectively. Each plasmid was prepared. Then, the base sequence analysis of the bait and prey sequences was performed by the dye terminator method. Both plasmids were introduced into yeast strain J692 having Gal7p-lacZ, which is the third promoter / reporter gene system, and the final confirmation of the interaction between bait and prey was carried out by β-galactosidase assay. Further, in order to detect false positives, a pool of 6 kinds of bait plasmids consisting of a protein derived from bacteria of Myriad and the prey plasmid were introduced into J692, and β-galactosidase assay was carried out.
When the above colonies developed blue, the above interaction was determined to be false positive.

As a result, two types of baits of KIAA1097 (the 70th to 169th amino acids and the 7th amino acid)
Yeast two-hybrid screening using 0th to 369th amino acids)
(X11β, Mint2) (749 amino acids in total length) 284th to 554th amino acids and 243rd to
The 415th overlapping play was selected. In other words, KIAA1097 is X11L (X11L
β, Mint 2).

[0092]

Example 2 (Isolation / Identification of AK024318) AK0
24318 cDNA is GenBank human cDNA.
From the library, bioinformatics (bi
It was extracted as a novel protease candidate gene by using the information technology. To isolate and identify this gene, human brain-derived mRNA was used as a template and GIBCO
BRL Super Script II Kit (Super
RT-P using r Script II Kit)
CR was performed. First, oligo (dT) primer [Ol
igo (dT) primer] (0.5 μg / μl) 1
μl, human brain-derived mRNA (1 μg / μl) 0.2 μm
1, H ₂ O (10.8 μl) were mixed, heated at 70 ° C. for 10 minutes and cooled with ice, and 5 × First Strand buffer (4 μl),
0.1 M DTT 2 μl, 10 mM dNTP Mi
x 1 μl was added. Then superscript
II (200 u / μl) (1 μl) was added, and the mixture was reacted at room temperature for 10 minutes at 42 ° C. for 50 minutes and at 70 ° C. for 15 minutes. RT
-The following primers for PCR were designed and produced based on the base sequence of AK024318 obtained from GenBank as information.

[0093] <Primer>   Pr-PLACE-S01 (SEQ ID NO: 32):     5'-AATATGGGCACCAATGCCCTC-3 '   Pr-PLACE-AS02 (SEQ ID NO: 33):     5'-TTACTCTCTTGACTGATAGA-3 '

The PCR reaction was performed by the Boehringer Expand High Fidelity PCR System (Expan).
d High Fidelity PCR System
m) was used. 25 of Mix 1 shown below
PCR reaction was performed by mixing 25 μl of Mix 2 with 25 μl of Mix 2.

(Mix 1) dNTP Mix (1.25 mM) 5 μl Pr-PLACE-S01 (38.11 pmol / μl) 0.6 μl Pr-PLACE-AS04 (32.24 pmol / μl) 0.6 μl cDNA 2 μl H ₂ O 16.8 μl 25 μl

(Mix 2) 10 × Expand HF Buffer 5 μl Expand HF (3.5 u / μl) 0.8 μl H ₂ O 19.2 μl 25 μl

[0097] (PCR operating conditions)     Pre-heat insulation (pre heat) 94 ℃ for 3 minutes     Step 1 94 ℃ for 30 seconds     Step 2 56 ℃ for 30 seconds     Step 3 72 ℃ 2 minutes                                     (30 cycles of steps 1 to 3)     Post heat 72 ℃ 3 minutes (post heat)

The resulting PCR product was used as BK for TaKaRa.
The L kit was used for blunting and phosphorylation. PCR reaction solution 2 μl, 10 × blunting
Kaination (Blunting Kinatio)
n) buffer 2 μl, blunting kaination enzyme mix 1 μl, H ₂ O 15 μl
Are mixed and reacted at 37 ° C for 10 minutes, MicroPure-EZ
After applying to a (micropure-EZ) filter cup, the mixture was centrifuged at 15 krpm for 30 seconds. PC passed
A part of the R product was ligated with a pBluescript (SK-) vector (BAP treatment after digestion with SmaI) and transformed into competent cells (JM109). After transformation, the E. coli colonies were confirmed by PCR (using Ampli-Taq, PE) for the presence and direction of the insert (PCR product), and the nucleotide sequences of two clones (# 12, # 16) were confirmed. As a result, AK024318c
The DNA was found to be a 3176 bp long gene encoding an amino acid of 355 residues as an ORF. In addition, AK024318 is the fourth from the 25th Gly.
Cys box for 0th Gln, T for 285th
It had a His box at the 303rd Tyr from yr. In the human USP used for expression, the 960th A was T, which was different from the base sequence of the library, but no amino acid substitution had occurred. Matched with.

(Expression of AK024318 in Escherichia coli) Amplification of the protein translation region of the target gene was carried out by using the above-mentioned cDNA as a template and reverse transcription of human brain-derived mRNA with an oligo (dT) primer as a template. I went the same way.

[0100] <Primer>   Pr-DONRplace-S01 (SEQ ID NO: 34):     5'-GGGACAAGTTTTGCATAAAAAAGCAGG                   CTATATGGGCACCAATGCCTCTGC-3 '   Pr-DONRplace-AS02 (SEQ ID NO: 35):     5'-GGGGACCACTTTGTACAAGAAAGCTG                   GGTGTTACTCTCTTTGACTGATAGA-3 '

Next, 45 μl of the PCR product was made up to 200 μl with TE buffer, and 30% PEG8000 / 30 mM was added.
Add 100 μl of MgCl ₂ and centrifuge at room temperature (15 krp
The pellet was washed with 70% ethanol, dried and dissolved in 10 μl of TE buffer. Then PCR
Product 1μ, entry vector (pDONR201: 1
50 ng / μl) 0.5 μl, BP clonase reaction buffer 1 μl, and TE buffer 1.5 μl were mixed on ice (total 4 μl), BP clonase enzyme mix 1 μl was added, and the mixture was reacted at 25 ° C. for 1 hour. After the reaction, proteinase K
0.5 μl was added and the enzyme mix was inactivated at 37 ° C. for 10 minutes. 1 μl of this reaction solution was transformed into competent cells (DH5α) and subcloned,
pDONR201 / UBP # 2 and pDONR201
/ UBP # 3 was obtained. After pDONR201 / UBP # 2 was digested with DraI and EcoRI to remove the mutation site by PCR, pDONR201 / UBP # 3 was added to D
An about 0.65 kbp fragment digested with raI and EcoRI was introduced to newly obtain pDONR201 / UBP # 6. 6 × using pDONR201 / UBP # 6
A vector was constructed that was expressed as a fusion protein with a His tag. pDONR201 / UBP # 6 (50 ng / μ
l) 1 μl, 6 × His tag expression vector (pDEST
17: 150 ng / μl) 0.5 μl, LR clonase reaction buffer 1 μl, and TE buffer 1.5 μl were mixed on ice (total 4 μl), and LR clonase enzyme mix 1 μl was added, followed by reaction at 25 ° C. for 1 hour.
After the reaction, proteinase K (proteinase K)
K) Add 0.5 μl of enzyme at 37 ° C for 10 minutes
Deactivated the mix. 1 μl of this reaction solution was transformed into competent cells (BL21-SI), and E. coli colonies were subjected to PCR (Ampli-Ta, PE).
2 clones (pDEST17-
hUBP # 3, pDEST17-hUBP # 5) was obtained.

LB-Amp (NaCl-) 2 was added to each E. coli.
The cells were shake-cultured in ml at 37 ° C. overnight. This pre-culture liquid 60
0 μl was transferred to 1.4 ml of LB-Amp (NaCl −) and cultured with shaking at 37 ° C. for 3 hours.
μl was added (final concentration 0.3 M), and the mixture was further incubated at 37 ° C. for 4 hours with shaking. As a negative control, 120 μl of H ₂ O was added and the cells were similarly cultured. After that, the culture solution was sampled 1 ml each 2 hours or 4 hours after induction and centrifuged (3 kr
pm 10 min 4 ° C), pellet 2% SDS / 50
mM Tris (pH 8.0) and PBS 100μ
suspension, freeze-thaw, sonicate and centrifuge (15 krp
m 10 minutes at 10 ° C or 4 ° C). Centrifuge supernatant 2-
2x sample buffer containing ME (2% SDS /
50 mM Tris (pH 6.8) / 30% glyc
erol / 0.01% BPB) and mix 2
SDS-on a 12% polyacrylamide gel after boiling for a minute
PAGE was performed in the same manner as in Example 1 to confirm the expressed protein. As a result, a band of about 41 kD, which is considered to be a fusion protein of human USP and 6 × His tag, was detected (FIG. 3).

(In-vivo deubiquitinating enzyme activity) Each E. coli was subjected to LB-Amp (NaCl-) at 37 ° C.
Pre-incubate overnight in 11 ml, add 5 ml of this pre-cultured solution, and OD ₆ with LB-Amp (NaCl −) total 50 ml.
Main culture was carried out until it became around ₀₀ = 1.0. Then 5M
Add NaCl to a final concentration of 0.3M, then add 1
Incubated for hours. TD of the cultured cells (for 5 ml of culture solution)
E buffer (50 mM Tris (pH 6.5) / 1
Suspend in 500 μl of mM EDTA / 1 mM DTT, sonicate and centrifuge (15 krpm, 10 at 4 ° C.).
Minutes), total protein mass 2000, 200, 20 μg / m
A sample was diluted with TDE buffer so that it became l. For this sample, as in Example 1, 1 μl or U of polyubiquitin chain (2 μg / μl) was used as a substrate.
In vitro deubiquitination activity was measured using 2 μl of b-CEP52 (1 μg / μl). However, no enzyme activity was observed in vitro

(Deubiquitinating enzyme activity in co-expression system with substrate) The substrate expression plasmid prepared in Example 1 and the above expression vector (pDEST17-hUBP # 5)
Were simultaneously transformed into BL21-SI that had been transformed into competent cells, and a co-expression system of the substrate and AK024318 was constructed in the same manner as in Example 1. After that, the vector was confirmed by colony PCR. Using the co-expression system, the deubiquitinase activity of AK024318 was examined in the same manner as in Example 1. As a result, AK024318 is Ub-
M-GST, Ub-R-GST, and Ub-I-GST showed enzymatic activity, and Ub was dissociated, but Ub-P-GST did not show any enzymatic activity, and it was active due to the difference in amino acid at the digestion site. It was suggested that there was a difference in
7).

(Confirmation of enzyme active center) AK024318
Was confirmed in the co-expression system with the substrate using the AK024318 mutant. First, in order to prepare a mutant (C33S) in which the 33rd Cys of the Cys box of AK024318 was converted to Ser (C33S), the target gene was mutated. For the introduction of mutation, the mutator shown below was designed, and the mutation was introduced using the reaction buffer of STRATAGENE.

[0106] <Mutator>   Pr-PLACE-S09 (SEQ ID NO: 36):     5'-TCAATTTTTGGAAACACATCC                       TACTGTAACTCCGTGC-3 '   Pr-PLACE-AS10 (SEQ ID NO: 37):     5'-GCACGGAGGTTACAGTAGTAGAT                       GTGTTTCCAAAAATTGA-3 '

10 × reaction buffer. 5 μl, Pr-
PLACE-S09 (200 ng / μl) 0.6 μl,
Pr-PLACE-AS10 (180 ng / μl) 0.
7 μl, dNTPmix 1 μl, entry vector pDONR201 / UBP # 6 (50 ng / μl) 1 μl
1 μl of Pfu turbo DNA polymerase (2.5 u / μl) was added to a final volume of 50 μl with H ₂ O, and PCR reaction was performed under the following conditions. <PCR operating conditions> Preliminary heat retention 95 ° C for 30 seconds Step 1 95 ° C for 30 seconds Step 2 55 ° C for 1 minute Step 3 68 ° C for 7 minutes (12 cycles of steps 1 to 3)

After the PCR reaction, 10 μl was electrophoresed and transferred to about 3.2 k.
After confirming the band of pDONR201 / UBP of bp, Dn
1 μl of pI was added and digested at 37 ° C. for 1 hour. Then, 1 μl of the reaction solution was transformed into JM109. The E. coli colonies that had grown after transformation were cultured in 2 ml of LB-Km at 37 ° C overnight,
Company P GFX microplasmid prep
After purification using kit, a sequence was performed to confirm the mutation site. As a result, 3 out of 3 clones were mutated (G
→ C) is confirmed, pDONR201 / UBPmut #
1, pDONR201 / UBPmut # 2, pDONR
201 / UBPmut # 3 was obtained. No mutation was observed in any of the three clones except at the mutation site. Then pD
ONR201 / UBPmut # 3 was used to construct a vector that was expressed as a fusion protein with a 6 × His tag.
pDONR201 / UBPmut # 3 (50 ng / μ
l) 1 μl, 6 × His tag expression vector (pDEST
17: 150 ng / μl) 0.5 μl, LR clonase reaction buffer 1 μl, TE buffer 1.5 μl were mixed on ice (total 4 μl), and LR clonase enzyme mix 1 μl was added and reacted at 25 ° C. for 1 hour.
After the reaction, proteinase K
0.5 μl was added and the enzyme mix was inactivated at 37 ° C. for 10 minutes. 1 μl of this reaction solution was transformed into a competent cell (BL21-SI) and the expression vector (pDEST17 / h-UBPmut) was transformed.
# 7, pDEST17 / h-UBPmut # 8) was obtained. This was transformed into the competent cell prepared above and selected with ampicillin and chloramphenicol to obtain recombinant Escherichia coli. The activity was lost in the C33S mutant, and it was revealed that this activity was due to ³³ Cys in the Cys box (FIGS. 4 to 7).

(Tissue Expression) The expression pattern of this gene in each organ was examined by Northern blotting. For labeling the probe, Alk Ph from AP Co.
os Direct, signal detection by the same company CDP-S
tar was used. DNA fragment (pBS / UB
P # 12 → BamHI, EcoRI digestion: target gene full length about 1 kbp) was diluted to 10 ng / ml with H ₂ O, 10 μl of which was heat-denatured (95 ° C. for 5 minutes) and then rapidly cooled on ice. Thereafter, 10 μl of reaction buffer and 2 μ of labeling buffer (labeling buffer)
1, cross-linker solution (cross-linker solution: H ₂ O
= 1: 4) 10 μl was added, mixed, and reacted at 37 ° C. for 30 minutes. Membrane (Human CLONTECH
multiissue northern bl
ot) was prehybridized for 45 minutes at 55 ° C. in the hybridization buffer provided with the kit. Subsequently, the labeled probe was entirely mixed with 13 ml of a hybridization buffer and hybridized at 55 ° C. overnight. Thereafter, the primary wash buffer was washed twice at 55 ° C. for 10 minutes, and then the secondary wash buffer was washed at room temperature for 5 minutes twice. After washing, 500 μl of the detection reagent was added, and the mixture was reacted at room temperature for 5 minutes, covered with a wrap, and exposed to a hyperfilm overnight. After exposure,
The film was developed.

As a result, bands of about 5.5 kb and 4 kb were detected in the heart, brain, placenta, skeletal muscle, kidney and pancreas,
Bands of about 7.5 kb and 6 kb were detected in the lung. The intensity of the signal was different depending on the organ, and it was strongly recognized especially in the brain and placenta (Fig. 8). The size was also slightly different.
No signal was detected in the liver. This suggested that AK024318 has a splicing variant.

[0111]

Example 3 (Isolation / Identification of KIAA1003) KIA
A1003 cDNA is obtained by bioinformatics from the human long chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
1003 is a USP consisting of 913 amino acid residues encoded by a gene having a base length of 4252 bp, and Cys box from the 146th Gly to the 161st Gln and the 626th Tyr to the It had a His box at the 643rd Tyr.

The existence of this KIAA1003 in human brain was confirmed. First, human brain polyA ⁽⁺⁾ RN
20 μl of reaction solution containing A (Clontech) (5
mMMgCl ₂ , 1 × PCR buffer, 1 mM d
NTPs, 20 units RNase inhibitor, 5 uni
ts AMV reverse transcriptase, 2.5 μM random 9me
r), 30 ° C, 10 minutes, 42 ° C, 30 minutes, 99
CDNA was prepared by treatment at 5 ° C for 5 minutes at 5 ° C. Using this cDNA as a template,
20 μl of reaction solution containing 1 (0.4 mM specific primer, 1 × PCR buffer, 0.2 mM dNTP
s, 1 × Advantage 2 polymerase mix (CLONTECH) was treated at 95 ° C. for 1.5 minutes, and then 95 ° C., 30 seconds, 63 ° C., 30 seconds, 72 ° C., 1.
The reaction of 5 minutes was performed for 30 cycles. The primer is
KIAA1003-01 (+) and KIAA1003-0
1 (-) and KIAA1003-02 (+) and KIA
A1003-02 (-) was used (Table 2). In addition, as primers, KIAA1003-02 (+) and KIAA
When 1003-02 (-) was used, the annealing temperature was 68 ° C. After the reaction, after confirming that the target region is amplified by 1% agarose gel electrophoresis,
The amplified PCR product was used as a TA cloning kit (CL
(ONTECH) was incorporated into the pCR2.1 vector, and the nucleotide sequence was confirmed by a sequence as in Example 1. As a result, an amplification product of the expected size was observed, and thus KIAA100 is certainly found in the human brain.
It was revealed that 3 genes were expressed at the mRNA level.

[0113]

[Table 2]

(Expression of KIAA1003 in E. coli) KIAA1003 obtained from Kazusa DNA Research Institute
Clone (pBluescript II SK (+)
Inserted into the SalI-NotI site) as a template and attB-KIAA1003 (+) and at as primers.
Using tB-KIAA1003 (-) (Table 2), the ORF region was amplified and then inserted into the entry vector (pDONR201) by a recombination reaction in the same manner as in Example 1 to prepare pDONR-K1003. Then pD
A His-tag-added KIAA1003 expression plasmid (pHis-K1003) was prepared using ONR-K1003 and pDEST17 as in Example 1. The expression plasmid is E. coli BL21-SI (Life Te
chnologies). It was confirmed by sequencing that mutations such as PCR errors did not occur in the ORF region.

LBON / Amp medium (LB medium containing 100 mg / ml ampicillin without NaCl)
1/25 volume of preculture solution was inoculated into OD and OD at 25 ℃
After culturing until ₆₀₀ reached about 1.0, NaCl was added to a final concentration of 0.3 M, and after culturing for about 4 hours,
The bacterial cells were collected. After suspending the cells in PBS, sonicate,
The supernatant was collected by centrifugation and used as an extract. Extract 10
After separation by% SDS-PAGE, the protein was detected in the same manner as in Example 1.

As a result, the expected molecular weight (105 kD
Since the expression of the protein of a) was observed in the soluble fraction (FIG. 9), it was revealed that KIAA1003 was inducibly expressed as a soluble protein.

(In vitro deubiquitinating enzyme activity) pHis-K1003 prepared above or Example 1
Escherichia coli BL retaining pHis-USP15 prepared in
21-SI was induced and cultured with NaCl in the same manner as above, and then the cells were collected. 1/10 amount of the culture solution is 50 mM
Tris-HCl, pH 7.6 / 5 mM EDTA / 1
After suspending in mM DTT, the cells were sonicated to destroy the cells, and the supernatant was collected by centrifugation. This was used as an Escherichia coli extract for activity measurement. Also, Ub-MG as a substrate
ST was used. Escherichia coli DH5α carrying the Ub-M-GST expression plasmid pTV118N / Ub-GST prepared in Example 1 was cultured in the same manner as in Example 1 to recover the cells, and 50 mM Tris-HCl, pH 7.6 /.
After suspending in 5 mM EDTA / 1 mM DTT, it was disrupted by lysozyme and ultrasonic treatment, and the supernatant was recovered by centrifugation. From this supernatant, glutathione sepharose 4B (Amersham pharmacia bio) was added.
tech) was used to purify Ub-M-GST.
The product is 50 mM Tris-HCl, pH 7.6 / 5.
mM EDTA / 1 mM DTT / 10% glycer
It was used after dialysis against ol.

10 μl of each E. coli extract and Ub-M-
GST 5 μl (2.5 μg) was added to 50 mM Tris-
HCl, pH 7.6 / 5 mM EDTA / 1 mM DT
After mixing in T (20 μl total), it was incubated at 37 ° C. for 16 hours. 15% or 5-20% of reaction mixture
After separation by SDS-PAGE, anti-GST antibody (Amersham pharmacia) was used as the primary antibody.
(Biotech), HRP-labeled anti-goat antibody (Alpha diagnostic antibody) as a secondary antibody
Ub-M-GST and released GST were detected by immunoblotting using National Co., Ltd.). In addition, the detection was carried out by ECL western blotting detection kit (ECL western blotting).
detection kit) (Amersham
(Pharmacia biotech) was used.

As a result, no activity was detected. However, since the activity was detected in USP15 which has been reported to have the deubiquitinating activity, it was confirmed that there is no problem in the test system.

(Deubiquitinating enzyme activity in co-expression system with substrate) Escherichia coli BL21-SI competent cells into which the substrate expression plasmid prepared in Example 1 was introduced,
The expression vectors pHis-K1003 and pHis-U
Various co-expressing strains were obtained in the same manner as in Example 1 by adding SP15 or pHis-Luc. pHis-USP15
Alternatively, the pHis-Luc used was that produced in Example 1. LBO containing various co-expressing strains containing 100 mg / ml ampicillin and 34 mg / ml chloramphenicol
After culturing in N medium at 25 ° C. until the OD ₆₀₀ reached about 0.7 to 1.0, NaCl was added to a final concentration of 0.3 M, and after culturing for about 9 hours, the bacterial cells were collected. did.
After preparing a bacterial cell extract in the same manner as above, the extract was subjected to SDS.
Separated by PAGE, Ub-X-GST and free GST were detected by immunoblotting using an anti-GST antibody as described above. Moreover, the expression of each co-expressed enzyme was determined by the anti-His-tag antibody (Penta × His).
It was detected by immunoblotting using TM antibody, QIAGEN).

As a result, KIAA1003 was Ub-M-
Although it showed deubiquitinating activity against GST, Ub-I-GST and Ub-R-GST, Ub-P-GS
No enzymatic activity on T was observed (Figures 10 and 11). Regarding USP15 which has been reported to show activity against Ub-P-GST, Ub-P-GST
The activity was observed for all substrates including (FIGS. 10 and 11). Moreover, when the expression of each enzyme was confirmed, all the enzymes were expressed.

(Confirmation of enzyme active center) KIAA1003
Was confirmed in the co-expression system with the substrate using the KIAA1003 mutant. First, 154-Cys, which is a putative active residue of KIAA1003, was replaced with Ser using a quick change site-directed mutagenesis kit. The primer used was TGC Se, which is the codon of 154-Cys.
It was designed to replace the rGC codon AGC (Table 2 above). Confirm the introduction of mutation by sequencing,
is--tagged ^{KIAA1003 C154S} expression plasmid, to obtain a ^{pHis-K1003 C1 54S.} This was transformed into Escherichia coli BL21-SI competent cells into which various substrate expression plasmids had been introduced in the same manner as above to obtain recombinant Escherichia coli. As a result, the activity disappeared in the C154S mutant, and this activity was detected in the Cys box.
It was revealed to be due to ¹⁵⁴ Cys (Fig. 12).

[0123]

Example 4 (Isolation / Identification of KIAA1372) KIA
A1372 cDNA is obtained by bioinformatics from a human long-chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
1372 is a USP consisting of 749 amino acid residues encoded by a gene having a base length of 3771 bp, and Cys box from the 173rd Gly to the 188th Gln and the 576th to Tyr as the active motif. It had a His box at the 594th Tyr.

The existence of KIAA1372 in human brain was confirmed in the same manner as in Example 3 except that the primers shown in Table 3 were used. As a result, an amplification product of the expected size was observed, so that KIAA1372 gene is certainly m in human brain.
It was found to be expressed at the RNA level.

[0125]

[Table 3]

(Expression of KIAA1372 in E. coli) KIAA1372 obtained from Kazusa DNA Research Institute
Clone (pBluescript II SK (+)
Inserted into the SalI-NotI site) as a template and attB-KIAA1372 (+) and at as primers.
Using tB-KIAA1372 (-) (Table 3 above)
His-tagged KIAA1 as in Example 3.
A 372 expression plasmid (pHia-K1372) was prepared and introduced into E. coli BL21-SI. It was confirmed by sequencing that mutations such as PCR errors did not occur in the ORF region.

Then, 100 ml of LBON / Amp medium (LB medium containing 100 μg / ml ampicillin without NaCl) was inoculated with 10 ml of the preculture medium, and 2
After culturing at 5 ° C until OD ₆₀₀ reached about 1.3, Na
Add Cl to a final concentration of 0.3M, and add about 6
After culturing for a period of time, the bacterial cells were collected. The cells were treated with 5 ml of buffer A (50 mM Sodium phosphate,
pH 7.0, 0.3 M NaCl, 1 mM PMSF) and suspended by sonication to destroy the cells, then Trit
on X-100 was added to a final concentration of 1%, and 1
After mixing by inversion for 5 minutes, the supernatant was recovered by centrifugation. Next, 100 μl of TALON resin (r
esin) (50% suspension, CLONTECH) was added and mixed by inversion at 4 ° C. for 1 hour. The resin was added to buffer B (50 mM Sodium phosphate, p
H7.0 / 0.3M NaCl / 1% Triton
After washing with X-100), 50 μl of elution buffer (50 mM Sodium phosphate, pH
7.0 / 0.3M NaCl / 1% Triton X-
100/150 mM imidazole) was added, and the mixture was allowed to stand for 3 minutes and then centrifuged to collect the supernatant, which was used as the eluent.
After separating the eluate by 10% SDS-PAGE,
Anti-His tag antibody (Penta-His ^™ antibody, QI
Hi by immunoblotting using AGEN)
KIAA1372 with s-tag was detected. In addition, the detection was carried out by the ECL Western blotting detection kit (Amersham pharmacia biot).
ech company) was used.

First AT of CDS at KIAA1372
Upstream from G (nucleotide number 627) in the same frame a stop codon (TAG, nucleotide number 55)
Since 2) exists, it was decided to express 627-ATG as a start codon. However, KIAA137
2 was inducibly expressed using Escherichia coli BL21-SI strain as a protein having a His-tag added to the N-terminus,
The expression of a protein having an expected molecular weight (86.8 kDa) was not detected by CBB staining and immunoblotting with an anti-His-tag antibody, and what was considered to be a degradation product was not detected. It was considered to be extremely unlikely. Therefore, increase the culture volume,
Furthermore, after concentrating the His-tagged protein with the His-tag affinity resin, an attempt was made to detect it by immunoblotting using an anti-His-tag antibody. It was detected (FIG. 13), and the expression of KIAA1372 was confirmed.

(In-Vitro Deubiquitinating Enzyme Activity) Escherichia coli BL21-SI carrying pHis-KIAA1372 prepared above or pHis-USP15 prepared in Example 1 was induced and cultured with NaCl in the same manner as above, and then the bacterial cells were Recovered. Using this cell, an Escherichia coli extract was obtained in the same manner as in Example 3. Further, Ub-M-GST prepared in the same manner as in Example 3 was used as the substrate. Furthermore, the substrate expression plasmid (pAC
Ub-P-GST, pACUb-I-GST and pA
E. coli BL21-SI carrying CUb-R-GST)
After culturing at 37 ° C. until the OD ₆₀₀ becomes about 1.0,
NaCl was added to a final concentration of 0.3 M, and after culturing for about 3 hours, the bacterial cells were collected. 1 / of the culture solution
10 volumes of 50 mM Tris-HCl, pH 7.6 / 5
After suspending in mM EDTA / 1 mM DTT, the cells were sonicated to destroy the cells, and the supernatant was collected by centrifugation.
This extract was used as a substrate for activity measurement (Ub-P-GST,
Ub-I-GST and Ub-R-GST) solution.

10 μl of each Escherichia coli extract and each substrate described above were mixed with 50 mM Tris-HCl, pH 7.6 / 5 mM.
After mixing in EDTA / 1mM DTT (total 20μ
l), incubated at 37 ° C. for 16 hours. In the case of Ub-M-GST, 5 μl of purified product (2.
5 μg), 10 μl of E. coli extract was used for Ub-P-GST, Ub-I-GST and Ub-R-GST. At this time, in order to examine the inhibitory effect of the cysteine protease inhibitor, N-ethylmaleimide (NEM, Sigma) was added to the reaction solution so that the final concentration was 20 mM. Next, add 15% or 5-2 of the reaction solution.
After separation by 0% SDS-PAGE, GST after Ub dissociation was detected as in Example 3.

As a result, KIAA1372 dissociated a larger amount of free GST from Ub-M-GST when the expression-inducing Escherichia coli extract was used as compared with the case of non-induction (FIG. 14). The slight release of GST in the case of non-induction was considered to be due to the leakage of expression. Furthermore, KIAA1372 is Ub-I-
It showed deubiquitinating enzyme activity against GST and Ub-R-GST, but no enzyme activity against Ub-P-GST was observed (Fig. 15). In addition, when an Escherichia coli extract expressing USP15, which is a positive control, was used, activity against all substrates including Ub-P-GST was observed (FIGS. 14 and 15).

Since USP is one of the cysteine proteases, it is a cysteine protease inhibitor N
As a result of examining the effect of EM, KIAA1 was detected in the presence of NEM.
The enzyme activity of 372 against Ub-M-GST completely disappeared (Fig. 16). Here, by adding NEM, Ub-M
The reason for the slower mobility of GST is probably due to the formation of a complex by cysteine-mediated binding with NEM.

(Confirmation of enzyme active center) KIAA1372
The enzyme active center of Escherichia coli was confirmed in the above in vitro system using the KIAA1372 mutant. First, KIAA1372
Substitution of 181-Cys, which is a putative active residue of Saccharomyces cerevisiae, into Ser was performed by using Quick Change Site-Directed Mutagenesis Kit
te-Directed Mutagenesis K
It). The primer used was 181
-The Cys codon TGC was designed to be replaced by the Ser codon AGC (Table 3 above). The introduction of the mutation was confirmed by sequencing, and the His-tagged KIAA1372 ^C181S expression plasmid, p
His-K1372 ^C181S was obtained. E. coli BL21 into which various substrate expression plasmids were introduced in the same manner as above
-Recombinant E. coli was obtained by transforming into SI competent cells. As a result, the activity was lost in the C181S mutant, and it was revealed that this activity was due to ¹⁸¹ Cys in the Cys box (FIG. 17). In addition, ^when the expression of KIAA1372 ^C181S was confirmed in the same manner as above, the same level of induction of expression as that of the wild type was observed, confirming that the loss of activity was not due to the non-expression of the protein (FIG. 17). .

[0134]

Example 5 (Isolation / Identification of KIAA1453) KIA
A1453 cDNA is obtained by bioinformatics from the human long-chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
1453 is a USP consisting of 1121 amino acid residues encoded by a gene having a base length of 5879 bp. As an active motif, 123th Gly to 138th Gln are Cys boxes and 365th Tyr are the active motifs.
To 383rd Tyr had a His box.

The presence of KIAA1453 in human brain was confirmed in the same manner as in Example 3 except that the primers shown in Table 4 were used. As a result, an amplification product of the expected size was observed, so it is true that the KIAA1453 gene is m in the human brain.
It was found to be expressed at the RNA level. here,
When a part of the nucleotide sequence of each amplification product was examined and compared with that in the published database, the 1029th G was A. Although this base substitution is accompanied by amino acid substitution from Val to Leu, it seems that it is not due to PCR error or the like, and the possibility of SNPs was considered.

(Expression of KIAA1453 in E. coli) KIAA1453 obtained from Kazusa DNA Research Institute
Clone (pBluescript II SK (+)
Of the entry vector (pDONR201) by a recombination reaction using BP clonase enzyme after amplifying the ORF region using the Expand High Fidelity PCR system (Boehringer Mannheim) as a template.
To prepare pDONR-K1453. The primer was 5'-ggg ACA AgTTTgTAC.
AAA AAA gCA ggC TTA ATg
CCAATA gTg gAT AAg TTg-3 '
(SEQ ID NO: 54) and 5'-ggg gAC CAC
TTT gTA CAA gAA AgC Tggg
TC CAg TCA gCg gCg ATA gC
TgAg-3 '(SEQ ID NO: 55) was used. next,
When the nucleotide sequence of the amplified ORF region was confirmed by sequencing, there was one mutation accompanied by amino acid substitution. Therefore, the quick change site-directed mutagenesis kit (Stratag)
ene) was used to repair this mutation. Then, using pDONR-K1453 and pDEST17 in which the mutation was repaired, a His-tagged KIAA1453 expression plasmid (pHis-K1453) was prepared by a recombination reaction with LR clonase enzyme, and E. coli BL21-SI (Life Technologies) was prepared.
Company). The prepared BL21-SI strain holding pHis-K1453 was treated with LBON / Amp medium (NaC
L containing 100 μg / ml ampicillin without L
After culturing in (B medium) at 25 ° C. until the OD ₆₀₀ became about 1.0, NaCl was added so as to have a final concentration of 0.3 M, and the cells were further collected after culturing for about 4 hours. P cells
After suspending in BS, sonicate and centrifuge to collect the supernatant,
The extract was used. The extract was separated by 10% SDS-PAGE, and then Rapid Stain CBB Kit.
(Nacalai Tesque) was used to detect the protein. As a result, expression of a protein having an expected molecular weight (130 kDa) was observed (Fig. 18). Since a part thereof was also found in the soluble fraction (Fig. 18), it was revealed that a part of the inducibly expressed KIAA1453 exists as a soluble protein.

(In-vivo deubiquitinating enzyme activity) pHis-K1453 or p prepared in Example 1
Escherichia coli BL21-SI carrying His-USP15 was induced and cultured with Nacl in the same manner as above, and then the bacterial cells were collected. Bacteria were added to 1/15 volume of 50 mM Tris
-HCl, pH 7.6 / 5 mM EDTA / 1 mM D
After suspending in TT, the cells were sonicated to destroy the cells, and the supernatant was collected by centrifugation. This was used as an Escherichia coli extract for activity measurement. 5 μl (2.5 μg) of the above E. coli extract and 5 μl (2.5 μg) of Ub-M-GST prepared in Example 3 were used.
0 mM Tris-HCl, pH 7.6 / 5 mM ED
After mixing in TA / 1 mM DTT (total 20 μl), 3
Incubated at 7 ° C for 16 hours. This reaction solution was subjected to electrophoresis in the same manner as in Example 3 to detect GST. However, the deubiquitinase activity of KIAA1453 against Ub-M-GST was not detected in vitro. However, since activity was detected in the positive control USP15, it was confirmed that there was no problem in the test system.

(Deubiquitinating enzyme activity in co-expression system with substrate) Escherichia coli BL21-SI competent cells into which the substrate expression plasmid prepared in Example 1 was introduced,
The expression vectors pHis-K1003 and pHis-U
Various co-expressing strains were obtained in the same manner as in Example 1 by adding SP15 or pHis-Luc. pHis-USP15
Alternatively, the pHis-Luc used was that produced in Example 1. LBO containing various co-expressing strains containing 100 mg / ml ampicillin and 34 mg / ml chloramphenicol
After culturing in N medium at 25 ° C. until the OD ₆₀₀ reached about 1.0, NaCl was added to a final concentration of 0.3 M, and after culturing for about 3 to 6 hours, the cells were recovered. After preparing a bacterial cell extract in the same manner as above, the extract was treated with SDS-
After separation by PAGE, Ub-X-GST and free GST were detected by immunoblotting using an anti-GST antibody as described above. In addition, the expression of each co-expressed enzyme was determined using the anti-His-tag antibody Penta × HisT.
It was detected by immunoblotting using M antibody, QIAGEN.

As a result, KIAA1453 was found to be used for all the substrates used in the studies, namely Ub-M-GST and Ub-I.
-GST, Ub-R-GST, and Ub-P-GST
It showed deubiquitinating activity against (Figs. 19 and 2).
0). Ub-P was also used for the positive control USP15.
-Activity was observed for all substrates including GST (Figures 19 and 20). Moreover, when the expression of each enzyme was confirmed, all the enzymes were expressed.

(Confirmation of enzyme active center) KIAA1453
Was confirmed in the co-expression system with the substrate using the KIAA1453 mutant. First, substitution of 131-Cys, which is a putative active residue of KIAA1453, with Ser was performed using a quick change site-directed mutagenesis kit. The primer used was TGC Se, which is the codon of 131-Cys.
It was designed to replace AGC, which is the codon of r (5′-C AAC CTT ggC AAC ACC.
AgC TTT CTC AAT gCC AC-
3 '(SEQ ID NO: 56) and 5'-gT ggCATT
gAg AAA gCT ggT gTT gCC
AAg gTTg-3 '(SEQ ID NO: 57)). Confirmation of introduction of mutation by sequencing, His-tagged KIAA1453 ^C131S expression plasmid, pH
The is-K1453 ^C131S was obtained. This was transformed into E. coli BL21-in which various substrate expression plasmids were introduced in the same manner as above.
Recombinant E. coli was obtained by transforming into SI competent cells. As a result, the activity was lost in the C131S mutant,
It was revealed that this activity was due to ¹³¹ Cys in the Cys box. Where KIAA1453
^When the expression of ^C131S was confirmed, it was confirmed that the wild type (KIAA
1453), the same level of expression was observed, indicating that the loss of activity was not the difference in expression level (FIG. 19).
And 20).

[0141]

Example 6 (Isolation / Identification of KIAA1063) KIA
A1063 cDNA is obtained by bioinformatics from the human long-chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
1063 is a USP consisting of 514 amino acid residues encoded by a gene having a base length of 5223 bp, and has Cys box from the 166th Gly to the 181st Gln and a 452nd Tyr to the first as an active motif. It had a His box at the 469th Tyr.

The existence of this KIAA1453 in the human brain was confirmed. The new gene KIAA1063 obtained at Kazusa DNA Research Institute was 5.2 kb, but the ORF was analyzed by bioinformatics analysis.
It was presumed that a part of the 5'-end side of L. The ORF required for the currently published sequence was approximately 1.7 kb. First, the consistency of the nucleotide sequences of this clone was examined. First, I tried RT-PCR, but ORF 1.7k
Since b was not amplified, it was fragmented and examined.
Human brain poly (A) + RNA (whole cere
bral brain, CLONTECH) 500 ng
Was used as a template to perform reverse transcription reaction using random hexamers to synthesize cDNA. PCR was performed using this cDNA as a template and combining various primers.
The expected size PCR product was purified (DNA pu
refining kit, BioRad), blunt-ended, and phosphorylated (BKL kit, Tak).
ara). This PCR product is designated as pBluescript.
It was ligated with a KS (+) vector (blunt-ended by decomposing the restriction enzyme SmaI and dephosphorylated) and transformed into competent cells (JM109). An E. coli colony containing a plasmid having the desired insert was selected and the plasmid was extracted.

The nucleotide sequence of the insert of the obtained plasmid was examined. For determination of the nucleotide sequence, Thermo Sequenase Cy5.5 dye terminator sequencing kit (Thermo Sequenase) was used.
Cy5.5 Dye Terminator Seq
uening kit) (Amersham pha)
Sequential reaction was carried out using 2 μg of each vector as a template using rmcia biotech). The reaction was performed under the following conditions using an arbitrary primer. Nucleotide sequence analysis is performed using a fluorescence sequencer (GeneRapid, Amersham pharm).
Acia biotech) was used. Each sample 2
μl was electrophoresed under the following conditions.

[0144] <PCR operating conditions>     Pre-heat 94 ° C for 2 minutes     Step 1 94 ℃ for 30 seconds     Step 2 60 ℃ for 30 seconds     Step 3 72 ° C for 1 minute                   (30 cycles of steps 1 to 3)     Post-heating (post-heat) 72 ° C for 2 minutes

[0145] <Sequence reaction conditions>     Pre-heat 94 ° C for 2 minutes     Step 1 94 ℃ for 30 seconds     Step 2 60 ℃ for 30 seconds     Step 3 72 ° C for 1 minute                   (30 cycles of steps 1 to 3)

<Electrophoresis conditions> Voltage 1250V Current 30mA Temperature 50 ℃ Laser 50%

[0147]   << Sequence of primer >>   KIAA1063-S1 (18-40) (SEQ ID NO: 58):     5'-GCG GTG CCT GCC                           TTG CAG CCT CC-3 '   KIAA1063-R2 (102-123) (SEQ ID NO: 59):     5'-CGG CTG GCC AGG                           CTG GCC AAG G-3 '   KIAA1063-S3 (1129-1149) (SEQ ID NO: 60):     5'-GTC AGG TGC CAA GTC TGC CAT-3 '   KIAA1063-R4 (517-540) (SEQ ID NO: 61):     5'-ACA GTA GAT GCC                           TCC ATA CAT CAG-3 '   KIAA1063-S5 (483-503) (SEQ ID NO: 62):     5'-TGC GAA GGC GAA GCG GCA CAA-3 '   KIAA1063-R6 (1767-1787) (SEQ ID NO: 63):     5'-TAA GGC TAC TCG TAT TCC AGG-3 '   KIAA1063-S7 (102-123) (SEQ ID NO: 64):     5'-CCT TGG CCA GCC                                   TGG CCA GCC G-3 '   KIAA1063-R8 (1177-1197) (SEQ ID NO: 65):     5'-GAG ATC CAA GCT GAT GTC CCA-3 '   KIAA1063-S9 (280-301) (SEQ ID NO: 66):     5'-CTG GGC AGC TTC                                   AAG GTG GAC A-3 '   KIAA1063-R10 (280-301) (SEQ ID NO: 67):     5'-TGT CCA CCT TGA                                   AGC TGC CCA G-3 '   KIAA1063-S11 (328-349) (SEQ ID NO: 68):     5'-TAC CAG TGC TTC                                   GTG TGG AGC G-3 '   KIAA1063-R12 (328-349) (SEQ ID NO: 69):     5'-CGC TCC ACA AGC                                   CGA ACT GGT A-3 '

For cloning the 5'-terminal deletion portion,
5'-race ready cDNA for human brain (M
axim Biotech, Inc. )It was used. For the primer, combine the primer attached to the kit with the primer prepared on the 5'end side, and use the Expand High Fidelity Tack DNA Polymerase (Expand High Fidelity T
aq DNA Polymerase (Roche) was used. First, 5AP1001 primer and KIAA
A PCR reaction was performed using a combination of 1063-R2 primers (PCR1). Then, using this PCR product as a template, 5AP1002 primer and KIAA
A re-PCR reaction was performed with the combination of 1063-R2 primers (PCR2). Then, a PCR reaction was performed using a combination of the 5AP1001 primer and the KIAA1063-R10 primer. PCR obtained respectively
After the product was purified, it was blunt-ended and phosphorylated. PCR
The product was ligated with a pBluescript KS (+) vector (blunt-ended by decomposing the restriction enzyme SmaI and dephosphorylated) to prepare a competent cell (JM1).
09). An E. coli colony containing a plasmid having the desired insert was selected and the plasmid was extracted.

The nucleotide sequence of the insert of the plasmid was examined. For determination of the nucleotide sequence, Thermo Sequenase Cy5.5 Dye Terminator Sequencing Kit (Amersham Pharmacia) was used.
Biotech) was used to carry out a sequence reaction using 2 μg of each vector as a template. The reaction was performed under the following conditions using an arbitrary primer. Nucleotide sequence analysis is performed using a fluorescent sequencer (GeneRapi).
d, Amersham pharmacia biot
ech) was used. 2 μl of each sample was electrophoresed under the following conditions.

[0150] <Operating conditions of PCR1>     Pre-heat 94 ° C for 2 minutes     Step 1 94 ° C for 1 minute     Step 2 55 ° C for 1 minute     Step 3 72 ℃ 1 minute 30 seconds                   (10 cycles of steps 1 to 3)     Step 4 94 ° C for 1 minute     Step 5 62 ℃ for 1 minute     Step 6 72 ℃ 2 minutes                   (20 cycles of steps 4 to 6)     Post-heating (post-heat) 72 ° C for 2 minutes

[0151] <Operating conditions of PCR2>     Pre-heat 94 ° C for 2 minutes     Step 1 94 ℃ for 30 seconds     Step 2 58 ℃ 30 seconds     Step 3 72 ℃ 2 minutes                   (30 cycles of steps 1 to 3)     Post-heating (post-heat) 72 ° C for 2 minutes

<Sequence reaction conditions> Same as above

<Electrophoresis conditions> Same as above

As described above, various primers were designed based on the nucleotide sequences determined by Kazusa DNA Research Institute,
S1-R4, S5-R8, S3-R6, S11-R4,
When each RT-PCR was examined with the combination of S9-R4, all PCR products were detected in the expected size. Examining the nucleotide sequences of these PCR products,
It was confirmed that the combination of S5-R8, S3-R6 and S11-R4 completely matched the sequence of KIAA1063. In the combination of S1-R4 and S9-R4,
PCR product was detected with expected size, but K
No sequence corresponding to the base sequence of IAA1063 was obtained. Subsequently, in order to clone the deleted portion at the 5'-end, 5'RACE method was performed, and R2 primer and R
By PCR using 10 primers, 3 types of PCR products were detected respectively. When the nucleotide sequences of these PCR products were examined, a PCR product containing the gene sequence on the 5'end side of KIAA1063 was not obtained.

(Expression of KIAA1063 in E. coli) pBS-KIAA obtained from Kazusa DNA Research Institute
PCR was performed by using 10ng of 1063 as a template and combining the following primers. Expand High Fidelity P for Taq DNA Polymerase
A CR system (Roche) was used. Amplified P
CR product is 30% PEG8000 / 30 mM MgC
₁₂ and purified using the pDONR 201 vector, B
1 at room temperature using P-Clonase Enzyme Mix
Reacted for hours. This reaction solution is used as a competent cell (DH
5α), seeded on LB-plate containing kanamycin (50 μg / ml), pD
Obtained ONR-KIAA1063.

[0156] <PCR operating conditions>     Pre-heat 95 ° C for 2 minutes     Step 1 95 ℃ 30 seconds     Step 2 60 ℃ 30 seconds     Step 3 72 ℃ 2 minutes                   (20 cycles of steps 1 to 3)     Post-heating (post-heat) 72 ° C for 2 minutes

[0157] <Primer sequence>   attB-KIAA1063-S1 (SEQ ID NO: 70):     5'-GGG ACA AGT TTG TAC AAA AAA GCA           GGC TCC ATG GAC GCC GAG CTG GAG           GTA-3 '   attB-KIAA1063-R2 (SEQ ID NO: 71):     5'-GGG GAC CAC TTT GTA CAA GAA AGC           TGG GTC CTA CTG GTA TTC CAG GAA           CTG-3 '

For determination of the nucleotide sequence of the pDONR-KIAA1063 vector, the Big Dye Terminator Cycle Sequencing (Applied Biosys) was used.
tems) using pDONR-KIAA1063
Sequence reaction was carried out using 500 ng of
The reaction was performed under the following conditions using 4 pmol of any primer. The reaction product was purified by ethanol precipitation and used for analysis. The base sequence is analyzed by a capillary sequencer (ABI PRISM 310, Applied).
Biosystems) was used.

<Preparation of reaction solution> Template DNA 500ng Primer 4 pmol Premix 8 μl

<Sequence reaction conditions> Step 1 96 ℃ 10 seconds Step 2 60 ℃ for 4 minutes (30 cycles of steps 1 and 2)

<Electrophoresis conditions> Voltage 12.2kV Current 4 μA Temperature 50 ℃ Running time 120 minutes

Using the pDONR-KIAA1063 vector and the pDEST17 vector, the reaction was carried out for 1 hour at room temperature using the LR clonase enzyme mix.
This reaction solution was transformed into competent cells (BL21-SI), and ampicillin (50 μg /
ml) containing LB-plate and His-tagged KIAA1063 expression vector (pDEST1).
7-KIAA1063).

Four types of arbitrary colonies of pDEST17-KIAA1063 were cultured in 1.5 ml of LBON-Amp medium at 37 ° C. for 4 hours, and then four (300 μl / tub)
Dispensed in e). 2 bottles each, NaCl addition group (5 M NaCl 20 μl addition, final concentration 0.3 M),
The non-addition group (sterile distilled water 20 μl) was used, and the NaCl addition group and the non-addition group were cultured at 37 ° C. and 25 ° C. for 2 hours, respectively. The cells were buffered with T (50 mM Tris (p
H8.0) / 150 mM NaCl / 1% Trito
n X-100) 50 μl, and the mixture was sonicated on ice and centrifuged to collect the supernatant. This supernatant was mixed with 2 × sample buffer (2% SDS / 50 mM Tris (p
H8.0) / 30% glycerol / 0.01%
BPB) 50 μl and β-mercaptoethanol 10 μ
1 was added (soluble fraction). Furthermore, suspend the precipitate in 50 μl of buffer T and
After adding 50 μl and ultrasonically crushing on ice, 10 μl of β-mercaptoethanol was added (insoluble fraction). Each sample was boiled for 3 minutes, electrophoresed on a 12% acrylamide gel, and detected by CBB staining.

When the H6-KIAA1063 protein was expressed in BL21-SI which expresses the target protein by addition of NaCl, production was induced only in the NaCl addition group, and a band was detected around 60 kDa estimated from the amino acid sequence ( FIG. 21A). But usually (37
The protein induced under the culture conditions of
It was not solubilized even in the presence of a surfactant such as -100, and was produced as an insoluble component. Therefore, H6-KIAA1
The culture conditions for transferring the 063 protein to the soluble fraction were examined. The temperature was changed to a low temperature (25 ° C) for examination, but no shift to the soluble fraction was observed (Fig. 2).
B of 1).

(Deubiquitinating enzyme activity in vitro) [0165] Any bacterial solution was added to 37 ml of LBON-Amp medium in 2 ml.
After culturing at 4 ° C. for 4 hours, the cells were dispensed into 2 tubes (800 μl / tube). NaCl addition group (52 μl of 5 M NaCl)
Addition, final concentration 0.3M, non-addition group (sterile distilled water 52μ
1) and further cultured at 37 ° C. for 3 hours. The cells were suspended in buffer T, sonicated on ice and centrifuged, and the supernatant was collected (soluble fraction). This soluble fraction (20 μl
And 2 μl) and the substrate Ub-MG prepared in Example 3
ST (5 μl) was reacted overnight at 37 ° C. The presence or absence of expression of the target protein was detected by His antibody, and the presence or absence of enzyme activity was detected by GST antibody in the same manner as in Example 3 by Western blotting. However, no deubiquitinase activity was observed in KIAA1063.
Therefore, when H6-KIAA1063 protein was purified and concentrated using a nickel affinity column, the activity of the purified sample was examined.
Most impurities are removed and H6-KIAA is obtained in high yield.
1063 protein was recovered. Of these, the refolded sample of the fraction eluted at the highest concentration was used for the examination of USP activity. However, USP activity was not observed in this purified preparation, although the presence of the H6-KIAA1063 protein was observed.

(Deubiquitinating Enzyme Activity in Substrate Co-Expression System) The expression vector pDEST17-KIAA1063 or pHis-USP15pHis-Luc was added to E. coli BL21-SI competent cells introduced with the substrate expression plasmid prepared in Example 1. In addition, various co-expression strains were obtained in the same manner as in Example 1. pHis-US
As P15, the one produced in Example 1 was used. Various co-expressing strains were cultured in 2 ml of LBON-Amp medium at 30 ° C. overnight. 200 ml of this bacterial solution was added to 2 ml of LBON-Amp medium.
After adding 3 μl and culturing at 30 ° C for 3 hours, 5 tubes (300μ)
1 / tube). Each of NaCl 0.
05M, 0.1M, 0.2M, 0.3M (5M each
NaCl 3 μl, 6 μl, 12 μl, 18 μl added), non-added group (sterilized distilled water 18 μl added), 30
Culturing was carried out at 3 ° C. for 3 hours or at 30 ° C. for 16 hours. The cells were suspended in 50 μl of buffer T, 50 μ of 2 × sample buffer was added, and the cells were ultrasonically disrupted on ice.
10 μl of β-mercaptoethanol was added. After boiling for 3 minutes, electrophoresis was performed on a 12% acrylamide gel. CBB staining or H for confirmation of protein production induction
It was analyzed by Western blotting using an is antibody. In addition, the presence or absence of enzyme activity was detected by Western blotting using a GST antibody.

The KIAA1063 protein had an increased production depending on the dose of NaCl (FIG. 22A). When the deubiquitinating enzyme activity at this time was examined using the Western blotting method, KIAA1063 protein was the substrate Ub-M-GST, Ub-R-GST, Ub.
It exhibited a deubiquitinating enzyme activity by acting on -I-GST (FIG. 22B and FIG. 23). However, Ub-PG
The enzyme activity against ST was weak. Regarding the enzyme activity of KIAA1063, the production amount of substrate and KIA were lower when NaCl was lower.
The activity of A1063 was well balanced, and good results were obtained (B in FIG. 22).

(Confirmation of enzyme active center) KIAA1453
Was confirmed in the co-expression system with the substrate using the KIAA1063 mutant. First, replacement of 174-Cys, which is a putative active residue of KIAA1063, with Ala was performed using a quick change site-directed mutagenesis kit. The primer used was TGC Al, which is the codon of 174-Cys.
It was designed to be replaced with GCC which is the codon of a (5'-GAT CAA CCT TGG GAA C
AC AGC CTTCAT GAA CTG CAT
CGT GC-3 '(SEQ ID NO: 72) and 5'-G
CA CGA TGC AGT TCA TGA AG
G CTGTGT TCC CAA GGT TGA
TC-3 '(SEQ ID NO: 73)). Using these primers, pDONR-KIAA1063 was used as a template, and Pfu turbo DNA polymerase was used as an enzyme to carry out a PCR reaction. After digesting this PCR product with a restriction enzyme (Dpn I), competent cells (J
M109), seeded on LB-plates containing kanamycin, pDONR-mut-
Obtained KIAA1063. The introduction of the mutation was confirmed by sequencing, and pDONR-mut-KIAA106
And pDEST17 vector were used to react for 1 hour at room temperature using LR clonase enzyme mix.
This reaction solution was transformed into competent cells (BL21-SI), seeded on an LB-plate containing ampicillin, and His-tagged KIAA1.
063 mutant protein expression vector (pDEST17-m
ut-KIAA1063). E. coli BL21 into which various substrate expression plasmids were introduced in the same manner as above
-Recombinant E. coli was obtained by transforming into SI competent cells. As a result, the activity was lost in the C174A mutant, and it was revealed that this activity was due to ¹⁷⁴ Cys in Cysbox (FIG. 24). Where KI
When the expression of AA1063 ^C174A was confirmed, the same level of expression as that of the wild type (KIAA1063) was observed, and it was revealed that the loss of activity was not the difference in the expression amount.

[0169]

Example 7 (Isolation / Identification of KIAA0190) KIA
A0190 cDNA was extracted as a novel protease candidate gene by bioinformatics from the human long chain cDNA analysis information database of Kazusa DNA Research Institute. KI
AA0190 is ORF predictive analysis software Gene Mark
Analysis by Gene Mark Analysis suggested that the N-terminal may be deleted (Kazusa DNA Research Institute, HUGE Data bas).
e, <http: // www. kazusa. or. j
p / huge / gfpage / KIAA0190>).
Therefore, an attempt was made to further extend the N-terminal by EST search using BLAST search, but the translation initiation site (Met) could not be confirmed in the upstream base sequence.
In addition, the upstream nucleotide sequence was not confirmed in the genome search. Based on the above, the cDNA provided by Kazusa DNA Research Institute was determined to be full-length, and 32ATG34 in the GenBank published base sequence was used as the translation initiation site, 2441.
TAA2443 was used as the translation termination site (ORF 2.4
Kb). KIAA0190 is a U consisting of 803 amino acid residues encoded by a gene having a base length of 3280 bp.
SP and the 421st Gly as an active motif
To the 436th Gln, the Cys box, the 73rd
His b from the 7th Tyr to the 755th Tyr
had ox. Further, in the homology search of the amino acid deduced from the ORF by BLAST search, it was found that P52479 which is mouse USP10 is Cys.
100% homology was found in the box and His box, and about 80% overall homology was observed.

(Expression of KIAA0190 in E. coli) KIAA0190 obtained from Kazusa DNA Research Institute
Clone (S of pBluescript II SK +
(inserted into alI-NotI site) as a template, and Pr-DONR0190 (+) 5′-GGGAC as a primer.
AAGTTTTGCATAAAAAAGCAGGCTCA
ATGAAACGGGCAGCCCATG-3 ′ (SEQ ID NO: 74) and Pr-DONR0190 (−) 5′-G
GGGACCACTTTGTACAAGAAAGCTG
GGTTTTACAGCAGGTCCACTCG-3 '
(SEQ ID NO: 75) and His-tagged KIAA0190 expression plasmid (pH
ia-K0190) was prepared and introduced into E. coli BL21-SI. It was confirmed by sequencing that mutations such as PCR errors did not occur in the ORF region.

Then, E. coli BL21-SI introduced with KIAA0190 as described above was treated with LBO containing ampicillin.
The cells were cultured in N medium (LB medium not containing NaCl) at 37 ° C. until the late logarithmic growth phase (OD ₆₀₀ = about 1.0). Then, NaCl was added to E. coli to induce T7 RNA polymerase to a final concentration of 0.3 M, and the cells were further cultured for 3 hours. After collecting the cells by centrifugation, 1/10 volume of TN buffer (5
0 mM Tris (pH 7.4), 150 mM NaC
1, 1 mM DTT, 0.25 mg / ml lysozyme), and the cells were disrupted by sonication (5 seconds × 2, under ice cooling) (whole cell extract). A part of the disrupted liquid was centrifuged (15000 rpm × 5 minutes), and the resulting supernatant was used as a soluble fraction crude extract and stored at −80 ° C. until use.

The crude extract was electrophoresed in the same manner as in Example 3 and the protein was detected by CBB staining in the same manner as in Example 3. In addition, the proteins that have been electrophoresed in the same manner are loaded onto a PVDF membrane (Pol
yscreen NEF-100, Daiichi Kagaku Co., Ltd., and anti-His antibody (Penta-His antibody, QIA)
His fusion protein was detected by (GEN). as a result,
The protein was expressed in a NaCl-dependent manner, and a positional band of about 100 kDa was detected on SDS-PAGE (Fig. 2).
5). Most of the wild-type and mutant-type expressed proteins were present in the insoluble fraction, and the distribution in the soluble fraction was small. Also, a low molecular weight protein was observed by Western blotting with His antibody, suggesting that the synthesized protein was degraded in the bacterial cells.

(In-vivo deubiquitinating enzyme activity) Escherichia coli BL21-SI containing pHis-KIAA0190 prepared above or pHis-USP15 prepared in Example 1 was induced and cultured in the same manner as above, and the cells were recovered. did. Using this cell, an Escherichia coli extract was obtained in the same manner as in Example 3. The substrate expression plasmid (pACUb-M) prepared in Example 1 was used as the substrate.
-GST, pACUb-P-GST, pACUb-I-
E. coli BL21-SI harboring GST and pACUb-R-GST) was cultured at 37 ° C. until the OD ₆₀₀ reached about 1.0, and then NaCl was added to a final concentration of 0.3 M, and further about 3 M. After culturing for a period of time, the bacterial cells were collected. After suspending the cells in PBS (-) of 1/10 volume of the culture solution, the cells were disrupted by ultrasonic treatment and centrifuged (15000 rp).
m, 10 minutes). The supernatant was used as a substrate for activity measurement (Ub-M-GST, Ub-P-GS).
T, Ub-I-GST and Ub-R-GST) solutions.

14 μl of each Escherichia coli extract and 14 μl of each substrate (protein mass: 39 μl) were mixed and incubated at 37 ° C. overnight. At this time, in order to examine the inhibitory effect of the cysteine protease inhibitor, N-ethylmaleimide (NEM, Sigma) was added to the reaction solution.
Was added to a final concentration of 20 mM. Next, after the reaction solution was separated by 15% SDS-PAGE, Example 3 was used.
GST after Ub dissociation was detected in the same manner as in.

In vitro, Ub-M-GST,
For each of the Ub-R-GST and Ub-I-GST substrates, Western blot showed GST release from the Ub-fused GST, showing that KIAA0190 has deubiquitinating enzyme activity against these substrates. (FIGS. 26 and 27). In addition, this deubiquitinating enzyme activity is N, which is a cysteine protease inhibitor.
Inhibited by the addition of EM, cysteine residues were shown to be important for activity. On the other hand, Ub-PG
When ST was used, GST release was not observed, and KIA
It was found that A0190 was difficult to cleave the Ub-P bond (Fig. 27).

Further, as a polyubiquitinated protein synthesis system, rabbit reticulocyte cell fluid (Rabbit Reti) was used.
Culocyte Lysate Systems / U
Using an embedded (RRL), Promega, 300 mL of Ub-mixed (33% (v / v) R)
RL, 50 mM Tris-HCl (pH 8.3), 5 m
M MgCl ₂ , 2 mM DTT, 20 mg Ub, 1
mM ATPγS, 97 mM lactocystin (proteasome inhibitor, Kyowa Medex Co., Ltd.) at 37 ° C
And incubated for 20 minutes. After the reaction was completed, NEM was added to a final concentration of 20 mM to stop the Ub formation reaction, and then the Centricon-3 concentrator (Ce
ntricon-3 Concentrator, Am
Ionic) was washed four times by centrifugation (7500 rpm × 30 minutes) to remove NEM, and this was used as a Ub protein.
De-Ub-ized reaction is Ub-ized protein (320 mg protein)
The crude extract of E. coli expressing KIAA0190 and KIAA0529 was added to the assay buffer (50 mM Tris-
HCl pH 8.3, 5 mM MgCl ₂ , ₂ mM DT
T) and incubated at 37 ° C. for 1 hour,
Western blot was performed and anti-Ub antibody (Sigma
The presence or absence of degradation of the Ubylated protein was detected in a). As a result, the positive control KIAA0529 showed the disappearance of the polyubiquitin chain and the deubiquitinating enzyme activity could be confirmed, but the activity could not be confirmed with KIAA0190.

(Deubiquitinating enzyme activity in co-expression system with substrate) E. coli BL21-SI competent cells into which the substrate expression plasmid prepared in Example 1 was introduced,
The expression vectors pHis-K1003 and pHis-U
Various co-expressing strains were obtained in the same manner as in Example 1 by adding SP15 or pHis-Luc. pHis-USP15
Alternatively, the pHis-Luc used was that produced in Example 1. A cell extract was prepared using various co-expressing strains in the same manner as in Example 5, the extract was separated by SDS-PAGE, and Ub-was subjected to immunoblotting using an anti-GST antibody in the same manner as in Example 5. X-GST and free GS
T was detected. In addition, the expression of each co-expressed enzyme was determined by the anti-His-tag antibody (Penta × His ^™ Ant).
It was detected by immunoblotting using ibody, QIAGEN).

As a result, KIAA0190 was used in the co-expression system in the same manner as in vitro, Ub-M-GST and Ub-.
GST release from Ub-fused GST was observed in R-GST and Ub-I-GST, and deubiquitinating enzyme activity against these substrates was observed. On the other hand, Ub
No deubiquitinating enzyme activity was observed for -P-GST (Fig. 28). Moreover, when the expression of each enzyme was confirmed, all the enzymes were expressed.

(Confirmation of enzyme active center) KIAA0190
The enzyme active center of Escherichia coli was confirmed in a co-expression system with a substrate using the KIAA0190 mutant. First, 429-Cys, which is a putative active residue of KIAA0190, was replaced with Ser using a quick change site-directed mutagenesis kit. That is, p
Primer 5'-G using ENTRY0190 as a template
GGAACTGGAGGCCATATTAATGCTAC
ACTGCAG-3 ′ (SEQ ID NO: 76) and 5′-C
TCGAGTGTAGCATTAATGTAGCTCC
Using AGTTCCCC-3 ′ (SEQ ID NO: 77), Pf
Cycling reactions were performed with u Turbo DNA polymerase. Then, the template plasmid was digested with DpnI, and the reaction solution was added to Escherichia coli DH5α competent cells for transformation. The introduction of the mutation was confirmed by sequencing, and His-tag was added to KIAA0190.
^C429-S expression plasmid, pHis-K0190
^C429S was obtained. This was transformed into Escherichia coli BL21-SI competent cells into which various substrate expression plasmids had been introduced in the same manner as above to obtain recombinant Escherichia coli. as a result,
In the ^C429- S mutant, the activity disappeared, and this activity was
It was revealed to be due to 429-Cys in the box. Here, when the expression of KIAA0190 ^C429S was confirmed, the same level of expression as that of the wild type (KIAA0190) was observed. Therefore, it was revealed that the loss of activity was not the difference in the expression level (FIG. 29).

[0180]

Example 8 (Isolation / Identification of KIAA0891) KIA
A0891 cDNA is obtained by bioinformatics from the human long-chain cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics.
It was extracted as a novel protease candidate gene. KIAA
0891 is a USP consisting of 1318 amino acid residues encoded by a gene having a base length of 4401 bp, and Cys box from the 498th Gly to the 513th Gln and the 1149th Ty as the active motif.
It had a His box at the 1166th Tyr from r.

(Expression of KIAA0891 in E. coli) KIAA0891 obtained from Kazusa DNA Research Institute
Primer: 0 using a gene-containing plasmid as a template
891Ex-s 5'-GGGACAAGTTTTGTA
CAAAAAAGCAGGCTTAGAAGGAGAT
AGAACCCATGTCTGGCGGGCGCCAGTG
CCAC-3 '(SEQ ID NO: 78), 0891Ex-a:
5'-GGGACCACTTTTGTACAAGAAAG
CTGGGTCCTAGTGATGGGTGATGGGTG
ATGTCTCCAGCGACTCTGGGA-3 '
Using (SEQ ID NO: 79), the ORF region in which His × 6 was added to the C terminus was subjected to PCR (AdvantageR-HF).
2, CLONTECH). The PCR product was transformed into the vector pD using BP clonase enzyme.
Recombinant with ONER201, E. coli DH5
By transforming into α, an entry clone was prepared.
A plasmid was extracted from this clone (WizardR
Plus SV Minipreps DNA Pu
refining system, Promega
Then, it was confirmed by performing a sequence whether the ORF region was inserted correctly. Also, using the same plasmid, L
A recombination reaction with the vector pDEST14 was carried out by R clonase, and Escherichia coli BL21-SI was transformed to prepare an expression clone.

In order to confirm the expression of KIAA0891 protein, 100 mL of recombinant E. coli was cultured under the following conditions for purification. BL21-SI transformed with the KIAA0891 expression vector was treated with ampicillin-containing LBO.
After culturing in N medium (LB medium not containing NaCl) at 37 ° C. until the late logarithmic growth phase (OD ₆₀₀ = about 1.0), NaCl was added to a final concentration of 0.15 M, The cells were cultured at 25 ° C for 3 hours. After culturing, the cells were collected by centrifugation. The obtained bacterial cells were treated with 1/20 volume of the buffer A (50 mM Sodium phosph).
ate, pH 7.0, 33 mM NaCl, 1 mM P
After suspending with MSF) and adding 10 mg / ml lysozyme (final concentration 0.9 mg / mL), the cells were ultrasonically disrupted on ice. Add Triton X-100 to the final concentration of 1
%, And the supernatant obtained by centrifugation was used as a crude extract, and TALONR metal affinity resin (Metal affinity Resin, CL) was used.
The target protein was purified by the batch method using ONTECH. That is, 1/50 replaced with buffer A
Add a volume of resin, mix by inversion at 4 ℃ for 1 hour, and add buffer B (50 mM Sodium phosphat
e, pH 7.0, 33 mM NaCl, 1% Trit
on X-100, 1 mM PMSF) resin
Was washed with 1/2 volume of elution buffer (50 mM
Sodium phosphate, pH 7.0,3
3 mM NaCl, 1% Triton X-100,
The protein chelated by the resin was eluted with 150 mM imidazole). This protein was detected by Western blotting.

As a result, as shown in FIG. 30, a band was detected near the estimated molecular weight of 146 kDa of this clone, although the expression level was weak. In addition, some bands with lower molecular weight than the estimated molecular weight were strongly detected by purification and concentration.

(In-Vitro Deubiquitinating Enzyme Activity) Escherichia coli BL21-SI carrying pHis-KIAA0891 prepared above or pHis-USP15 prepared in Example 1 was induced and cultured with NaCl in the same manner as above, and then the bacterial cells were Recovered. Using this cell, an Escherichia coli extract was obtained in the same manner as in Example 3. Further, as substrates, Ub-M-GST and Ub-R- prepared in the same manner as in Example 7 were used.
GST, Ub-I-GST, and Ub-P-GST were used.

Each substrate and enzyme was used in a crude extract volume of 1:
Mix in 1 and react at 37 ° C. for 16 hr, add 2 × SDS sample buffer containing mercaptoethanol in an equal amount, and heat at 100 ° C. for 5 minutes, then 4-12% SD
S-polyacrylamide gradient gel (Nu-
PAGE 4-12% Bis-Tris Gel,
Electrophoresis was performed by Invitrogen). Proteins were detected by CBB staining (Rapid Stain CBB).
Kit, Nacalai Tesque) and Western blotting. Western blotting after electrophoresis,
PVDF membrane (PolyScreen ^{registered trademark} PVDF
Transferred to Transfer Membrane (Daiichi Pure Chemicals), anti-GST antibody (Amersham Ph)
Ub fusion GST and de-Ub free GST were detected at Armia Biotech Inc.).

As a result, the substrates (Ub-I-GST, Ub-R-GST, U) used in examining the confirmation of enzyme activity were obtained.
b-M-GST, Ub-P-GST)
KIAA0891 protein was confirmed to have de-Ub synthase activity, although it was weaker overall than USP15 as a positive control, and the cleavage activity was 0.15M at 0.35M, which is the optimum NaCl concentration of T7 promoter. It showed strong activity (Fig. 31). Substrate selectivity of the enzyme activity was also observed, and particularly for Ub-P-GST, it tended to be more difficult to cleave than other substrates.

(Confirmation of enzyme active center) KIAA0891
The enzyme active center of Escherichia coli was confirmed in the above in vitro system using the KIAA0891 mutant. First, KIAA0891
Substitution of 506-Cys, which is a putative active residue of S. cerevisiae, into Ser was carried out using a Quick Change Site-Directed Mutagenesis Kit. The primer used was TGC Ser, which is the codon of 506-Cys.
Was designed to be replaced with AGC, which is the codon of (primer: 5'-TTTAGGGCAACACCTCTCTT
CATGAACAGCG-3 '(SEQ ID NO: 80) and 5'-CGCTGTTTCATGAAGGAGGTGTT
GCCTAAA-3 '(SEQ ID NO: 81)). The introduction of the mutation was confirmed by sequencing, and the His-tagged KIAA0891 ^C506S expression plasmid, ^pHi
s-K0891 ^C506S was obtained. E. coli BL21-S into which various substrate expression plasmids were introduced in the same manner as above
Recombinant E. coli was obtained by transforming into I competent cells. As a result, the activity was lost in the C506S mutant, and it was revealed that this activity was due to ⁵⁰⁶ Cys in the Cys box. In addition, as in the above, KIAA0
As a ^result of confirming the expression of 891 ^C506S, the same level of induction of expression as that of the wild type was observed, and it was confirmed that the loss of activity was not due to the non-expression of the protein (FIG. 31).

[0188]

EFFECT OF THE INVENTION Human long-chain cDNA of Kazusa DNA Research Institute
From the analytical information database and GenBank, bioinformatics (bioinformatics)
8) cDNA clones extracted as novel protease candidate genes by s), KIAA1097, AK
024318, KIAA1003, KIAA1372,
KIAA1453, KIAA1063, KIAA019
0 and KIAA0891 were confirmed to be expressed in Escherichia coli, and these gene products deubiquitinate ubiquitinated proteins.
It was confirmed to be SP). INDUSTRIAL APPLICABILITY The present invention enables elucidation of biological functions involved in USP, for example, elucidation of carcinogenic process, muscular atrophy, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and prevention, treatment and diagnosis thereof. It is very useful for doing.

[0189]

[Sequence list]                                SEQUENCE LISTING            <110> DAIICHI PHARMACEUTICAL CO., LTD.    <120> Novel ubiquitin specific proteases    <130> NP02-1103    <140> <141>    <150> JP P2001-304709 <151> 2001-09-28 <160> 81    <170> PatentIn Ver. 2.1    <210> 1 <211> 911 <212> PRT <213> Homo sapiens    <400> 1 Met Ser Ala Phe Arg Asn His Cys Pro His Leu Asp Ser Val Gly Glu   1 5 10 15 Ile Thr Lys Glu Asp Leu Ile Gln Lys Ser Leu Gly Thr Cys Gln Asp              20 25 30 Cys Lys Val Gln Gly Pro Asn Leu Trp Ala Cys Leu Glu Asn Arg Cys          35 40 45 Ser Tyr Val Gly Cys Gly Glu Ser Gln Val Asp His Ser Thr Ile His      50 55 60 Ser Gln Glu Thr Lys His Tyr Leu Thr Val Asn Leu Thr Thr Leu Arg  65 70 75 80 Val Trp Cys Tyr Ala Cys Ser Lys Glu Val Phe Leu Asp Arg Lys Leu                  85 90 95 Gly Thr Gln Pro Ser Leu Pro His Val Arg Gln Pro His Gln Ile Gln             100 105 110 Glu Asn Ser Val Gln Asp Phe Lys Ile Pro Ser Asn Thr Thr Leu Lys         115 120 125 Thr Pro Leu Val Ala Val Phe Asp Asp Leu Asp Ile Glu Ala Asp Glu     130 135 140 Glu Asp Glu Leu Arg Ala Arg Gly Leu Thr Gly Leu Lys Asn Ile Gly 145 150 155 160 Asn Thr Cys Tyr Met Asn Ala Ala Leu Gln Ala Leu Ser Asn Cys Pro                 165 170 175 Pro Leu Thr Gln Phe Phe Leu Asp Cys Gly Gly Leu Ala Arg Thr Asp             180 185 190 Lys Lys Pro Ala Ile Cys Lys Ser Tyr Leu Lys Leu Met Thr Glu Leu         195 200 205 Trp Tyr Lys Ser Arg Pro Gly Ser Val Val Pro Thr Thr Leu Phe Gln     210 215 220 Gly Ile Lys Thr Val Asn Pro Thr Phe Arg Gly Tyr Ser Gln Gln Asp 225 230 235 240 Ala Gln Glu Phe Leu Arg Cys Leu Met Asp Leu Leu His Glu Glu Leu                 245 250 255 Lys Glu Gln Val Met Glu Val Glu Glu Asp Pro Gln Thr Ile Thr Thr             260 265 270 Glu Glu Thr Met Glu Glu Asp Lys Ser Gln Ser Asp Val Asp Phe Gln         275 280 285 Ser Cys Glu Ser Cys Ser Asn Ser Asp Arg Ala Glu Asn Glu Asn Gly     290 295 300 Ser Arg Cys Phe Ser Glu Asp Asn Asn Glu Thr Thr Met Leu Ile Gln 305 310 315 320 Asp Asp Glu Asn Asn Ser Glu Met Ser Lys Asp Trp Gln Lys Glu Lys                 325 330 335 Met Cys Asn Lys Ile Asn Lys Val Asn Ser Glu Gly Glu Phe Asp Lys             340 345 350 Asp Arg Asp Ser Ile Ser Glu Thr Val Asp Leu Asn Asn Gln Glu Thr         355 360 365 Val Lys Val Gln Ile His Ser Arg Ala Ser Glu Tyr Ile Thr Asp Val     370 375 380 His Ser Asn Asp Leu Ser Thr Pro Gln Ile Leu Pro Ser Asn Glu Gly 385 390 395 400 Val Asn Pro Arg Leu Ser Ala Ser Pro Pro Lys Ser Gly Asn Leu Trp                 405 410 415 Pro Gly Leu Ala Pro Pro His Lys Lys Ala Gln Ser Ala Ser Pro Lys             420 425 430 Arg Lys Lys Gln His Lys Lys Tyr Arg Ser Val Ile Ser Asp Ile Phe         435 440 445 Asp Gly Thr Ile Ile Ser Ser Val Gln Cys Leu Thr Cys Asp Arg Val     450 455 460 Ser Val Thr Leu Glu Thr Phe Gln Asp Leu Ser Leu Pro Ile Pro Gly 465 470 475 480 Lys Glu Asp Leu Ala Lys Leu His Ser Ser Ser His Pro Thr Ser Ile                 485 490 495 Val Lys Ala Gly Ser Cys Gly Glu Ala Tyr Ala Pro Gln Gly Trp Ile             500 505 510 Ala Phe Phe Met Glu Tyr Val Lys Arg Phe Val Val Ser Cys Val Pro         515 520 525 Ser Trp Phe Trp Gly Pro Val Val Thr Leu Gln Asp Cys Leu Ala Ala     530 535 540 Phe Phe Ala Arg Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys Glu 545 550 555 560 Lys Cys Lys Lys Leu Arg Asn Gly Val Lys Phe Cys Lys Val Gln Asn                 565 570 575 Phe Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Leu             580 585 590 Met Phe Ser Thr Lys Ile Ser Thr His Val Ser Phe Pro Leu Glu Gly         595 600 605 Leu Asp Leu Gln Pro Phe Leu Ala Lys Asp Ser Pro Ala Gln Ile Val     610 615 620 Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Ser Ser 625 630 635 640 Gly His Tyr Ile Ala Tyr Cys Arg Asn Asn Leu Asn Asn Leu Trp Tyr                 645 650 655 Glu Phe Asp Asp Gln Ser Val Thr Glu Val Ser Glu Ser Thr Val Gln             660 665 670 Asn Ala Glu Ala Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala         675 680 685 Gln Lys Glu Arg Arg Arg Ile Ser Asn Leu Leu Asn Ile Met Glu Pro     690 695 700 Ser Leu Leu Gln Phe Tyr Ile Ser Arg Gln Trp Leu Asn Lys Phe Lys 705 710 715 720 Thr Phe Ala Glu Pro Gly Pro Ile Ser Asn Asn Asp Phe Leu Cys Ile                 725 730 735 His Gly Gly Val Pro Pro Arg Lys Ala Gly Tyr Ile Glu Asp Leu Val             740 745 750 Leu Met Leu Pro Gln Asn Ile Trp Asp Asn Leu Tyr Ser Arg Tyr Gly         755 760 765 Gly Gly Pro Ala Val Asn His Leu Tyr Ile Cys His Thr Cys Gln Ile     770 775 780 Glu Ala Glu Lys Ile Glu Lys Arg Arg Lys Thr Glu Leu Glu Ile Phe 785 790 795 800 Ile Arg Leu Asn Arg Ala Phe Gln Lys Glu Asp Ser Pro Ala Thr Phe                 805 810 815 Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ser Phe Val Lys             820 825 830 Gly Lys Asp Gly Asp Pro Pro Gly Pro Ile Asp Asn Thr Lys Ile Ala         835 840 845 Val Thr Lys Cys Gly Asn Val Met Leu Arg Gln Gly Ala Asp Ser Gly     850 855 860 Gln Ile Ser Glu Glu Thr Trp Asn Phe Leu Gln Ser Ile Tyr Gly Gly 865 870 875 880 Gly Pro Glu Val Ile Leu Arg Pro Pro Val Val His Val Asp Pro Asp                 885 890 895 Ile Leu Gln Ala Glu Glu Lys Ile Glu Val Glu Thr Arg Ser Leu             900 905 910    <210> 2 <211> 4271 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (210) .. (2945)    <400> 2 cggacccccc gagggcagcg ctgcggggcc gttttccggc cctcctgacg cgacactgcc 60    cctctccgag agctgagaag gaaaagagga gcttgcggag gtgcggctgc aggccgttgt 120    tggtcgagtt ggcgggtccc gcgggccagg ccgtggaggt gttacctcat tttgaaagtc 180    ttgggaaaca ggaaaaaatt cctaacaaa atg tca gct ttt cga aat cat tgt 233                                 Met Ser Ala Phe Arg Asn His Cys                                   1 5    cca cat ttg gat tca gtt ggt gaa ata aca aaa gaa gat ttg ata caa 281 Pro His Leu Asp Ser Val Gly Glu Ile Thr Lys Glu Asp Leu Ile Gln      10 15 20    aaa tcc ctt ggt act tgt cag gat tgt aaa gtc caa gga cca aat ctt 329 Lys Ser Leu Gly Thr Cys Gln Asp Cys Lys Val Gln Gly Pro Asn Leu  25 30 35 40    tgg gca tgt ctg gag aat aga tgt tca tat gtt ggc tgt ggt gaa tca 377 Trp Ala Cys Leu Glu Asn Arg Cys Ser Tyr Val Gly Cys Gly Glu Ser                  45 50 55    caa gta gat cac agc acc ata cat tct cag gag aca aag cat tat cta 425 Gln Val Asp His Ser Thr Ile His Ser Gln Glu Thr Lys His Tyr Leu              60 65 70    act gtg aac ctt acc act ctt cga gta tgg tgt tat gct tgc agc aaa 473 Thr Val Asn Leu Thr Thr Leu Arg Val Trp Cys Tyr Ala Cys Ser Lys          75 80 85    gaa gta ttt ttg gat agg aaa tta gga act cag cct tca ttg cct cat 521 Glu Val Phe Leu Asp Arg Lys Leu Gly Thr Gln Pro Ser Leu Pro His      90 95 100    gta aga caa cct cac caa ata caa gaa aac agt gtc cag gat ttt aaa 569 Val Arg Gln Pro His Gln Ile Gln Glu Asn Ser Val Gln Asp Phe Lys 105 110 115 120    ata ccc agt aat aca aca tta aaa act cct ctg gtt gcc gta ttt gat 617 Ile Pro Ser Asn Thr Thr Leu Lys Thr Pro Leu Val Ala Val Phe Asp                 125 130 135    gat ctg gat ata gaa gcg gat gaa gaa gat gaa ctt agg gcc aga ggt 665 Asp Leu Asp Ile Glu Ala Asp Glu Glu Asp Glu Leu Arg Ala Arg Gly             140 145 150    ctt aca ggt ttg aaa aat att gga aat act tgt tac atg aat gca gct 713 Leu Thr Gly Leu Lys Asn Ile Gly Asn Thr Cys Tyr Met Asn Ala Ala         155 160 165    ttg cag gct ctt tct aat tgc cca cct ttg aca cag ttt ttt ctt gat 761 Leu Gln Ala Leu Ser Asn Cys Pro Pro Leu Thr Gln Phe Phe Leu Asp     170 175 180    tgt gga gga cta gct cga aca gat aag aaa cct gcc att tgt aaa agt 809 Cys Gly Gly Leu Ala Arg Thr Asp Lys Lys Pro Ala Ile Cys Lys Ser 185 190 195 200    tat ctc aaa cta atg aca gag ctg tgg tat aaa agc agg cca gga tct 857 Tyr Leu Lys Leu Met Thr Glu Leu Trp Tyr Lys Ser Arg Pro Gly Ser                 205 210 215    gtt gtg cct act act ctg ttt caa gga att aaa act gta aat cca aca 905 Val Val Pro Thr Thr Leu Phe Gln Gly Ile Lys Thr Val Asn Pro Thr             220 225 230    ttt cgg ggg tat tct cag cag gat gct caa gaa ttc ctt cga tgt tta 953 Phe Arg Gly Tyr Ser Gln Gln Asp Ala Gln Glu Phe Leu Arg Cys Leu         235 240 245    atg gat ttg ctt cat gaa gaa ttg aaa gag caa gtc atg gaa gta gaa 1001 Met Asp Leu Leu His Glu Glu Leu Lys Glu Gln Val Met Glu Val Glu     250 255 260    gaa gat ccg caa acc ata acc act gag gag aca atg gaa gaa gac aag 1049 Glu Asp Pro Gln Thr Ile Thr Thr Glu Glu Thr Met Glu Glu Asp Lys 265 270 275 280    agc cag tcg gat gta gat ttt cag tct tgt gaa tct tgt agc aac agt 1097 Ser Gln Ser Asp Val Asp Phe Gln Ser Cys Glu Ser Cys Ser Asn Ser                 285 290 295    gat aga gca gaa aat gaa aat ggc tct aga tgc ttt tct gaa gat aat 1145 Asp Arg Ala Glu Asn Glu Asn Gly Ser Arg Cys Phe Ser Glu Asp Asn             300 305 310    aat gaa aca aca atg tta att cag gat gat gaa aac aat tca gaa atg 1193 Asn Glu Thr Thr Met Leu Ile Gln Asp Asp Glu Asn Asn Ser Glu Met         315 320 325    tca aag gat tgg caa aaa gag aag atg tgc aat aag att aat aaa gta 1241 Ser Lys Asp Trp Gln Lys Glu Lys Met Cys Asn Lys Ile Asn Lys Val     330 335 340    aat tct gaa ggc gaa ttt gat aaa gat aga gac tct ata tct gaa aca 1289 Asn Ser Glu Gly Glu Phe Asp Lys Asp Arg Asp Ser Ile Ser Glu Thr 345 350 355 360    gtc gac tta aac aac cag gaa act gtc aaa gtg caa ata cac agc aga 1337 Val Asp Leu Asn Asn Gln Glu Thr Val Lys Val Gln Ile His Ser Arg                 365 370 375    gct tca gaa tat atc act gat gtc cat tcg aat gac ctg tct aca cca 1385 Ala Ser Glu Tyr Ile Thr Asp Val His Ser Asn Asp Leu Ser Thr Pro             380 385 390    cag atc ctt cca tca aat gaa ggt gtt aat cca cgt tta tcg gca agc 1433 Gln Ile Leu Pro Ser Asn Glu Gly Val Asn Pro Arg Leu Ser Ala Ser         395 400 405    cct cct aaa tca ggc aat ttg tgg cca gga ttg gca cca cca cac aaa 1481 Pro Pro Lys Ser Gly Asn Leu Trp Pro Gly Leu Ala Pro Pro His Lys     410 415 420    aaa gct cag tct gca tct cca aag aga aaa aaa cag cac aag aaa tac 1529 Lys Ala Gln Ser Ala Ser Pro Lys Arg Lys Lys Gln His Lys Lys Tyr 425 430 435 440    aga agt gtt att tca gac ata ttt gat gga aca atc att agt tca gtg 1577 Arg Ser Val Ile Ser Asp Ile Phe Asp Gly Thr Ile Ile Ser Ser Val                 445 450 455    cag tgt ctg act tgt gac agg gtg tct gta acc ctc gag acc ttt caa 1625 Gln Cys Leu Thr Cys Asp Arg Val Ser Val Thr Leu Glu Thr Phe Gln             460 465 470    gat ctg tcc ttg cca att cct ggc aag gaa gac ctt gct aag ctg cat 1673 Asp Leu Ser Leu Pro Ile Pro Gly Lys Glu Asp Leu Ala Lys Leu His         475 480 485    tca tca agt cat cca act tct ata gtc aaa gca gga tca tgt ggc gaa 1721 Ser Ser Ser His Pro Thr Ser Ile Val Lys Ala Gly Ser Cys Gly Glu     490 495 500    gca tat gct cca caa ggg tgg ata gct ttt ttc atg gaa tat gtg aag 1769 Ala Tyr Ala Pro Gln Gly Trp Ile Ala Phe Phe Met Glu Tyr Val Lys 505 510 515 520    agg ttt gtt gtc tca tgt gtc cct agc tgg ttt tgg ggt cca gta gta 1817 Arg Phe Val Val Ser Cys Val Pro Ser Trp Phe Trp Gly Pro Val Val                 525 530 535    acc ttg caa gat tgt ctt gct gcc ttc ttt gcc aga gat gaa cta aaa 1865 Thr Leu Gln Asp Cys Leu Ala Ala Phe Phe Ala Arg Asp Glu Leu Lys             540 545 550    ggt gac aat atg tac agt tgt gaa aaa tgc aaa aag ttg aga aat gga 1913 Gly Asp Asn Met Tyr Ser Cys Glu Lys Cys Lys Lys Leu Arg Asn Gly         555 560 565    gtg aag ttt tgt aaa gta caa aac ttt cct gag att ttg tgc atc cac 1961 Val Lys Phe Cys Lys Val Gln Asn Phe Pro Glu Ile Leu Cys Ile His     570 575 580    ctt aaa aga ttc aga cat gaa cta atg ttt tcc acc aaa atc agt acc 2009 Leu Lys Arg Phe Arg His Glu Leu Met Phe Ser Thr Lys Ile Ser Thr 585 590 595 600    cat gtt tca ttt ccg cta gaa ggc ttg gat ctt cag cca ttt ctt gct 2057 His Val Ser Phe Pro Leu Glu Gly Leu Asp Leu Gln Pro Phe Leu Ala                 605 610 615    aag gat agt cca gct caa att gtg aca tat gat ctt ctg tca gtc att 2105 Lys Asp Ser Pro Ala Gln Ile Val Thr Tyr Asp Leu Leu Ser Val Ile             620 625 630    tgc cat cat gga act gca agt agt gga cac tat ata gcc tac tgc cga 2153 Cys His His Gly Thr Ala Ser Ser Gly His Tyr Ile Ala Tyr Cys Arg         635 640 645    aac aat cta aat aat ctc tgg tat gaa ttt gat gat cag agt gtc act 2201 Asn Asn Leu Asn Asn Leu Trp Tyr Glu Phe Asp Asp Gln Ser Val Thr     650 655 660    gaa gtt tca gaa tct act gta caa aat gca gaa gct tac gtt ctt ttc 2249 Glu Val Ser Glu Ser Thr Val Gln Asn Ala Glu Ala Tyr Val Leu Phe 665 670 675 680    tat agg aag agc agc gaa gag gca caa aaa gag agg aga agg ata tca 2297 Tyr Arg Lys Ser Ser Glu Glu Ala Gln Lys Glu Arg Arg Arg Ile Ser                 685 690 695    aat tta ttg aac ata atg gaa cca agc ctc ctt cag ttt tat att tct 2345 Asn Leu Leu Asn Ile Met Glu Pro Ser Leu Leu Gln Phe Tyr Ile Ser             700 705 710    cga cag tgg ctt aat aaa ttt aag acc ttt gcc gaa cct ggc cct att 2393 Arg Gln Trp Leu Asn Lys Phe Lys Thr Phe Ala Glu Pro Gly Pro Ile         715 720 725    tca aat aat gac ttt ctt tgt att cat gga ggt gtt cct cca aga aaa 2441 Ser Asn Asn Asp Phe Leu Cys Ile His Gly Gly Val Pro Pro Arg Lys     730 735 740    gct ggt tat att gaa gac ctg gtt ttg atg ctg cct cag aac att tgg 2489 Ala Gly Tyr Ile Glu Asp Leu Val Leu Met Leu Pro Gln Asn Ile Trp 745 750 755 760    gat aac cta tat agc agg tat ggt gga gga cca gct gtc aac cat ctg 2537 Asp Asn Leu Tyr Ser Arg Tyr Gly Gly Gly Pro Ala Val Asn His Leu                 765 770 775    tac att tgt cat act tgc caa att gag gcg gag aaa att gaa aaa aga 2585 Tyr Ile Cys His Thr Cys Gln Ile Glu Ala Glu Lys Ile Glu Lys Arg             780 785 790    aga aaa act gaa ttg gaa att ttt att cgg ctt aac aga gcg ttc caa 2633 Arg Lys Thr Glu Leu Glu Ile Phe Ile Arg Leu Asn Arg Ala Phe Gln         795 800 805    aaa gag gac tct cca gct act ttt tat tgc atc agt atg cag tgg ttt 2681 Lys Glu Asp Ser Pro Ala Thr Phe Tyr Cys Ile Ser Met Gln Trp Phe     810 815 820    aga gaa tgg gaa agt ttt gtg aag ggt aaa gat gga gat cct cca ggt 2729 Arg Glu Trp Glu Ser Phe Val Lys Gly Lys Asp Gly Asp Pro Pro Gly 825 830 835 840    cct att gac aat act aag att gca gtc act aaa tgt ggt aat gtg atg 2777 Pro Ile Asp Asn Thr Lys Ile Ala Val Thr Lys Cys Gly Asn Val Met                 845 850 855    ctt agg caa gga gca gat tct ggc cag att tct gaa gaa aca tgg aat 2825 Leu Arg Gln Gly Ala Asp Ser Gly Gln Ile Ser Glu Glu Thr Trp Asn             860 865 870    ttt ctg cag tct att tat ggt gga ggg cct gaa gtt atc ctg cga cct 2873 Phe Leu Gln Ser Ile Tyr Gly Gly Gly Pro Glu Val Ile Leu Arg Pro         875 880 885    ccg gtt gtt cat gtt gat cca gat ata ctt caa gca gaa gaa aaa att 2921 Pro Val Val His Val Asp Pro Asp Ile Leu Gln Ala Glu Glu Lys Ile     890 895 900    gaa gta gaa act cgg tct ttg taa tttttaggat gtagagagtt ctaatgagga 2975 Glu Val Glu Thr Arg Ser Leu 905 910    atcattttca tgtgccctga catgtacaca tgcgaaaaca ttcctaaaag cgtgtttatt 3035    tgctttattt tttttcatca tttatcccat ttatttcttc ttagtgggca ttatggaaga 3095    atatattaaa atgtgtaata taccacaggt tggtatattt agttttaaat acttaccata 3155    aagtctttca gtgtaatttt tttttgagac agagtcttgc tttgtcaccc aggctggagt 3215    gctgtggtgt tacctcagct cactgcagcc tccacctcct gggttcaagc gattctcctg 3275    cctcagcctc tcgagtagct gggattacag gcacctgcca ccatgcccgg ctaatttttg 3335    tattttagta gagatggggt ttcaccatgt tggccaggct agtctcaaac tcctgacctc 3395    aggtgatcca cccacctcgg cctcccaaag tgctgggatt acaggtgtga gccacagcgc 3455    ctggcccagt gtaatatttt tgaaagagga gggacaattg tgaaatcagt aggttatctt 3515    taatctttac actacatgca gatccatagt atcctttgta gtgttgtaaa tacttttgct 3575    ttgaaaactt tttcattgtc ctaaatcacc ctgactctga ccagtctttc agttctccaa 3635    aagcccaatt taattgtata gttttgtcat ggcttcatat aataaagagc ctattttaag 3695    ttgaaagtag tagtcagaaa attgttaatt tcctaaagct caggaaacta gggtgtcact 3755    ttttttgcac tgcagcatat acactaacta gcttattaaa atttacaaaa tgtctttttg 3815    aatgtatcaa ggatatattt agtttgagtg gaatttgtca gcagatatca gtaacttatt 3875    gccgcttata ttgtacaatg ttaaacttca attcctgtaa cctggttagt attaatgtca 3935    gtgactaaaa aacttagagt tagttttagg gcacttttta ttttgagagc atgaagtgtg 3995    gaatgtgtca ctacgattgt tgataaagct gaggccactt gcaacttgat tttttaaatg 4055    aaatagataa agtctttttg aataatatag tatgcactgc tatttgcttg attatgtaat 4115    gtcaaaagtt taactatatt ccaagtacaa aaacatactg gattacattg aggatgttga 4175    atagcattca tgatggcttt gttttggttt ggggcagctg tcaccagcta aagcaatgtt 4235    gttaaaatta gctcaataaa aatgtcttta aaatgt 4271       <210> 3 <211> 355 <212> PRT <213> Homo sapiens    <400> 3 Met Gly Thr Asn Ala Ser Ala Leu Glu Lys Asp Ile Gly Pro Glu Gln   1 5 10 15 Phe Pro Ile Asn Glu His Tyr Phe Gly Leu Val Asn Phe Gly Asn Thr              20 25 30 Cys Tyr Cys Asn Ser Val Leu Gln Ala Leu Tyr Phe Cys Arg Pro Phe          35 40 45 Arg Glu Asn Val Leu Ala Tyr Lys Ala Gln Gln Lys Lys Lys Glu Asn      50 55 60 Leu Leu Thr Cys Leu Ala Asp Leu Phe His Ser Ile Ala Thr Gln Lys  65 70 75 80 Lys Lys Val Gly Val Ile Pro Pro Lys Lys Phe Ile Ser Arg Leu Arg                  85 90 95 Lys Glu Asn Asp Leu Phe Asp Asn Tyr Met Gln Gln Asp Ala His Glu             100 105 110 Phe Leu Asn Tyr Leu Leu Asn Thr Ile Ala Asp Ile Leu Gln Glu Glu         115 120 125 Lys Lys Gln Glu Lys Gln Asn Gly Lys Leu Lys Asn Gly Asn Met Asn     130 135 140 Glu Pro Ala Glu Asn Asn Lys Pro Glu Leu Thr Trp Val His Glu Ile 145 150 155 160 Phe Gln Gly Thr Leu Thr Asn Glu Thr Arg Cys Leu Asn Cys Glu Thr                 165 170 175 Val Ser Ser Lys Asp Glu Asp Phe Leu Asp Leu Ser Val Asp Val Glu             180 185 190 Gln Asn Thr Ser Ile Thr His Cys Leu Arg Asp Phe Ser Asn Thr Glu         195 200 205 Thr Leu Cys Ser Glu Gln Lys Tyr Tyr Cys Glu Thr Cys Cys Ser Lys     210 215 220 Gln Glu Ala Gln Lys Arg Met Arg Val Lys Lys Leu Pro Met Ile Leu 225 230 235 240 Ala Leu His Leu Lys Arg Phe Lys Tyr Met Glu Gln Leu His Arg Tyr                 245 250 255 Thr Lys Leu Ser Tyr Arg Val Val Phe Pro Leu Glu Leu Arg Leu Phe             260 265 270 Asn Thr Ser Ser Asp Ala Val Asn Leu Asp Arg Met Tyr Asp Leu Val         275 280 285 Ala Val Val Val His Cys Gly Ser Gly Pro Asn Arg Gly His Tyr Ile     290 295 300 Thr Ile Val Lys Ser His Gly Phe Trp Leu Leu Phe Asp Asp Asp Ile 305 310 315 320 Val Glu Lys Ile Asp Ala Gln Ala Ile Glu Glu Phe Tyr Gly Leu Thr                 325 330 335 Ser Asp Ile Ser Lys Asn Ser Glu Ser Gly Tyr Ile Leu Phe Tyr Gln             340 345 350 Ser Arg Glu         355       <210> 4 <211> 1068 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (1) .. (1068)    <400> 4 atg ggc acc aat gcc tct gct ctg gaa aaa gac att ggt cca gag cag 48 Met Gly Thr Asn Ala Ser Ala Leu Glu Lys Asp Ile Gly Pro Glu Gln   1 5 10 15    ttt cca atc aat gaa cac tat ttc gga ttg gtc aat ttt gga aac aca 96 Phe Pro Ile Asn Glu His Tyr Phe Gly Leu Val Asn Phe Gly Asn Thr              20 25 30    tgc tac tgt aac tcc gtg ctt cag gca ttg tac ttc tgc cgt cca ttc 144 Cys Tyr Cys Asn Ser Val Leu Gln Ala Leu Tyr Phe Cys Arg Pro Phe          35 40 45    cgg gag aat gtg ttg gca tac aag gcc cag caa aag aag aag gaa aac 192 Arg Glu Asn Val Leu Ala Tyr Lys Ala Gln Gln Lys Lys Lys Glu Asn      50 55 60    ttg ctg acg tgc ctg gcg gac ctt ttc cac agc att gcc aca cag aag 240 Leu Leu Thr Cys Leu Ala Asp Leu Phe His Ser Ile Ala Thr Gln Lys  65 70 75 80    aag aag gtt ggc gtc atc cca cca aag aag ttc att tca agg ctg aga 288 Lys Lys Val Gly Val Ile Pro Pro Lys Lys Phe Ile Ser Arg Leu Arg                  85 90 95    aaa gag aat gat ctc ttt gat aac tac atg cag cag gat gct cat gaa 336 Lys Glu Asn Asp Leu Phe Asp Asn Tyr Met Gln Gln Asp Ala His Glu             100 105 110    ttt tta aat tat ttg cta aac act att gcg gac atc ctt cag gag gag 384 Phe Leu Asn Tyr Leu Leu Asn Thr Ile Ala Asp Ile Leu Gln Glu Glu         115 120 125    aag aaa cag gaa aaa caa aat gga aaa tta aaa aat ggc aac atg aac 432 Lys Lys Gln Glu Lys Gln Asn Gly Lys Leu Lys Asn Gly Asn Met Asn     130 135 140    gaa cct gcg gaa aat aat aaa cca gaa ctc acc tgg gtc cat gag att 480 Glu Pro Ala Glu Asn Asn Lys Pro Glu Leu Thr Trp Val His Glu Ile 145 150 155 160    ttt cag gga acg ctt acc aat gaa act cga tgc ttg aac tgt gaa act 528 Phe Gln Gly Thr Leu Thr Asn Glu Thr Arg Cys Leu Asn Cys Glu Thr                 165 170 175    gtt agt agc aaa gat gaa gat ttt ctt gac ctt tct gtt gat gtg gag 576 Val Ser Ser Lys Asp Glu Asp Phe Leu Asp Leu Ser Val Asp Val Glu             180 185 190    cag aat aca tcc att acc cac tgt cta aga gac ttc agc aac aca gaa 624 Gln Asn Thr Ser Ile Thr His Cys Leu Arg Asp Phe Ser Asn Thr Glu         195 200 205    aca ctg tgt agt gaa caa aaa tat tat tgt gaa aca tgc tgc agc aaa 672 Thr Leu Cys Ser Glu Gln Lys Tyr Tyr Cys Glu Thr Cys Cys Ser Lys     210 215 220    caa gaa gcc cag aaa agg atg agg gta aaa aag ctg ccc atg atc ttg 720 Gln Glu Ala Gln Lys Arg Met Arg Val Lys Lys Leu Pro Met Ile Leu 225 230 235 240    gcc ctg cac cta aag cgg ttc aag tac atg gag cag ctg cac aga tac 768 Ala Leu His Leu Lys Arg Phe Lys Tyr Met Glu Gln Leu His Arg Tyr                 245 250 255    acc aag ctg tct tac cgt gtg gtc ttc cct ctg gaa ctc cgg ctc ttc 816 Thr Lys Leu Ser Tyr Arg Val Val Phe Pro Leu Glu Leu Arg Leu Phe             260 265 270    aac acc tcc agt gat gca gtg aac ctg gac cgc atg tat gac ttg gtt 864 Asn Thr Ser Ser Asp Ala Val Asn Leu Asp Arg Met Tyr Asp Leu Val         275 280 285    gcg gtg gtc gtt cac tgt ggc agt ggt cct aat cgt ggg cat tat atc 912 Ala Val Val Val His Cys Gly Ser Gly Pro Asn Arg Gly His Tyr Ile     290 295 300    act att gtg aaa agt cac ggc ttc tgg ctt ttg ttt gat gat gac att 960 Thr Ile Val Lys Ser His Gly Phe Trp Leu Leu Phe Asp Asp Asp Ile 305 310 315 320    gta gag aaa ata gat gct caa gct att gaa gaa ttc tat ggc ctg acg 1008 Val Glu Lys Ile Asp Ala Gln Ala Ile Glu Glu Phe Tyr Gly Leu Thr                 325 330 335    tca gat ata tca aaa aat tca gaa tct gga tat att tta ttc tat cag 1056 Ser Asp Ile Ser Lys Asn Ser Glu Ser Gly Tyr Ile Leu Phe Tyr Gln             340 345 350    tca aga gag taa 1068 Ser Arg Glu         355       <210> 5 <211> 913 <212> PRT <213> Homo sapiens    <400> 5 Met Gly Asp Ser Arg Asp Leu Cys Pro His Leu Asp Ser Ile Gly Glu   1 5 10 15 Val Thr Lys Glu Asp Leu Leu Leu Lys Ser Lys Gly Thr Cys Gln Ser              20 25 30 Cys Gly Val Thr Gly Pro Asn Leu Trp Ala Cys Leu Gln Val Ala Cys          35 40 45 Pro Tyr Val Gly Cys Gly Glu Ser Phe Ala Asp His Ser Thr Ile His      50 55 60 Ala Gln Ala Lys Lys His Asn Leu Thr Val Asn Leu Thr Thr Phe Arg  65 70 75 80 Leu Trp Cys Tyr Ala Cys Glu Lys Glu Val Phe Leu Glu Gln Arg Leu                  85 90 95 Ala Ala Pro Leu Leu Gly Ser Ser Ser Lys Phe Ser Glu Gln Asp Ser             100 105 110 Pro Pro Pro Ser His Pro Leu Lys Ala Val Pro Ile Ala Val Ala Asp         115 120 125 Glu Gly Glu Ser Glu Ser Glu Asp Asp Asp Leu Lys Pro Arg Gly Leu     130 135 140 Thr Gly Met Lys Asn Leu Gly Asn Ser Cys Tyr Met Asn Ala Ala Leu 145 150 155 160 Gln Ala Leu Ser Asn Cys Pro Pro Leu Thr Gln Phe Phe Leu Glu Cys                 165 170 175 Gly Gly Leu Val Arg Thr Asp Lys Lys Pro Ala Leu Cys Lys Ser Tyr             180 185 190 Gln Lys Leu Val Ser Glu Val Trp His Lys Lys Arg Pro Ser Tyr Val         195 200 205 Val Pro Thr Ser Leu Ser His Gly Ile Lys Leu Val Asn Pro Met Phe     210 215 220 Arg Gly Tyr Ala Gln Gln Asp Thr Gln Glu Phe Leu Arg Cys Leu Met 225 230 235 240 Asp Gln Leu His Glu Glu Leu Lys Glu Pro Val Val Ala Thr Val Ala                 245 250 255 Leu Thr Glu Ala Arg Asp Ser Asp Ser Ser Asp Thr Asp Glu Lys Arg             260 265 270 Glu Gly Asp Arg Ser Pro Ser Glu Asp Glu Phe Leu Ser Cys Asp Ser         275 280 285 Ser Ser Asp Arg Gly Glu Gly Asp Gly Gln Gly Arg Gly Gly Gly Ser     290 295 300 Ser Gln Ala Glu Thr Glu Leu Leu Ile Pro Asp Glu Ala Gly Arg Val 305 310 315 320 Ile Ser Glu Lys Glu Arg Met Lys Asp Arg Lys Phe Ser Trp Gly Gln                 325 330 335 Gln Arg Thr Asn Ser Glu Gln Val Asp Glu Asp Ala Asp Val Asp Thr             340 345 350 Ala Met Ala Ala Leu Asp Gln Pro Ala Glu Ala Gln Pro Pro Ser Pro         355 360 365 Arg Ser Ser Ser Pro Cys Arg Thr Pro Glu Pro Asp Asn Asp Ala His     370 375 380 Leu Arg Ser Ser Ser Arg Pro Cys Ser Pro Val His His His Glu Gly 385 390 395 400 His Ala Lys Leu Ser Ser Ser Pro Pro Arg Ala Ser Pro Val Arg Met                 405 410 415 Ala Pro Ser Tyr Val Leu Lys Lys Ala Gln Val Leu Ser Ala Gly Ser             420 425 430 Arg Arg Arg Lys Glu Gln Arg Tyr Arg Ser Val Ile Ser Asp Ile Phe         435 440 445 Asp Gly Ser Ile Leu Ser Leu Val Gln Cys Leu Thr Cys Asp Arg Val     450 455 460 Ser Thr Thr Val Glu Thr Phe Gln Asp Leu Ser Leu Pro Ile Pro Gly 465 470 475 480 Lys Glu Asp Leu Ala Lys Leu His Ser Ala Ile Tyr Gln Asn Val Pro                 485 490 495 Ala Lys Pro Gly Ala Cys Gly Asp Ser Tyr Ala Ala Gln Gly Trp Leu             500 505 510 Ala Phe Ile Val Glu Tyr Ile Arg Arg Phe Val Val Ser Cys Thr Pro         515 520 525 Ser Trp Phe Trp Gly Pro Val Val Thr Leu Glu Asp Cys Leu Ala Ala     530 535 540 Phe Phe Ala Ala Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys Glu 545 550 555 560 Arg Cys Lys Lys Leu Arg Asn Gly Val Lys Tyr Cys Lys Val Leu Arg                 565 570 575 Leu Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Val             580 585 590 Met Tyr Ser Phe Lys Ile Asn Ser His Val Ser Phe Pro Leu Glu Gly         595 600 605 Leu Asp Leu Arg Pro Phe Leu Ala Lys Glu Cys Thr Ser Gln Ile Thr     610 615 620 Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Gly Ser 625 630 635 640 Gly His Tyr Ile Ala Tyr Cys Gln Asn Val Ile Asn Gly Gln Trp Tyr                 645 650 655 Glu Phe Asp Asp Gln Tyr Val Thr Glu Val His Glu Thr Val Val Gln             660 665 670 Asn Ala Glu Gly Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala         675 680 685 Met Arg Glu Arg Gln Gln Val Val Ser Leu Ala Ala Met Arg Glu Pro     690 695 700 Ser Leu Leu Arg Phe Tyr Val Ser Arg Glu Trp Leu Asn Lys Phe Asn 705 710 715 720 Thr Phe Ala Glu Pro Gly Pro Ile Thr Asn Gln Thr Phe Leu Cys Ser                 725 730 735 His Gly Gly Ile Pro Pro His Lys Tyr His Tyr Ile Asp Asp Leu Val             740 745 750 Val Ile Leu Pro Gln Asn Val Trp Glu His Leu Tyr Asn Arg Phe Gly         755 760 765 Gly Gly Pro Ala Val Asn His Leu Tyr Val Cys Ser Ile Cys Gln Val     770 775 780 Glu Ile Glu Ala Leu Ala Lys Arg Arg Arg Ile Glu Ile Asp Thr Phe 785 790 795 800 Ile Lys Leu Asn Lys Ala Phe Gln Ala Glu Glu Ser Pro Gly Val Ile                 805 810 815 Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ala Phe Val Lys             820 825 830 Gly Lys Asp Asn Glu Pro Pro Gly Pro Ile Asp Asn Ser Arg Ile Ala         835 840 845 Gln Val Lys Gly Ser Gly His Val Gln Leu Lys Gln Gly Ala Asp Tyr     850 855 860 Gly Gln Ile Ser Glu Glu Thr Trp Thr Tyr Leu Asn Ser Leu Tyr Gly 865 870 875 880 Gly Gly Pro Glu Ile Ala Ile Arg Gln Ser Val Ala Gln Pro Leu Gly                 885 890 895 Pro Glu Asn Leu His Gly Glu Gln Lys Ile Glu Ala Glu Thr Arg Ala             900 905 910 Val    <210> 6 <211> 4252 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (133) .. (2874)    <400> 6 gcaggctcct tgccagaggc ctccactcac tccagacccc tatagcccgt cgctgtcagc 60    tgtcaacaaa ggatgcgaat gctggccgct tcctgtgggc ttcgtgtcac ccagaggtga 120    gcccaggcca gg atg ggg gac tcc agg gac ctt tgc cct cac ctt gac tcc 171               Met Gly Asp Ser Arg Asp Leu Cys Pro His Leu Asp Ser                 1 5 10    ata gga gag gtg acc aaa gag gac ttg ctg ctc aaa tct aag gga acc 219 Ile Gly Glu Val Thr Lys Glu Asp Leu Leu Leu Lys Ser Lys Gly Thr      15 20 25    tgt cag tcg tgt ggg gtc acc gga cca aac cta tgg gcc tgt ctg cag 267 Cys Gln Ser Cys Gly Val Thr Gly Pro Asn Leu Trp Ala Cys Leu Gln  30 35 40 45    gtt gcc tgc ccc tat gtt ggc tgc gga gaa tcc ttc gct gac cac agc 315 Val Ala Cys Pro Tyr Val Gly Cys Gly Glu Ser Phe Ala Asp His Ser                  50 55 60    acc att cat gca cag gca aaa aag cac aac ttg acc gtg aac ctg acc 363 Thr Ile His Ala Gln Ala Lys Lys His Asn Leu Thr Val Asn Leu Thr              65 70 75    acg ttc cga ctg tgg tgt tac gcc tgt gag aag gag gta ttc ctg gag 411 Thr Phe Arg Leu Trp Cys Tyr Ala Cys Glu Lys Glu Val Phe Leu Glu          80 85 90    cag cgg ctg gca gcc cct ctg ctg ggc tcc tct tcc aag ttc tct gaa 459 Gln Arg Leu Ala Ala Pro Leu Leu Gly Ser Ser Ser Lys Phe Ser Glu      95 100 105    cag gac tcc ccg cca ccc tcc cac cct ctg aaa gct gtt cct att gct 507 Gln Asp Ser Pro Pro Pro Ser His Pro Leu Lys Ala Val Pro Ile Ala 110 115 120 125    gtg gct gat gaa gga gag tct gag tca gag gat gat gac ctg aaa cct 555 Val Ala Asp Glu Gly Glu Ser Glu Ser Glu Asp Asp Asp Leu Lys Pro                 130 135 140    cga ggc ctc acg ggc atg aag aac ctc ggg aac tcc tgc tac atg aac 603 Arg Gly Leu Thr Gly Met Lys Asn Leu Gly Asn Ser Cys Tyr Met Asn             145 150 155    gcc gcc ctg cag gcc ctg tcc aat tgc ccg ccg ctg act cag ttc ttc 651 Ala Ala Leu Gln Ala Leu Ser Asn Cys Pro Pro Leu Thr Gln Phe Phe         160 165 170    ttg gag tgt ggc ggc ctg gtg cgc aca gat aag aag cca gcc ctg tgc 699 Leu Glu Cys Gly Gly Leu Val Arg Thr Asp Lys Lys Pro Ala Leu Cys     175 180 185    aag agc tac cag aag ctg gtc tct gag gtc tgg cat aag aaa cgg cca 747 Lys Ser Tyr Gln Lys Leu Val Ser Glu Val Trp His Lys Lys Arg Pro 190 195 200 205    agc tac gtg gtc ccc acc agt ctg tct cat ggg atc aag ttg gtc aac 795 Ser Tyr Val Val Pro Thr Ser Leu Ser His Gly Ile Lys Leu Val Asn                 210 215 220    cca atg ttc cga ggc tat gcc cag cag gac acc caa gag ttc ctt cgc 843 Pro Met Phe Arg Gly Tyr Ala Gln Gln Asp Thr Gln Glu Phe Leu Arg             225 230 235    tgc ctg atg gac cag ctg cac gag gag ctc aag gag ccg gtg gtg gcc 891 Cys Leu Met Asp Gln Leu His Glu Glu Leu Lys Glu Pro Val Val Ala         240 245 250    acg gtg gcg ctg acg gag gct cgg gac tca gat tcg agt gac acg gat 939 Thr Val Ala Leu Thr Glu Ala Arg Asp Ser Asp Ser Ser Asp Thr Asp     255 260 265    gag aaa cgg gag ggt gac cgg agc cca tca gaa gat gag ttc ttg tcc 987 Glu Lys Arg Glu Gly Asp Arg Ser Pro Ser Glu Asp Glu Phe Leu Ser 270 275 280 285    tgt gac tcg agc agt gac cgg ggt gag ggt gac ggg cag ggg cgt ggc 1035 Cys Asp Ser Ser Ser Asp Arg Gly Glu Gly Asp Gly Gln Gly Arg Gly                 290 295 300    ggg ggc agc tcg cag gcc gag acg gag ctg ctg atc cca gat gag gcg 1083 Gly Gly Ser Ser Gln Ala Glu Thr Glu Leu Leu Ile Pro Asp Glu Ala             305 310 315    ggc cga gtc atc tct gag aag gag cgg atg aag gac cgc aag ttc tcc 1131 Gly Arg Val Ile Ser Glu Lys Glu Arg Met Lys Asp Arg Lys Phe Ser         320 325 330    tgg ggc cag cag cgt aca aac tcg gag caa gtg gac gag gac gct gat 1179 Trp Gly Gln Gln Arg Thr Asn Ser Glu Gln Val Asp Glu Asp Ala Asp     335 340 345    gtg gac act gcc atg gct gcc ctt gac cag ccc gcg gag gcc cag ccc 1227 Val Asp Thr Ala Met Ala Ala Leu Asp Gln Pro Ala Glu Ala Gln Pro 350 355 360 365    ccg tca cca cgg tcc tcc agc ccc tgc cgg acg cca gag ccg gac aat 1275 Pro Ser Pro Arg Ser Ser Ser Pro Cys Arg Thr Pro Glu Pro Asp Asn                 370 375 380    gat gct cac cta cgc agc tcc tct cgc ccc tgc agc ccc gtc cac cac 1323 Asp Ala His Leu Arg Ser Ser Ser Arg Pro Cys Ser Pro Val His His             385 390 395    cac gag ggc cat gcc aag ctg tct agc agc ccc cct cgt gca agc ccc 1371 His Glu Gly His Ala Lys Leu Ser Ser Ser Pro Pro Arg Ala Ser Pro         400 405 410    gtg agg atg gca ccg tcg tac gtg ctc aag aaa gcc cag gta ttg agt 1419 Val Arg Met Ala Pro Ser Tyr Val Leu Lys Lys Ala Gln Val Leu Ser     415 420 425    gct ggc agc cgg agg cgg aag gag cag cgc tac cgc agc gtc atc tca 1467 Ala Gly Ser Arg Arg Arg Lys Glu Gln Arg Tyr Arg Ser Val Ile Ser 430 435 440 445    gac atc ttt gac ggc tcc att ctc agc ctc gtg cag tgt ctc acc tgt 1515 Asp Ile Phe Asp Gly Ser Ile Leu Ser Leu Val Gln Cys Leu Thr Cys                 450 455 460    gac cgg gta tcc acc aca gtg gaa acg ttc cag gac tta tca ctg ccc 1563 Asp Arg Val Ser Thr Thr Val Glu Thr Phe Gln Asp Leu Ser Leu Pro             465 470 475    att cct gga aag gag gac ctg gcc aag ctc cat tca gcc atc tac cag 1611 Ile Pro Gly Lys Glu Asp Leu Ala Lys Leu His Ser Ala Ile Tyr Gln         480 485 490    aat gtg ccg gcc aag cca ggc gcc tgt ggg gac agc tat gcc gcc cag 1659 Asn Val Pro Ala Lys Pro Gly Ala Cys Gly Asp Ser Tyr Ala Ala Gln     495 500 505    ggc tgg ctg gcc ttc att gtg gag tac atc cga cgg ttt gtg gta tcc 1707 Gly Trp Leu Ala Phe Ile Val Glu Tyr Ile Arg Arg Phe Val Val Ser 510 515 520 525    tgt acc ccc agc tgg ttt tgg ggg cct gtc gtc acc ctg gaa gac tgc 1755 Cys Thr Pro Ser Trp Phe Trp Gly Pro Val Val Thr Leu Glu Asp Cys                 530 535 540    ctt gct gcc ttc ttt gcc gct gat gag tta aag ggt gac aac atg tac 1803 Leu Ala Ala Phe Phe Ala Ala Asp Glu Leu Lys Gly Asp Asn Met Tyr             545 550 555    agc tgt gag cgg tgt aag aag ctg cgg aac gga gtg aag tac tgc aaa 1851 Ser Cys Glu Arg Cys Lys Lys Leu Arg Asn Gly Val Lys Tyr Cys Lys         560 565 570    gtc ctg cgg ttg ccc gag atc ctg tgc att cac cta aag cgc ttt cgg 1899 Val Leu Arg Leu Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg     575 580 585    cac gag gtg atg tac tca ttc aag atc aac agc cac gtc tcc ttc ccc 1947 His Glu Val Met Tyr Ser Phe Lys Ile Asn Ser His Val Ser Phe Pro 590 595 600 605    ctc gag ggg ctc gac ctg cgc ccc ttc ctt gcc aag gag tgc aca tcc 1995 Leu Glu Gly Leu Asp Leu Arg Pro Phe Leu Ala Lys Glu Cys Thr Ser                 610 615 620    cag atc acc acc tac gac ctc ctc tcg gtc atc tgc cac cac ggc acg 2043 Gln Ile Thr Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr             625 630 635    gca ggc agt ggg cac tac atc gcc tac tgc cag aac gtg att aat ggg 2091 Ala Gly Ser Gly His Tyr Ile Ala Tyr Cys Gln Asn Val Ile Asn Gly         640 645 650    cag tgg tac gag ttt gat gac cag tac gtc aca gaa gtc cac gag acg 2139 Gln Trp Tyr Glu Phe Asp Asp Gln Tyr Val Thr Glu Val His Glu Thr     655 660 665    gtg gtg cag aac gcc gag ggc tac gta ctc ttc tac agg aag agc agc 2187 Val Val Gln Asn Ala Glu Gly Tyr Val Leu Phe Tyr Arg Lys Ser Ser 670 675 680 685    gag gag gcc atg cgg gag cga cag cag gtg gtg tcc ctg gcc gcc atg 2235 Glu Glu Ala Met Arg Glu Arg Gln Gln Val Val Ser Leu Ala Ala Met                 690 695 700    cgg gag ccc agc ctg ctg cgg ttc tac gtg tcc cgc gag tgg ctc aac 2283 Arg Glu Pro Ser Leu Leu Arg Phe Tyr Val Ser Arg Glu Trp Leu Asn             705 710 715    aag ttc aac acc ttc gca gag cca ggc ccc atc acc aac cag acc ttc 2331 Lys Phe Asn Thr Phe Ala Glu Pro Gly Pro Ile Thr Asn Gln Thr Phe         720 725 730    ctc tgc tcc cac gga ggc atc ccg ccc cac aaa tac cac tac atc gac 2379 Leu Cys Ser His Gly Gly Ile Pro Pro His Lys Tyr His Tyr Ile Asp     735 740 745    gac ctg gtg gtc atc ctg ccc cag aac gtc tgg gag cac ctg tac aac 2427 Asp Leu Val Val Ile Leu Pro Gln Asn Val Trp Glu His Leu Tyr Asn 750 755 760 765    aga ttc ggg ggt ggc ccc gcc gtg aac cac ctg tac gtg tgc tcc atc 2475 Arg Phe Gly Gly Gly Pro Ala Val Asn His Leu Tyr Val Cys Ser Ile                 770 775 780    tgc cag gtg gag atc gag gca ctg gcc aag cgc agg agg atc gag atc 2523 Cys Gln Val Glu Ile Glu Ala Leu Ala Lys Arg Arg Arg Ile Glu Ile             785 790 795    gac acc ttc atc aag ttg aac aag gcc ttc cag gcc gag gag tcg ccg 2571 Asp Thr Phe Ile Lys Leu Asn Lys Ala Phe Gln Ala Glu Glu Ser Pro         800 805 810    ggc gtc atc tac tgc atc agc atg cag tgg ttc cgg gag tgg gag gcg 2619 Gly Val Ile Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ala     815 820 825    ttc gtc aag ggg aag gac aac gag ccc ccc ggg ccc att gac aac agc 2667 Phe Val Lys Gly Lys Asp Asn Glu Pro Pro Gly Pro Ile Asp Asn Ser 830 835 840 845    agg att gca cag gtc aaa gga agc ggc cat gtc cag ctg aag cag gga 2715 Arg Ile Ala Gln Val Lys Gly Ser Gly His Val Gln Leu Lys Gln Gly                 850 855 860    gct gac tac ggg cag att tcg gag gag acc tgg acc tac ctg aac agc 2763 Ala Asp Tyr Gly Gln Ile Ser Glu Glu Thr Trp Thr Tyr Leu Asn Ser             865 870 875    ctg tat gga ggt ggc ccc gag att gcc atc cgc cag agt gtg gcg cag 2811 Leu Tyr Gly Gly Gly Pro Glu Ile Ala Ile Arg Gln Ser Val Ala Gln         880 885 890    ccg ctg ggc cca gag aac ctg cac ggg gag cag aag atc gaa gcc gag 2859 Pro Leu Gly Pro Glu Asn Leu His Gly Glu Gln Lys Ile Glu Ala Glu     895 900 905    acg cgg gcc gtg tga tctgctgggc tagtctcccc atgtgcccca ccccgcggaa 2914 Thr Arg Ala Val 910    ggcgtgtttg tgcccagaag agaggccggg ctgctgcaga accccgccgt gtaaagaggc 2974    agaaaagttg gtttggtttg cagtaacgct gcaactagaa aatatatgca cttcaggctt 3034    gttgaaacga ccaagactct gtgacgttaa tttgggtctt tgtcctggca gtgcctctgc 3094    cagtcactgt catcgttgtg tcccccacaa ctgtcctctt gctagctcgg cccagctttg 3154    tccctggagc ccgatgctac ccctgtcaga cagaggctgc ggcctgggcc agagtcaggg 3214    agtagctgct gcttcacggc gtctccactg tgcgattggc ccggagcccc gaagactcgg 3274    agggagctgc tcagggccgg tgagcgcagc cagaagccct ggccagtgag gagctcacag 3334    gtcctccctg gtggtcccgc cgcacctctg catctcctgg gcgtcaccag gaaggctctg 3394    aagtcccggg ctgctctcag cacttctcct gcagactgaa gactctggac tcattgctga 3454    ttggaacacc aggaggaggt tggatttctg ccagtggggg atgtttctgg aggcagctgg 3514    tcccccacac cgcgtcctgc tgagcctgcc ccctggattg gctgtaattt gcctcgaagt 3574    tcagcagttc atcttcatgg gaaatttgct gagcccccac cagggaaccg gatgatgaaa 3634    cagggatacc tcacagcttg gccatttgag gcaaaggcag cttcccgagc tgatgctaaa 3694    gaagacagac tttcccttcc tcccagcagc agcagtgcag agcccgcctg gagggatgtg 3754    ggggctgtgc agggtgcagc gctcaggtgg atcctgggaa gcagcctctg gatgctgagt 3814    ggagggagcc actgagcaca gcaaggcacc aaagcccctg gagaaaccgc cagggcgagg 3874    tgcgaccatc atcaggatca aagcagacgg ggcgtgggtg gggaaggggc tctgggacca 3934    gaccccccac actactgcgt ctttgtttct atcagtcttt gtagaagcag gtggtggtgg 3994    aaattccagc aggtgggtcc cgcagaggcc ctgaggcctc acttttcgga tcttctgtcc 4054    cagatcctgc tccctccctg ctgagcctgg ggttcccctg gcattggccc cagccttctg 4114    aaagccggcg ctgcagccag aggccgcacg ctgcactgtc gcgacgcaga gaggcttctg 4174    tgcaggctgg gatcgggccc catgtctgtg ctgtctagtt tgtgttcaaa atgtcagaat 4234    aaacacagaa taaatgtt 4252       <210> 7 <211> 749 <212> PRT <213> Homo sapiens    <400> 7 Met Ile Asp Trp Val Ser Trp Pro Leu Gly Lys Asn Ile Asp Lys Trp   1 5 10 15 Ile Ile Ala Leu Leu Lys Gly Leu Ala Ala Val Lys Lys Phe Ser Ile              20 25 30 Leu Ile Glu Val Ser Leu Thr Lys Ile Glu Lys Val Phe Ser Lys Leu          35 40 45 Leu Tyr Pro Ile Val Arg Gly Ala Ala Leu Ser Val Leu Lys Tyr Met      50 55 60 Leu Leu Thr Phe Gln His Ser His Glu Ala Phe His Leu Leu Leu Pro  65 70 75 80 His Ile Pro Pro Met Val Ala Ser Leu Val Lys Glu Asp Ser Asn Ser                  85 90 95 Gly Thr Ser Cys Leu Glu Gln Leu Ala Glu Leu Val His Cys Met Val             100 105 110 Phe Arg Phe Pro Gly Phe Pro Asp Leu Tyr Glu Pro Val Met Glu Ala         115 120 125 Ile Lys Asp Leu His Val Pro Asn Glu Asp Arg Ile Lys Gln Leu Leu     130 135 140 Gly Gln Asp Ala Trp Thr Ser Gln Lys Ser Glu Leu Ala Gly Phe Tyr 145 150 155 160 Pro Arg Leu Met Ala Lys Ser Asp Thr Gly Lys Ile Gly Leu Ile Asn                 165 170 175 Leu Gly Asn Thr Cys Tyr Val Asn Ser Ile Leu Gln Ala Leu Phe Met             180 185 190 Ala Ser Asp Phe Arg His Cys Val Leu Arg Leu Thr Glu Asn Asn Ser         195 200 205 Gln Pro Leu Met Thr Lys Leu Gln Trp Leu Phe Gly Phe Leu Glu His     210 215 220 Ser Gln Arg Pro Ala Ile Ser Pro Glu Asn Phe Leu Ser Ala Ser Trp 225 230 235 240 Thr Pro Trp Phe Ser Pro Gly Thr Gln Gln Asp Cys Ser Glu Tyr Leu                 245 250 255 Lys Tyr Leu Leu Asp Arg Leu His Glu Glu Glu Lys Thr Gly Thr Arg             260 265 270 Ile Cys Gln Lys Leu Lys Gln Ser Ser Ser Pro Ser Pro Pro Glu Glu         275 280 285 Pro Pro Ala Pro Ser Ser Thr Ser Val Glu Lys Met Phe Gly Gly Lys     290 295 300 Ile Val Thr Arg Ile Cys Cys Leu Cys Cys Leu Asn Val Ser Ser Arg 305 310 315 320 Glu Glu Ala Phe Thr Asp Leu Ser Leu Ala Phe Pro Pro Pro Glu Arg                 325 330 335 Cys Arg Arg Arg Arg Leu Gly Ser Val Met Arg Pro Thr Glu Asp Ile             340 345 350 Thr Ala Arg Glu Leu Pro Pro Pro Thr Ser Ala Gln Gly Pro Gly Arg         355 360 365 Val Gly Pro Arg Arg Gln Arg Lys His Cys Ile Thr Glu Asp Thr Pro     370 375 380 Pro Thr Ser Leu Tyr Ile Glu Gly Leu Asp Ser Lys Glu Ala Gly Gly 385 390 395 400 Gln Ser Ser Gln Glu Glu Arg Ile Glu Arg Glu Glu Glu Gly Lys Glu                 405 410 415 Glu Arg Thr Glu Lys Glu Glu Val Gly Glu Glu Glu Glu Ser Thr Arg             420 425 430 Gly Glu Gly Glu Arg Glu Lys Glu Glu Glu Val Glu Glu Glu Glu Glu         435 440 445 Lys Val Glu Lys Glu Thr Glu Lys Glu Ala Glu Gln Glu Lys Glu Glu     450 455 460 Asp Ser Leu Gly Ala Gly Thr His Pro Asp Ala Ala Ile Pro Ser Gly 465 470 475 480 Glu Arg Thr Cys Gly Ser Glu Gly Ser Arg Ser Val Leu Asp Leu Val                 485 490 495 Asn Tyr Phe Leu Ser Pro Glu Lys Leu Thr Ala Glu Asn Arg Tyr Tyr             500 505 510 Cys Glu Ser Cys Ala Ser Leu Gln Asp Ala Glu Lys Val Val Glu Leu         515 520 525 Ser Gln Gly Pro Cys Tyr Leu Ile Leu Thr Leu Leu Arg Phe Ser Phe     530 535 540 Asp Leu Arg Thr Met Arg Arg Arg Lys Ile Leu Asp Asp Val Ser Ile 545 550 555 560 Pro Leu Leu Leu Arg Leu Pro Leu Ala Gly Gly Arg Gly Gln Ala Tyr                 565 570 575 Asp Leu Cys Ser Val Val Val His Ser Gly Val Ser Ser Glu Ser Gly             580 585 590 His Tyr Tyr Cys Tyr Ala Arg Glu Gly Ala Ala Arg Pro Ala Ala Ser         595 600 605 Leu Gly Thr Ala Asp Arg Pro Glu Pro Glu Asn Gln Trp Tyr Leu Phe     610 615 620 Asn Asp Thr Arg Val Ser Phe Ser Ser Phe Glu Ser Val Ser Asn Val 625 630 635 640 Thr Ser Phe Phe Pro Lys Asp Thr Ala Tyr Val Leu Phe Tyr Arg Gln                 645 650 655 Arg Pro Arg Glu Gly Pro Glu Ala Glu Leu Gly Ser Ser Arg Val Arg             660 665 670 Thr Glu Pro Thr Leu His Lys Asp Leu Met Glu Ala Ile Ser Lys Asp         675 680 685 Asn Ile Leu Tyr Leu Gln Glu Gln Glu Lys Glu Ala Arg Ser Arg Ala     690 695 700 Ala Tyr Ile Ser Ala Leu Pro Thr Ser Pro His Trp Gly Arg Gly Phe 705 710 715 720 Asp Glu Asp Lys Asp Glu Asp Glu Gly Ser Pro Gly Gly Cys Asn Pro                 725 730 735 Ala Gly Gly Asn Gly Gly Asp Phe His Arg Leu Val Phe             740 745    <210> 8 <211> 3771 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (627) .. (2876)    <400> 8 cagtctgctg agtctaggct gttttcctca gcactgactg catggcagct gtgtgctcag 60    cccgcacagg caggctgtgt tgtggggtcc agaggggctg atacatgtaa agttgatgga 120    atatcctgct caatggtgaa tacgacctaa gtgttagctg ctgttatctc ctttaatcca 180    tacaagaacc ctctgaggga ggtattatcc ccattttaca gaggaagaaa ctgaggccca 240    gagcggggaa gtggcttgct ctgggtcaca cagcaaggac ttgctggagc caaagcttct 300    ggcccagggg cagtgccctt ccaggcagaa gcagctgaaa gccttgttct ggcattgcag 360    cccagtgtgg aggggcgaga cttggggtgc tgagagggcc tggctccctt cttggtgggc 420    tgggagcctc agtgtctggc aggtggctgt gctgggcttg tgcatttccc agccatgtct 480    ggttcagccc acagaccccg gggctgccct ttgcaggtgt gagtcaatgt gggaacccag 540    gacattacgg atagcccctg ggctgccctc accccagcat tttgggtgtc ccttgtccct 600    gtgtcactgt cactcccctc accagg atg att gac tgg gtg tcc tgg ccc ctg 653                              Met Ile Asp Trp Val Ser Trp Pro Leu                                1 5    ggg aag aat att gac aag tgg atc att gca ctg ctg aag ggc ctg gct 701 Gly Lys Asn Ile Asp Lys Trp Ile Ile Ala Leu Leu Lys Gly Leu Ala  10 15 20 25    gct gtt aag aag ttc agc atc ttg atc gag gtt tcg ctc acc aaa att 749 Ala Val Lys Lys Phe Ser Ile Leu Ile Glu Val Ser Leu Thr Lys Ile                  30 35 40    gag aag gtt ttc tct aag ctg ctg tac ccc atc gtc cgg gga gct gcc 797 Glu Lys Val Phe Ser Lys Leu Leu Tyr Pro Ile Val Arg Gly Ala Ala              45 50 55    ttg tct gtg ctc aag tac atg ctc ctg acc ttc cag cac tcc cac gaa 845 Leu Ser Val Leu Lys Tyr Met Leu Leu Thr Phe Gln His Ser His Glu          60 65 70    gcc ttc cac ctg ctc ctc cct cac atc ccc ccc atg gtg gcc tct ctg 893 Ala Phe His Leu Leu Leu Pro His Ile Pro Pro Met Val Ala Ser Leu      75 80 85    gtc aag gag gac tcg aac tcg ggg acc agc tgc ctg gag cag ctg gcg 941 Val Lys Glu Asp Ser Asn Ser Gly Thr Ser Cys Leu Glu Gln Leu Ala  90 95 100 105    gag ctg gtc cac tgc atg gtg ttc cgg ttc ccg ggc ttc ccg gat ctg 989 Glu Leu Val His Cys Met Val Phe Arg Phe Pro Gly Phe Pro Asp Leu                 110 115 120    tat gag cct gtc atg gag gcc atc aag gac ctc cat gtt ccc aat gag 1037 Tyr Glu Pro Val Met Glu Ala Ile Lys Asp Leu His Val Pro Asn Glu             125 130 135    gac cgc atc aag cag ctg ctg ggg cag gat gcc tgg act tcg cag aag 1085 Asp Arg Ile Lys Gln Leu Leu Gly Gln Asp Ala Trp Thr Ser Gln Lys         140 145 150    agc gag ctg gcg ggt ttc tat ccc cgg ctc atg gcc aag tca gac acg 1133 Ser Glu Leu Ala Gly Phe Tyr Pro Arg Leu Met Ala Lys Ser Asp Thr     155 160 165    ggc aag att ggt ctc atc aac ctg ggc aac aca tgc tat gtc aac agc 1181 Gly Lys Ile Gly Leu Ile Asn Leu Gly Asn Thr Cys Tyr Val Asn Ser 170 175 180 185    atc ctt cag gcc tta ttc atg gcg tct gac ttc aga cat tgt gtg ctc 1229 Ile Leu Gln Ala Leu Phe Met Ala Ser Asp Phe Arg His Cys Val Leu                 190 195 200    cgc ttg act gag aac aac tca cag ccc ctg atg acc aag ctg cag tgg 1277 Arg Leu Thr Glu Asn Asn Ser Gln Pro Leu Met Thr Lys Leu Gln Trp             205 210 215    ctc ttt ggc ttc cta gaa cac agc cag cgg cct gcc att tcc cca gag 1325 Leu Phe Gly Phe Leu Glu His Ser Gln Arg Pro Ala Ile Ser Pro Glu         220 225 230    aac ttc ctc tcc gca tcc tgg acg ccc tgg ttc agc cct ggc acc cag 1373 Asn Phe Leu Ser Ala Ser Trp Thr Pro Trp Phe Ser Pro Gly Thr Gln     235 240 245    cag gac tgc tcg gag tat ctg aag tac ctg ctg gat cgg ctg cac gaa 1421 Gln Asp Cys Ser Glu Tyr Leu Lys Tyr Leu Leu Asp Arg Leu His Glu 250 255 260 265    gag gag aaa acg ggc aca agg atc tgc cag aaa ctc aag cag tcc agc 1469 Glu Glu Lys Thr Gly Thr Arg Ile Cys Gln Lys Leu Lys Gln Ser Ser                 270 275 280    tcg ccc tct ccg ccc gag gag ccc ccg gcc cca agt tca acc tct gtg 1517 Ser Pro Ser Pro Pro Glu Glu Pro Pro Ala Pro Ser Ser Thr Ser Val             285 290 295    gaa aaa atg ttt gga ggc aag ata gtg act cgg atc tgc tgt ctc tgc 1565 Glu Lys Met Phe Gly Gly Lys Ile Val Thr Arg Ile Cys Cys Leu Cys         300 305 310    tgc ctc aac gtc tcc tcc cgg gag gag gcc ttc acg gac ctc tct ctc 1613 Cys Leu Asn Val Ser Ser Arg Glu Glu Ala Phe Thr Asp Leu Ser Leu     315 320 325    gcc ttc cct cct cct gag cgc tgt cgc cgc cgc cgc ctg ggc tct gtg 1661 Ala Phe Pro Pro Pro Glu Arg Cys Arg Arg Arg Arg Leu Gly Ser Val 330 335 340 345    atg cgc ccc aca gaa gac atc aca gcc cgg gag ttg ccc cca cca acc 1709 Met Arg Pro Thr Glu Asp Ile Thr Ala Arg Glu Leu Pro Pro Pro Thr                 350 355 360    agt gca cag ggg cca ggc agg gtg ggt cct cgg agg caa agg aaa cac 1757 Ser Ala Gln Gly Pro Gly Arg Val Gly Pro Arg Arg Gln Arg Lys His             365 370 375    tgc atc aca gag gac acc ccc ccc acc agc ctg tac atc gaa ggc ctg 1805 Cys Ile Thr Glu Asp Thr Pro Pro Thr Ser Leu Tyr Ile Glu Gly Leu         380 385 390    gac tcc aag gaa gct ggt ggg cag agc agt cag gag gaa agg ata gag 1853 Asp Ser Lys Glu Ala Gly Gly Gln Ser Ser Gln Glu Glu Arg Ile Glu     395 400 405    agg gag gaa gaa ggg aag gag gag aga acg gag aag gaa gaa gtg ggg 1901 Arg Glu Glu Glu Gly Lys Glu Glu Arg Thr Glu Lys Glu Glu Val Gly 410 415 420 425    gag gag gag gaa agc acc aga ggg gaa gga gag agg gag aaa gag gag 1949 Glu Glu Glu Glu Ser Thr Arg Gly Glu Gly Glu Arg Glu Lys Glu Glu                 430 435 440    gag gtg gaa gag gaa gaa gag aag gtg gag aag gag aca gaa aag gag 1997 Glu Val Glu Glu Glu Glu Glu Lys Val Glu Lys Glu Thr Glu Lys Glu             445 450 455    gct gag cag gaa aag gaa gaa gac agc ctg gga gcg ggg acc cac ccg 2045 Ala Glu Gln Glu Lys Glu Glu Asp Ser Leu Gly Ala Gly Thr His Pro         460 465 470    gat gct gcc atc ccc tcc ggg gag cgg aca tgt ggc tct gag ggc tcc 2093 Asp Ala Ala Ile Pro Ser Gly Glu Arg Thr Cys Gly Ser Glu Gly Ser     475 480 485    cgc tcc gtc ctg gac ctg gtt aac tac ttc ctg tcc ccc gag aag ctg 2141 Arg Ser Val Leu Asp Leu Val Asn Tyr Phe Leu Ser Pro Glu Lys Leu 490 495 500 505    aca gca gaa aac cgc tac tac tgc gag tcg tgt gcc tcc ctg cag gat 2189 Thr Ala Glu Asn Arg Tyr Tyr Cys Glu Ser Cys Ala Ser Leu Gln Asp                 510 515 520    gcc gag aag gtg gtg gag ctg agc caa ggg ccg tgc tac ctc atc ctc 2237 Ala Glu Lys Val Val Glu Leu Ser Gln Gly Pro Cys Tyr Leu Ile Leu             525 530 535    aca ctg ctg cgc ttc tct ttc gac ctg cgc acc atg cgg cgc cgc aag 2285 Thr Leu Leu Arg Phe Ser Phe Asp Leu Arg Thr Met Arg Arg Arg Lys         540 545 550    atc ctg gat gac gtc tcc atc ccc ctg ctg ctc cgc ctg cca ctg gct 2333 Ile Leu Asp Asp Val Ser Ile Pro Leu Leu Leu Arg Leu Pro Leu Ala     555 560 565    ggt ggc cgt ggc cag gcc tat gac ctc tgc agt gtg gtg gtg cac tct 2381 Gly Gly Arg Gly Gln Ala Tyr Asp Leu Cys Ser Val Val Val His Ser 570 575 580 585    gga gtg tct tcg gag agt ggt cac tac tac tgc tat gcc cgt gag ggc 2429 Gly Val Ser Ser Glu Ser Gly His Tyr Tyr Cys Tyr Ala Arg Glu Gly                 590 595 600    gct gcc cgc cct gcc gct tct ctg gga act gcc gat agg cca gag ccc 2477 Ala Ala Arg Pro Ala Ala Ser Leu Gly Thr Ala Asp Arg Pro Glu Pro             605 610 615    gag aac cag tgg tac ctg ttc aat gac act cgg gtg tcc ttc tct tcc 2525 Glu Asn Gln Trp Tyr Leu Phe Asn Asp Thr Arg Val Ser Phe Ser Ser         620 625 630    ttc gaa tct gtc agc aac gtc acc tcc ttc ttc cct aag gac aca gcc 2573 Phe Glu Ser Val Ser Asn Val Thr Ser Phe Phe Pro Lys Asp Thr Ala     635 640 645    tat gtg ctg ttt tac cgg cag cgg ccc agg gag ggg ccc gag gct gag 2621 Tyr Val Leu Phe Tyr Arg Gln Arg Pro Arg Glu Gly Pro Glu Ala Glu 650 655 660 665    ttg ggc tct tct aga gtc cgg aca gag ccc acc ctg cac aag gac ttg 2669 Leu Gly Ser Ser Arg Val Arg Thr Glu Pro Thr Leu His Lys Asp Leu                 670 675 680    atg gaa gcc att tcc aaa gac aac atc ctt tac cta cag gag cag gag 2717 Met Glu Ala Ile Ser Lys Asp Asn Ile Leu Tyr Leu Gln Glu Gln Glu             685 690 695    aag gag gcc cgg agc agg gcg gcc tac atc tct gca ctc ccc aca tct 2765 Lys Glu Ala Arg Ser Arg Ala Ala Tyr Ile Ser Ala Leu Pro Thr Ser         700 705 710    ccg cac tgg ggg agg ggc ttt gat gaa gac aag gat gag gat gaa ggc 2813 Pro His Trp Gly Arg Gly Phe Asp Glu Asp Lys Asp Glu Asp Glu Gly     715 720 725    tct cca ggg ggc tgc aat cct gca ggt ggc aat ggt ggt gac ttc cac 2861 Ser Pro Gly Gly Cys Asn Pro Ala Gly Gly Asn Gly Gly Asp Phe His 730 735 740 745    aga ctg gtc ttc taa tgtgaacctg ctgccaacct gaccccttcc ctccaggagc 2916 Arg Leu Val Phe                 750    caggtagggc ctgagggaag ctgtggaggc aggccctacc aagaggaagg atggtacagc 2976    tcatggcacc ttagtcctca gcctgatgaa gggtacacag agattctctc agatatggaa 3036    gtaagaccta agtccctttc attggggatc agtcccatta aaactttaca cccaagtgtc 3096    ctggttaact tgaagcagcc gagatgggca cacacgggtc tttgcctccc cctccttccc 3156    tagcaggctc cccatgcggg aagatctgat gatgttcagg aaacaggcta gacctcagct 3216    ccaatgtttt gacatcaagt actattttcc ttccgactgc tgtacggtat aaagcacagc 3276    aggatccaag ccttgcacaa aggggtgggg ggggcagtgt ctcctctggc tgtcctgttt 3336    gtttgtttct catatggggg tggggggtac ctgcactgtc tgtacctttc tgagaagaac 3396    agagaccgag acctgccccc ttaccaagcg ccactgcatg gtttgggggg gggggggcgg 3456    ggggctagct tctcacagca ggaggccttt gcccccacag cccctccacg cctgcctcag 3516    ggcctgagaa gccaccactt gtatccccct tgtggttaga gtcctgattt tactgcaaag 3576    gtgttcatgt tccttgtgaa gtgtgggctc ttaggaagcc tgtgggctcc tctgagcagt 3636    tggcctttgt agctgcaaca gcagccacct gcaggttggg tgaagtgccc tgacactgct 3696    gtagccccct tctaacttct aaccgaagac aagacagaca cccatgttca taaataaata 3756    aaagtaagcc taagc 3771       <210> 9 <211> 1121 <212> PRT <213> Homo sapiens    <400> 9 Met Pro Ile Val Asp Lys Leu Lys Glu Ala Leu Lys Pro Gly Arg Lys   1 5 10 15 Asp Ser Ala Asp Asp Gly Glu Leu Gly Lys Leu Leu Ala Ser Ser Ala              20 25 30 Lys Lys Val Leu Leu Gln Lys Ile Glu Phe Glu Pro Ala Ser Lys Ser          35 40 45 Phe Ser Tyr Gln Leu Glu Ala Leu Lys Ser Lys Tyr Val Leu Leu Asn      50 55 60 Pro Lys Thr Glu Gly Ala Ser Arg His Lys Ser Gly Asp Asp Pro Pro  65 70 75 80 Ala Arg Arg Gln Gly Ser Glu His Thr Tyr Glu Ser Cys Gly Asp Gly                  85 90 95 Val Pro Ala Pro Gln Lys Val Leu Phe Pro Thr Glu Arg Leu Ser Leu             100 105 110 Arg Trp Glu Arg Val Phe Arg Val Gly Ala Gly Leu His Asn Leu Gly         115 120 125 Asn Thr Cys Phe Leu Asn Ala Thr Ile Gln Cys Leu Thr Tyr Thr Pro     130 135 140 Pro Leu Ala Asn Tyr Leu Leu Ser Lys Glu His Ala Arg Ser Cys His 145 150 155 160 Gln Gly Ser Phe Cys Met Leu Cys Val Met Gln Asn His Ile Val Gln                 165 170 175 Ala Phe Ala Asn Ser Gly Asn Ala Ile Lys Pro Val Ser Phe Ile Arg             180 185 190 Asp Leu Lys Lys Ile Ala Arg His Phe Arg Phe Gly Asn Gln Glu Asp         195 200 205 Ala His Glu Phe Leu Arg Tyr Thr Ile Asp Ala Met Gln Lys Ala Cys     210 215 220 Leu Asn Gly Cys Ala Lys Leu Asp Arg Gln Thr Gln Ala Thr Thr Leu 225 230 235 240 Val His Gln Ile Phe Gly Gly Tyr Leu Arg Ser Arg Val Lys Cys Ser                 245 250 255 Val Cys Lys Ser Val Ser Asp Thr Tyr Asp Pro Tyr Leu Asp Val Ala             260 265 270 Leu Glu Ile Arg Gln Ala Ala Asn Ile Val Arg Ala Leu Glu Leu Phe         275 280 285 Val Lys Ala Asp Val Leu Ser Gly Glu Asn Ala Tyr Met Cys Ala Lys     290 295 300 Cys Lys Lys Lys Val Pro Ala Ser Lys Arg Phe Thr Ile His Arg Thr 305 310 315 320 Ser Asn Val Leu Thr Leu Ser Leu Lys Arg Phe Ala Asn Phe Ser Gly                 325 330 335 Gly Lys Ile Thr Lys Asp Val Gly Tyr Pro Glu Phe Leu Asn Ile Arg             340 345 350 Pro Tyr Met Ser Gln Asn Asn Gly Asp Pro Val Met Tyr Gly Leu Tyr         355 360 365 Ala Val Leu Val His Ser Gly Tyr Ser Cys His Ala Gly His Tyr Tyr     370 375 380 Cys Tyr Val Lys Ala Ser Asn Gly Gln Trp Tyr Gln Met Asn Asp Ser 385 390 395 400 Leu Val His Ser Ser Asn Val Lys Val Val Leu Asn Gln Gln Ala Tyr                 405 410 415 Val Leu Phe Tyr Leu Arg Ile Pro Gly Ser Lys Lys Ser Pro Glu Gly             420 425 430 Leu Ile Ser Arg Thr Gly Ser Ser Ser Leu Pro Gly Arg Pro Ser Val         435 440 445 Ile Pro Asp His Ser Lys Lys Asn Ile Gly Asn Gly Ile Ile Ser Ser     450 455 460 Pro Leu Thr Gly Lys Arg Gln Asp Ser Gly Thr Met Lys Lys Pro His 465 470 475 480 Thr Thr Glu Glu Ile Gly Val Pro Ile Ser Arg Asn Gly Ser Thr Leu                 485 490 495 Gly Leu Lys Ser Gln Asn Gly Cys Ile Pro Pro Lys Leu Pro Ser Gly             500 505 510 Ser Pro Ser Pro Lys Leu Ser Gln Thr Pro Thr His Met Pro Thr Ile         515 520 525 Leu Asp Asp Pro Gly Lys Lys Val Lys Lys Pro Ala Pro Pro Gln His     530 535 540 Phe Ser Pro Arg Thr Ala Gln Gly Leu Pro Gly Thr Ser Asn Ser Asn 545 550 555 560 Ser Ser Arg Ser Gly Ser Gln Arg Gln Gly Ser Trp Asp Ser Arg Asp                 565 570 575 Val Val Leu Ser Thr Ser Pro Lys Leu Leu Ala Thr Ala Thr Ala Asn             580 585 590 Gly His Gly Leu Lys Gly Asn Asp Glu Ser Ala Gly Leu Asp Arg Arg         595 600 605 Gly Ser Ser Ser Ser Ser Pro Glu His Ser Ala Ser Ser Asp Ser Thr     610 615 620 Lys Ala Pro Gln Thr Pro Arg Ser Gly Ala Ala His Leu Cys Asp Ser 625 630 635 640 Gln Glu Thr Asn Cys Ser Thr Ala Gly His Ser Lys Thr Pro Pro Ser                 645 650 655 Gly Ala Asp Ser Lys Thr Val Lys Leu Lys Ser Pro Val Leu Ser Asn             660 665 670 Thr Thr Thr Glu Pro Ala Ser Thr Met Ser Pro Pro Pro Ala Lys Lys         675 680 685 Leu Ala Leu Ser Ala Lys Lys Ala Ser Thr Leu Trp Arg Ala Thr Gly     690 695 700 Asn Asp Leu Arg Pro Pro Pro Pro Ser Pro Ser Ser Asp Leu Thr His 705 710 715 720 Pro Met Lys Thr Ser His Pro Val Val Ala Ser Thr Trp Pro Val His                 725 730 735 Arg Ala Arg Ala Val Ser Pro Ala Pro Gln Ser Ser Ser Arg Leu Gln             740 745 750 Pro Pro Phe Ser Pro His Pro Thr Leu Leu Ser Ser Thr Pro Lys Pro         755 760 765 Pro Gly Thr Ser Glu Pro Arg Ser Cys Ser Ser Ile Ser Thr Ala Leu     770 775 780 Pro Gln Val Asn Glu Asp Leu Val Ser Leu Pro His Gln Leu Pro Glu 785 790 795 800 Ala Ser Glu Pro Pro Arg Ser Pro Ser Glu Lys Arg Lys Lys Thr Phe                 805 810 815 Val Gly Glu Pro Gln Arg Leu Gly Ser Glu Thr Cys Leu Pro Gln His             820 825 830 Ile Arg Glu Ala Thr Ala Ala Pro His Gly Lys Arg Lys Arg Lys Lys         835 840 845 Lys Lys Arg Pro Glu Asp Thr Ala Ala Ser Ala Leu Gln Glu Gly Gln     850 855 860 Thr Gln Arg Gln Pro Gly Ser Pro Met Tyr Arg Arg Glu Gly Gln Ala 865 870 875 880 Gln Leu Pro Ala Val Arg Arg Gln Glu Asp Gly Thr Gln Pro Gln Val                 885 890 895 Asn Gly Gln Gln Val Gly Cys Val Thr Asp Gly His His Ala Ser Ser             900 905 910 Arg Lys Arg Arg Arg Lys Gly Ala Glu Gly Leu Gly Glu Glu Gly Gly         915 920 925 Leu His Gln Asp Pro Leu Arg His Ser Cys Ser Pro Met Gly Asp Gly     930 935 940 Asp Pro Glu Ala Met Glu Glu Ser Pro Arg Lys Lys Lys Lys Arg Lys 945 950 955 960 Gln Glu Thr Gln Arg Ala Val Glu Glu Asp Gly His Leu Lys Cys Pro                 965 970 975 Arg Ser Ala Lys Pro Gln Asp Ala Val Val Pro Glu Ser Ser Ser Cys             980 985 990 Ala Pro Ser Ala Asn Gly Trp Cys Pro Gly Asp Arg Met Gly Leu Ser         995 1000 1005 Gln Ala Pro Pro Val Ser Trp Asn Gly Glu Arg Glu Ser Asp Val Val    1010 1015 1020 Gln Glu Leu Leu Lys Tyr Ser Ser Asp Lys Ala Tyr Gly Arg Lys Val 1025 1030 1035 1040 Leu Thr Trp Asp Gly Lys Met Ser Ala Val Ser Gln Asp Ala Ile Glu                1045 1050 1055 Asp Ser Arg Gln Ala Arg Thr Glu Thr Val Val Asp Asp Trp Asp Glu            1060 1065 1070 Glu Phe Asp Arg Gly Lys Glu Lys Lys Ile Lys Lys Phe Lys Arg Glu        1075 1080 1085 Lys Arg Arg Asn Phe Asn Ala Phe Gln Lys Leu Gln Thr Arg Arg Asn    1090 1095 1100 Phe Trp Ser Val Thr His Pro Ala Lys Ala Ala Ser Leu Ser Tyr Arg 1105 1110 1115 1120 Arg    <210> 10 <211> 5879 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (219) .. (3584)    <400> 10 cgccagggct gcgtaggctt gtggcgcgcc cgcggagagg ccggggctct gacgcccgct 60    ctgcggcttc ggtgtttgaa caggccacag tccaggagcg cttacattca ggagctccgc 120    gtagcacctg cccaaccaaa ctcagccctc cgttaagatc ctggttccat gccgcagtag 180    gacagcaggc ccaagtctgc acatcccagt gatgcacc atg cca ata gtg gat aag 236                                           Met Pro Ile Val Asp Lys                                             1 5    ttg aag gag gcc ctg aaa ccc ggc cgc aag gac tcg gct gat gat gga 284 Leu Lys Glu Ala Leu Lys Pro Gly Arg Lys Asp Ser Ala Asp Asp Gly              10 15 20    gaa ctg ggg aag ctt ctt gcc tcc tct gcc aag aag gtc ctt tta cag 332 Glu Leu Gly Lys Leu Leu Ala Ser Ser Ala Lys Lys Val Leu Leu Gln          25 30 35    aaa atc gag ttc gag cca gcc agc aag agc ttc tcc tac cag ctg gag 380 Lys Ile Glu Phe Glu Pro Ala Ser Lys Ser Phe Ser Tyr Gln Leu Glu      40 45 50    gcc tta aag agc aaa tat gtg ttg ctc aac ccc aaa aca gag gga gct 428 Ala Leu Lys Ser Lys Tyr Val Leu Leu Asn Pro Lys Thr Glu Gly Ala  55 60 65 70    agt cgc cac aag agt gga gat gac cca ccg gcc agg aga cag ggc agt 476 Ser Arg His Lys Ser Gly Asp Asp Prop Ala Arg Arg Gln Gly Ser                  75 80 85    gag cac acg tat gag agc tgt ggt gac gga gtc cca gcc ccg cag aaa 524 Glu His Thr Tyr Glu Ser Cys Gly Asp Gly Val Pro Ala Pro Gln Lys              90 95 100    gtg ctt ttc ccc acg gag cga ctg tct ctg agg tgg gag cgg gtc ttc 572 Val Leu Phe Pro Thr Glu Arg Leu Ser Leu Arg Trp Glu Arg Val Phe         105 110 115    cgc gtg ggc gca gga ctc cac aac ctt ggc aac acc tgc ttt ctc aat 620 Arg Val Gly Ala Gly Leu His Asn Leu Gly Asn Thr Cys Phe Leu Asn     120 125 130    gcc acc atc cag tgc ttg acc tac aca cca cct cta gcc aac tac ctg 668 Ala Thr Ile Gln Cys Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Leu 135 140 145 150    ctc tcc aag gag cat gct cgc agc tgc cac cag gga agc ttc tgc atg 716 Leu Ser Lys Glu His Ala Arg Ser Cys His Gln Gly Ser Phe Cys Met                 155 160 165    ctg tgt gtc atg cag aac cac att gtc cag gcc ttc gcc aac agc ggc 764 Leu Cys Val Met Gln Asn His Ile Val Gln Ala Phe Ala Asn Ser Gly             170 175 180    aac gcc atc aag ccc gtc tcc ttc atc cga gac ctg aaa aag atc gcc 812 Asn Ala Ile Lys Pro Val Ser Phe Ile Arg Asp Leu Lys Lys Ile Ala         185 190 195    cga cac ttc cgc ttt ggg aac cag gag gac gcg cat gag ttc ctg cgg 860 Arg His Phe Arg Phe Gly Asn Gln Glu Asp Ala His Glu Phe Leu Arg     200 205 210    tac acc atc gac gcc atg cag aaa gcc tgc ctg aat ggc tgt gcc aag 908 Tyr Thr Ile Asp Ala Met Gln Lys Ala Cys Leu Asn Gly Cys Ala Lys 215 220 225 230    ttg gat cgt caa acg cag gct act acc ttg gtc cat caa att ttt gga 956 Leu Asp Arg Gln Thr Gln Ala Thr Thr Leu Val His Gln Ile Phe Gly                 235 240 245    ggg tat ctc aga tca cgc gtg aag tgc tcc gtg tgc aag agc gtc tcg 1004 Gly Tyr Leu Arg Ser Arg Val Lys Cys Ser Val Cys Lys Ser Val Ser             250 255 260    gac acc tac gac ccc tac ttg gac gtc gcg ctg gag atc cgg caa gct 1052 Asp Thr Tyr Asp Pro Tyr Leu Asp Val Ala Leu Glu Ile Arg Gln Ala         265 270 275    gcg aat att gtg cgt gct ctg gaa ctt ttt gtg aaa gca gat gtc ctg 1100 Ala Asn Ile Val Arg Ala Leu Glu Leu Phe Val Lys Ala Asp Val Leu     280 285 290    agt gga gag aat gcc tac atg tgt gct aaa tgc aag aag aag gtt cca 1148 Ser Gly Glu Asn Ala Tyr Met Cys Ala Lys Cys Lys Lys Lys Val Pro 295 300 305 310    gcc agc aag cgc ttc acc atc cac aga aca tcc aac gtc tta acc ctt 1196 Ala Ser Lys Arg Phe Thr Ile His Arg Thr Ser Asn Val Leu Thr Leu                 315 320 325    tcc ctc aag cgt ttt gcc aac ttc agc ggg ggg aag atc acc aag gat 1244 Ser Leu Lys Arg Phe Ala Asn Phe Ser Gly Gly Lys Ile Thr Lys Asp             330 335 340    gta ggc tat ccg gaa ttc ctc aac ata cgt ccg tat atg tcc cag aat 1292 Val Gly Tyr Pro Glu Phe Leu Asn Ile Arg Pro Tyr Met Ser Gln Asn         345 350 355    aat ggt gat cct gtc atg tat gga ctc tat gct gtc ctg gtg cac tcg 1340 Asn Gly Asp Pro Val Met Tyr Gly Leu Tyr Ala Val Leu Val His Ser     360 365 370    ggc tac agc tgc cat gcc ggg cac tat tac tgc tac gtg aag gca agc 1388 Gly Tyr Ser Cys His Ala Gly His Tyr Tyr Cys Tyr Val Lys Ala Ser 375 380 385 390    aat gga cag tgg tac cag atg aat gat tcc ttg gtc cat tcc agc aac 1436 Asn Gly Gln Trp Tyr Gln Met Asn Asp Ser Leu Val His Ser Ser Asn                 395 400 405    gtc aag gtg gtt ctg aac cag cag gcc tac gtg ctg ttc tat ctg cga 1484 Val Lys Val Val Leu Asn Gln Gln Ala Tyr Val Leu Phe Tyr Leu Arg             410 415 420    att cca ggc tct aag aaa agt ccc gag ggc ctc atc tcc agg aca ggc 1532 Ile Pro Gly Ser Lys Lys Ser Pro Glu Gly Leu Ile Ser Arg Thr Gly         425 430 435    tcc tcc tcc ctt ccc ggc cgc ccg agt gtg att cca gat cac tcc aag 1580 Ser Ser Ser Leu Pro Gly Arg Pro Ser Val Ile Pro Asp His Ser Lys     440 445 450    aag aac atc ggc aat ggg att att tcc tcc cca ctg act gga aag cga 1628 Lys Asn Ile Gly Asn Gly Ile Ile Ser Ser Pro Leu Thr Gly Lys Arg 455 460 465 470    caa gac tct ggg acg atg aag aag ccg cac acc act gaa gag att ggt 1676 Gln Asp Ser Gly Thr Met Lys Lys Pro His Thr Thr Glu Glu Ile Gly                 475 480 485    gtg ccc ata tcc agg aat ggc tcc acc ctg ggc ctg aag tcc cag aac 1724 Val Pro Ile Ser Arg Asn Gly Ser Thr Leu Gly Leu Lys Ser Gln Asn             490 495 500    ggc tgc att cct cca aag ctg ccc tcg ggg tcc cct tcc ccc aaa ctc 1772 Gly Cys Ile Pro Pro Lys Leu Pro Ser Gly Ser Pro Ser Pro Lys Leu         505 510 515    tcc cag aca ccc aca cac atg cca acc atc cta gac gac cct gga aag 1820 Ser Gln Thr Pro Thr His Met Pro Thr Ile Leu Asp Asp Pro Gly Lys     520 525 530    aag gtg aag aag cca gct cct cca cag cac ttt tcc ccc aga act gct 1868 Lys Val Lys Lys Pro Ala Pro Pro Gln His Phe Ser Pro Arg Thr Ala 535 540 545 550    cag ggg ctg cct ggg acc agc aac tcg aat agc agc aga tct ggg agc 1916 Gln Gly Leu Pro Gly Thr Ser Asn Ser Asn Ser Ser Arg Ser Gly Ser                 555 560 565    caa agg cag ggc tcc tgg gac agc agg gat gtt gtc ctc tct acc tca 1964 Gln Arg Gln Gly Ser Trp Asp Ser Arg Asp Val Val Leu Ser Thr Ser             570 575 580    cct aag ctc ctg gct aca gcc act gcc aac ggg cat ggg ctg aag ggg 2012 Pro Lys Leu Leu Ala Thr Ala Thr Ala Asn Gly His Gly Leu Lys Gly         585 590 595    aac gac gag agc gct ggc ctc gac agg agg ggc tcc agc agc tcc agc 2060 Asn Asp Glu Ser Ala Gly Leu Asp Arg Arg Gly Ser Ser Ser Ser Ser     600 605 610    cca gag cac tcg gcc agc agc gac tcc acc aag gcc ccc cag acc ccc 2108 Pro Glu His Ser Ala Ser Ser Asp Ser Thr Lys Ala Pro Gln Thr Pro 615 620 625 630    agg agt gga gcg gcc cat ctc tgc gat tct cag gaa acg aac tgt tcc 2156 Arg Ser Gly Ala Ala His Leu Cys Asp Ser Gln Glu Thr Asn Cys Ser                 635 640 645    acc gct ggc cac tcc aaa acg ccg cca agt gga gca gat tct aag acg 2204 Thr Ala Gly His Ser Lys Thr Pro Pro Ser Gly Ala Asp Ser Lys Thr             650 655 660    gtg aag ctg aag tcc cct gtc ctg agc aac acc acc act gag cct gca 2252 Val Lys Leu Lys Ser Pro Val Leu Ser Asn Thr Thr Thr Glu Pro Ala         665 670 675    agc acc atg tct cct cca cca gcc aaa aaa ctg gcc ctt tct gcc aag 2300 Ser Thr Met Ser Pro Pro Pro Ala Lys Lys Leu Ala Leu Ser Ala Lys     680 685 690    aag gcc agc acc ctg tgg agg gcg acc ggc aat gac ctc cgt cca cct 2348 Lys Ala Ser Thr Leu Trp Arg Ala Thr Gly Asn Asp Leu Arg Pro Pro 695 700 705 710    ccc ccc tca cca tcc tcc gac ctc acc cac ccc atg aaa acc tct cac 2396 Pro Pro Ser Pro Ser Ser Asp Leu Thr His Pro Met Lys Thr Ser His                 715 720 725    ccc gtc gtt gcc tcc act tgg ccc gtc cat aga gcc agg gct gtg tca 2444 Pro Val Val Ala Ser Thr Trp Pro Val His Arg Ala Arg Ala Val Ser             730 735 740    cct gct ccc caa tca tcc agc cgc ctg caa ccc ccc ttc agc ccc cac 2492 Pro Ala Pro Gln Ser Ser Ser Arg Leu Gln Pro Pro Phe Ser Pro His         745 750 755    ccc aca ttg ctg tcc agt acc ccc aag ccc cca ggg acg tca gaa cca 2540 Pro Thr Leu Leu Ser Ser Thr Pro Lys Pro Pro Gly Thr Ser Glu Pro     760 765 770    cgg agc tgc tcc tcc atc tcg acg gcg ctg cct cag gtc aac gag gac 2588 Arg Ser Cys Ser Ser Ile Ser Thr Ala Leu Pro Gln Val Asn Glu Asp 775 780 785 790    ctt gtg tcc ctt cca cac cag ttg cca gag gcc agt gag ccc ccc cgg 2636 Leu Val Ser Leu Pro His Gln Leu Pro Glu Ala Ser Glu Pro Pro Arg                 795 800 805    agc ccc tct gag aag agg aaa aag acc ttt gtg gga gag ccg cag agg 2684 Ser Pro Ser Glu Lys Arg Lys Lys Thr Phe Val Gly Glu Pro Gln Arg             810 815 820    ctg ggc tca gag acg tgc ctc cca cag cac atc agg gag gcc act gcg 2732 Leu Gly Ser Glu Thr Cys Leu Pro Gln His Ile Arg Glu Ala Thr Ala         825 830 835    gct ccc cac ggg aag agg aag agg aag aag aag aag cgc ccg gag gac 2780 Ala Pro His Gly Lys Arg Lys Arg Lys Lys Lys Lys Arg Pro Glu Asp     840 845 850    aca gct gcc agc gcc ctg cag gag ggg cag aca cag aga cag cct ggg 2828 Thr Ala Ala Ser Ala Leu Gln Glu Gly Gln Thr Gln Arg Gln Pro Gly 855 860 865 870    agc ccc atg tac agg agg gag ggc cag gca cag ctg ccc gct gtc aga 2876 Ser Pro Met Tyr Arg Arg Glu Gly Gln Ala Gln Leu Pro Ala Val Arg                 875 880 885    cgg cag gaa gat ggc aca cag cca cag gtg aat ggc cag cag gtg gga 2924 Arg Gln Glu Asp Gly Thr Gln Pro Gln Val Asn Gly Gln Gln Val Gly             890 895 900    tgt gtt acg gac ggc cac cac gcg agc agc agg aag cgg agg agg aaa 2972 Cys Val Thr Asp Gly His His Ala Ser Ser Arg Lys Arg Arg Arg Lys         905 910 915    gga gca gaa ggt ctt ggt gaa gaa ggc ggc ctg cac cag gac cca ctt 3020 Gly Ala Glu Gly Leu Gly Glu Glu Gly Gly Leu His Gln Asp Pro Leu     920 925 930    cgg cac agc tgc tct ccc atg ggt gat ggt gat cca gag gcc atg gaa 3068 Arg His Ser Cys Ser Pro Met Gly Asp Gly Asp Pro Glu Ala Met Glu 935 940 945 950    gag tct cca agg aaa aag aaa aaa aga aag cag gag aca cag cgg gca 3116 Glu Ser Pro Arg Lys Lys Lys Lys Arg Lys Gln Glu Thr Gln Arg Ala                 955 960 965    gta gaa gag gat ggg cat ctc aaa tgc cca agg agt gcc aag ccc caa 3164 Val Glu Glu Asp Gly His Leu Lys Cys Pro Arg Ser Ala Lys Pro Gln             970 975 980    gat gct gtt gtc ccc gag tcc agc agc tgc gca cca tcc gcg aat ggc 3212 Asp Ala Val Val Pro Glu Ser Ser Ser Cys Ala Pro Ser Ala Asn Gly         985 990 995    tgg tgt cct ggg gac cgc atg ggg ctg agc cag gcc cct cct gtg tct 3260 Trp Cys Pro Gly Asp Arg Met Gly Leu Ser Gln Ala Pro Pro Val Ser    1000 1005 1010    tgg aat gga gag cgg gag tct gat gtg gtc cag gaa ctg ctc aaa tac 3308 Trp Asn Gly Glu Arg Glu Ser Asp Val Val Gln Glu Leu Leu Lys Tyr 1015 1020 1025 1030    tca tct gat aaa gct tac ggg aga aaa gtt ctg acc tgg gat ggc aag 3356 Ser Ser Asp Lys Ala Tyr Gly Arg Lys Val Leu Thr Trp Asp Gly Lys                1035 1040 1045    atg tcg gcg gtc agt cag gat gct att gaa gac agc aga cag gcc cgg 3404 Met Ser Ala Val Ser Gln Asp Ala Ile Glu Asp Ser Arg Gln Ala Arg            1050 1055 1060    act gag acc gtg gtt gat gac tgg gac gaa gag ttt gac cga ggg aag 3452 Thr Glu Thr Val Val Asp Asp Trp Asp Glu Glu Phe Asp Arg Gly Lys        1065 1070 1075    gaa aag aaa att aaa aaa ttt aag aga gag aag agg aga aac ttc aac 3500 Glu Lys Lys Ile Lys Lys Phe Lys Arg Glu Lys Arg Arg Asn Phe Asn    1080 1085 1090    gcc ttc cag aaa ctt cag act cga cgg aac ttc tgg tct gtg act cac 3548 Ala Phe Gln Lys Leu Gln Thr Arg Arg Asn Phe Trp Ser Val Thr His 1095 1100 1105 1110    cca gca aag gct gcc agc ctc agc tat cgc cgc tga ctgtgcccct 3594 Pro Ala Lys Ala Ala Ser Leu Ser Tyr Arg Arg                1115 1120    gtggaaggag gtcggttccg agggggtggg tgtaagggtg aggtgggggt gtgtgtgccg 3654    tgtatgtgtg taggggtgtg gtggggtgtg gcgtgtgtgt atgatgtatg tgggctgtgt 3714    atctggcaca tgtgtgttgg gtggtgtgtg tggtgtgggc gcgtctgtgg tgtgtgtcct 3774    agtcacttgg agaagggtgt gtgtgggatg cgtgtgtata agggggtgtgtgtgggatgc 3834    gtgtgtataa gggggtgtgt gtagtgtgtg ttgggtgtgg ggtgtgtaca cctggcatct 3894    gtggcatgtg ttctggtcac ttggagaagg gcatgtgtgg ggtgtgtgtgtg ggatgtgggt 3954    tgtgtgtata tctggcatgt gtcccagtca cttgcagaag ggtgacttct tgccagccgc 4014    atcgagatgc catgcattgg gttcctaggt tggactcata cccgagggtg gcagtgggaa 4074    gattcgggtc tcgtttctct ctgtcaggac taccgtggtt tgttctgcag cctcctggag 4134    acaaggcgtc ccttcccggg agctgtcggt ctggatctga gggagctctc tgtgtgggct 4194    ctgctgtgct gggagcctgt cacggtagga gctctcccgg taccagtgtc cacagaccgc 4254    ccaacataga ggctttgagg cttctctaga tcggaacctc tttggtgaca ttcccgacca 4314    gccctgcaag agaaacgaca gtgtgtgtgt gagcagaggt ggctgcacac ctgctggaca 4374    tctttgccag gctgtgcctt ctcatgtttc atagacagtg gtctgtgctg gcagaggctg 4434    ctgcccctgg ttggggctat caggagagtg ggggatggtg gccacatgtc ctccaggtgg 4494    tctcccggtg catagctggt ggctctgggc aagccatccc ttgcttctcg gggctgacgc 4554    caccgttgtg tccgagcccg ccctcccctg cttcctcagc gggacccctt catctgttgg 4614    ccttacctgt cctcagaaag gaagaggtga ccccacccag ccacctctcc cttttatgga 4674    actcgagagg gtggccctac tgtgcacccc ttccttgtga gtagctctca actgtcctgg 4734    agagcagagg ctatttgggg tcggaggagc cctcgatacc tgcgaataca tctgctttcc 4794    aggctgctgt ttattctgag acgactgtgc tgtagcttcc cttgcagctg caataacccg 4854    caggtcttca ctgaggtgga ggctttgggg tagaattctc catttatttt actacttaat 4914    acaaaacatt tatttttgac cagtcctgtg gcttccatta gcaatatgtt tcctttccca 4974    aatatgcaaa tagtggcttt gtttgctcaa ttttgtgagt gctttggaat ttaaatgatt 5034    gtataactca agaagattac ttttctatgt tgctcaagct gtgcctgcca acttgtaact 5094    taataaatac aggaaatcct cagagaaggt gatattttca ggaaaaagac aaatgccctc 5154    atagtagtgg gaagtgtgaa ggtgaccgtg aacatccttc ctcatcgggt ctgtccccgt 5214    catttcctcc cggagtcgtc gcaggtggag atggacaacg tggtgttgga cttagacctc 5274    cttcagtgtg gctctgctgg gccagaggca tcctgctgtc ccgggtggct gcctcgctgt 5334    ctgcaccccc tctccctggg gcagctttgc ttcctgcccc tgtgctcggg gcctgggtgg 5394    ttactggcgt gtagatggaa ttgctttttt aatatgggaa gatacattta tttttttcca 5454    tgtgggtggg tgtctctttt tggattttct tctgttttta cgtttctctt cttagaaggg 5514    tgggagagaa tcaagctcct gtggccacct gtgtcccagc agcagtgagt ggagctgctc 5574    agggtgccct ctcctgcgga ccagtctctg aatgttcaaa gatgagggcc tggcttccgt 5634    gctctggctt tgtaacttat ctggaaggga aagcacatgc cttcacgggc aggatgatca 5694    agcggtgctg attcacgaga gtggaagcct ccagagcttg gggctttctg gctgctcttc 5754    attgacctgt gtgttcccag cacacgaaca gcgcccctaa cggagatttg ttcagcgact 5814    gaatatacac ctgtaaacga gtagcatgta tacattgatt ttgattacaa atggttctgt 5874    attat 5879       <210> 11 <211> 514 <212> PRT <213> Homo sapiens    <400> 11 Met Asp Ala Glu Leu Ala Val Ala Pro Pro Gly Cys Ser His Leu Gly   1 5 10 15 Ser Phe Lys Val Asp Asn Trp Lys Gln Asn Leu Arg Ala Ile Tyr Gln              20 25 30 Cys Phe Val Trp Ser Gly Thr Ala Glu Ala Arg Lys Arg Lys Ala Lys          35 40 45 Ser Cys Ile Cys His Val Cys Gly Val His Leu Asn Arg Leu His Ser      50 55 60 Cys Leu Tyr Cys Val Phe Phe Gly Cys Phe Thr Lys Lys His Ile His  65 70 75 80 Glu His Ala Lys Ala Lys Arg His Asn Leu Ala Ile Asp Leu Met Tyr                  85 90 95 Gly Gly Ile Tyr Cys Phe Leu Cys Gln Asp Tyr Ile Tyr Asp Lys Asp             100 105 110 Met Glu Ile Ile Ala Lys Glu Glu Gln Arg Lys Ala Trp Lys Met Gln         115 120 125 Gly Val Gly Glu Lys Phe Ser Thr Trp Glu Pro Thr Lys Arg Glu Leu     130 135 140 Glu Leu Leu Lys His Asn Pro Lys Arg Arg Lys Ile Thr Ser Asn Cys 145 150 155 160 Thr Ile Gly Leu Arg Gly Leu Ile Asn Leu Gly Asn Thr Cys Phe Met                 165 170 175 Asn Cys Ile Val Gln Ala Leu Thr His Thr Pro Leu Leu Arg Asp Phe             180 185 190 Phe Leu Ser Asp Arg His Arg Cys Glu Met Gln Ser Pro Ser Ser Cys         195 200 205 Leu Val Cys Glu Met Ser Ser Leu Phe Gln Glu Phe Tyr Ser Gly His     210 215 220 Arg Ser Pro His Ile Pro Tyr Lys Leu Leu His Leu Val Trp Thr His 225 230 235 240 Ala Arg His Leu Ala Gly Tyr Glu Gln Gln Asp Ala His Glu Phe Leu                 245 250 255 Ile Ala Ala Leu Asp Val Leu His Arg His Cys Lys Gly Asp Asp Asn             260 265 270 Gly Lys Lys Ala Asn Asn Pro Asn His Cys Asn Cys Ile Ile Asp Gln         275 280 285 Ile Phe Thr Gly Gly Leu Gln Ser Asp Val Thr Cys Gln Val Cys His     290 295 300 Gly Val Ser Thr Thr Ile Asp Pro Phe Trp Asp Ile Ser Leu Asp Leu 305 310 315 320 Pro Gly Ser Ser Thr Pro Phe Trp Pro Leu Ser Pro Gly Ser Glu Gly                 325 330 335 Asn Val Val Asn Gly Glu Ser His Val Ser Gly Thr Thr Thr Leu Thr             340 345 350 Asp Cys Leu Arg Arg Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala         355 360 365 Lys Ile Lys Cys Ser Gly Cys His Ser Tyr Gln Glu Ser Thr Lys Gln     370 375 380 Leu Thr Met Lys Lys Leu Pro Ile Val Ala Cys Phe His Leu Lys Arg 385 390 395 400 Phe Glu His Ser Ala Lys Leu Arg Arg Lys Ile Thr Thr Tyr Val Ser                 405 410 415 Phe Pro Leu Glu Leu Asp Met Thr Pro Phe Met Ala Ser Ser Lys Glu             420 425 430 Ser Arg Met Asn Gly Gln Tyr Gln Gln Pro Thr Asp Ser Leu Asn Asn         435 440 445 Asp Asn Lys Tyr Ser Leu Phe Ala Val Val Asn His Gln Gly Thr Leu     450 455 460 Glu Ser Gly His Tyr Thr Ser Phe Ile Arg Gln His Lys Asp Gln Trp 465 470 475 480 Phe Lys Cys Asp Asp Ala Ile Ile Thr Lys Ala Ser Ile Lys Asp Val                 485 490 495 Leu Asp Ser Glu Gly Tyr Leu Leu Phe Tyr His Lys Gln Phe Leu Glu             500 505 510 Tyr Glu       <210> 12 <211> 1545 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (1) .. (1545)    <400> 12 atg gac gcc gag ctg gcg gta gcg ccg ccg ggc tgc tcg cac ctg ggc 48 Met Asp Ala Glu Leu Ala Val Ala Pro Pro Gly Cys Ser His Leu Gly   1 5 10 15    agc ttc aag gtg gac aac tgg aag cag aac ctg cgg gcc atc tac cag 96 Ser Phe Lys Val Asp Asn Trp Lys Gln Asn Leu Arg Ala Ile Tyr Gln              20 25 30    tgc ttc gtg tgg agc ggc acg gct gag gcc cgc aag cgc aag gcc aag 144 Cys Phe Val Trp Ser Gly Thr Ala Glu Ala Arg Lys Arg Lys Ala Lys          35 40 45    tcc tgt atc tgc cat gtc tgt ggc gtc cac ctc aac agg ctg cat tcc 192 Ser Cys Ile Cys His Val Cys Gly Val His Leu Asn Arg Leu His Ser      50 55 60    tgc ctc tac tgt gtc ttc ttc ggc tgt ttc aca aag aag cat att cac 240 Cys Leu Tyr Cys Val Phe Phe Gly Cys Phe Thr Lys Lys His Ile His  65 70 75 80    gag cat gcg aag gcg aag cgg cac aac ctg gcc att gat ctg atg tat 288 Glu His Ala Lys Ala Lys Arg His Asn Leu Ala Ile Asp Leu Met Tyr                  85 90 95    gga ggc atc tac tgt ttt ctg tgc cag gac tac atc tat gac aaa gac 336 Gly Gly Ile Tyr Cys Phe Leu Cys Gln Asp Tyr Ile Tyr Asp Lys Asp             100 105 110    atg gaa ata atc gcc aag gag gag cag cga aaa gct tgg aaa atg caa 384 Met Glu Ile Ile Ala Lys Glu Glu Gln Arg Lys Ala Trp Lys Met Gln         115 120 125    ggc gtt gga gag aag ttt tca act tgg gaa cca acc aaa cgg gag ctt 432 Gly Val Gly Glu Lys Phe Ser Thr Trp Glu Pro Thr Lys Arg Glu Leu     130 135 140    gaa ctg ctg aag cac aac ccg aaa agg aga aag atc acc tcg aac tgc 480 Glu Leu Leu Lys His Asn Pro Lys Arg Arg Lys Ile Thr Ser Asn Cys 145 150 155 160    acc ata ggt ctg cgt ggg ctg atc aac ctt ggg aac aca tgc ttc atg 528 Thr Ile Gly Leu Arg Gly Leu Ile Asn Leu Gly Asn Thr Cys Phe Met                 165 170 175    aac tgc atc gtg cag gcc ctg acc cac acg cca ctt ctg cgg gac ttc 576 Asn Cys Ile Val Gln Ala Leu Thr His Thr Pro Leu Leu Arg Asp Phe             180 185 190    ttc ctg tct gac agg cac cgc tgt gag atg cag agc ccc agc tcc tgt 624 Phe Leu Ser Asp Arg His Arg Cys Glu Met Gln Ser Pro Ser Ser Cys         195 200 205    ctg gtc tgt gag atg tcc tca ctg ttt cag gag ttt tac tct gga cac 672 Leu Val Cys Glu Met Ser Ser Leu Phe Gln Glu Phe Tyr Ser Gly His     210 215 220    cgg tcc cct cac atc ccg tat aag ttg ctg cac ctg gtg tgg acc cac 720 Arg Ser Pro His Ile Pro Tyr Lys Leu Leu His Leu Val Trp Thr His 225 230 235 240    gcg agg cac cta gca ggc tac gag cag cag gac gcc cac gag ttc ctc 768 Ala Arg His Leu Ala Gly Tyr Glu Gln Gln Asp Ala His Glu Phe Leu                 245 250 255    atc gcg gcc ctg gac gtg ctc cac cga cac tgc aaa ggt gat gac aat 816 Ile Ala Ala Leu Asp Val Leu His Arg His Cys Lys Gly Asp Asp Asn             260 265 270    ggg aag aag gcc aac aac ccc aac cac tgc aac tgc atc ata gac cag 864 Gly Lys Lys Ala Asn Asn Pro Asn His Cys Asn Cys Ile Ile Asp Gln         275 280 285    atc ttc aca ggc ggg ttg cag tca gac gtc acc tgc caa gtc tgc cat 912 Ile Phe Thr Gly Gly Leu Gln Ser Asp Val Thr Cys Gln Val Cys His     290 295 300    gga gtc tcc acc acc atc gac ccc ttc tgg gac atc agc ttg gat ctc 960 Gly Val Ser Thr Thr Ile Asp Pro Phe Trp Asp Ile Ser Leu Asp Leu 305 310 315 320    ccc ggc tct tcc acc cca ttc tgg ccc ctg agc cca ggg agc gag ggc 1008 Pro Gly Ser Ser Thr Pro Phe Trp Pro Leu Ser Pro Gly Ser Glu Gly                 325 330 335    aac gtg gta aac ggg gaa agc cac gtg tcg gga acc acc acg ctc acg 1056 Asn Val Val Asn Gly Glu Ser His Val Ser Gly Thr Thr Thr Leu Thr             340 345 350    gac tgc ctg cga cga ttc acc aga cca gag cac ttg ggc agc agc gcc 1104 Asp Cys Leu Arg Arg Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala         355 360 365    aag atc aag tgc agc ggt tgc cat agc tac cag gag tcc aca aag cag 1152 Lys Ile Lys Cys Ser Gly Cys His Ser Tyr Gln Glu Ser Thr Lys Gln     370 375 380    ctc act atg aag aaa ctg ccc atc gta gcc tgt ttt cat ctc aaa cga 1200 Leu Thr Met Lys Lys Leu Pro Ile Val Ala Cys Phe His Leu Lys Arg 385 390 395 400    ttt gaa cac tca gcc aag ctg cgg cgg aag atc acc acg tat gtg tcc 1248 Phe Glu His Ser Ala Lys Leu Arg Arg Lys Ile Thr Thr Tyr Val Ser                 405 410 415    ttc ccc ctg gag ctg gac atg acc cct ttc atg gcc tcc agc aaa gag 1296 Phe Pro Leu Glu Leu Asp Met Thr Pro Phe Met Ala Ser Ser Lys Glu             420 425 430    agc agg atg aat gga cag tac cag cag ccc acg gac agt ctc aac aat 1344 Ser Arg Met Asn Gly Gln Tyr Gln Gln Pro Thr Asp Ser Leu Asn Asn         435 440 445    gac aac aag tat tcc ctg ttt gct gtt gtt aac cat caa ggg acc ttg 1392 Asp Asn Lys Tyr Ser Leu Phe Ala Val Val Asn His Gln Gly Thr Leu     450 455 460    gag agt ggc cac tac acc agc ttt atc cgg cag cac aaa gac cag tgg 1440 Glu Ser Gly His Tyr Thr Ser Phe Ile Arg Gln His Lys Asp Gln Trp 465 470 475 480    ttc aag tgt gac gat gcc atc atc acc aag gcc agc atc aag gac gtc 1488 Phe Lys Cys Asp Asp Ala Ile Ile Thr Lys Ala Ser Ile Lys Asp Val                 485 490 495    ctg gac agc gaa ggg tac ttg ctg ttc tat cac aaa cag ttc ctg gaa 1536 Leu Asp Ser Glu Gly Tyr Leu Leu Phe Tyr His Lys Gln Phe Leu Glu             500 505 510    tac gag tag 1545 Tyr Glu         515       <210> 13 <211> 803 <212> PRT <213> Homo sapiens    <400> 13 Met Lys Arg Ala Ala Met Ala Leu His Ser Pro Gln Tyr Ile Phe Gly   1 5 10 15 Asp Phe Ser Pro Asp Glu Phe Asn Gln Phe Phe Val Thr Pro Arg Ser              20 25 30 Ser Val Glu Leu Pro Pro Tyr Ser Gly Thr Val Leu Cys Gly Thr Gln          35 40 45 Ala Val Asp Lys Leu Pro Asp Gly Gln Glu Tyr Gln Arg Ile Glu Phe      50 55 60 Gly Val Asp Glu Val Ile Glu Pro Ser Asp Thr Leu Pro Arg Thr Pro  65 70 75 80 Ser Tyr Ser Ile Ser Ser Thr Leu Asn Pro Gln Ala Pro Glu Phe Ile                  85 90 95 Leu Gly Cys Thr Ala Ser Lys Ile Thr Pro Asp Gly Ile Thr Lys Glu             100 105 110 Ala Ser Tyr Gly Ser Ile Asp Cys Gln Tyr Pro Gly Ser Ala Leu Ala         115 120 125 Leu Asp Gly Ser Ser Asn Val Glu Ala Glu Val Leu Glu Asn Asp Gly     130 135 140 Val Ser Gly Gly Leu Gly Gln Arg Glu Arg Lys Lys Lys Lys Lys Arg 145 150 155 160 Pro Pro Gly Tyr Tyr Ser Tyr Leu Lys Asp Gly Gly Asp Asp Ser Ile                 165 170 175 Ser Thr Glu Ala Leu Val Asn Gly His Ala Asn Ser Ala Val Pro Asn             180 185 190 Ser Val Ser Ala Glu Asp Ala Glu Phe Met Gly Asp Met Pro Pro Ser         195 200 205 Val Thr Pro Arg Thr Cys Asn Ser Pro Gln Asn Ser Thr Asp Ser Val     210 215 220 Ser Asp Ile Val Pro Asp Ser Pro Phe Pro Gly Ala Leu Gly Ser Asp 225 230 235 240 Thr Arg Thr Ala Gly Gln Pro Glu Gly Gly Pro Gly Ala Asp Phe Gly                 245 250 255 Gln Ser Cys Phe Pro Ala Glu Ala Gly Arg Asp Thr Leu Ser Arg Thr             260 265 270 Ala Gly Ala Gln Pro Cys Val Gly Thr Asp Thr Thr Glu Asn Leu Gly         275 280 285 Val Ala Asn Gly Gln Ile Leu Glu Ser Ser Gly Glu Gly Thr Ala Thr     290 295 300 Asn Gly Val Glu Leu His Thr Thr Glu Ser Ile Asp Leu Asp Pro Thr 305 310 315 320 Lys Pro Glu Ser Ala Ser Pro Pro Ala Asp Gly Thr Gly Ser Ala Ser                 325 330 335 Gly Thr Leu Pro Val Ser Gln Pro Lys Ser Trp Ala Ser Leu Phe His             340 345 350 Asp Ser Lys Pro Ser Ser Ser Ser Pro Val Ala Tyr Val Glu Thr Lys         355 360 365 Tyr Ser Pro Pro Ala Ile Ser Pro Leu Val Ser Glu Lys Gln Val Glu     370 375 380 Val Lys Glu Gly Leu Val Pro Val Ser Glu Asp Pro Val Ala Ile Lys 385 390 395 400 Ile Ala Glu Leu Leu Glu Asn Val Thr Leu Ile His Lys Pro Val Ser                 405 410 415 Leu Gln Pro Arg Gly Leu Ile Asn Lys Gly Asn Trp Cys Tyr Ile Asn             420 425 430 Ala Thr Leu Gln Ala Leu Val Ala Cys Pro Pro Met Tyr His Leu Met         435 440 445 Lys Phe Ile Pro Leu Tyr Ser Lys Val Gln Arg Pro Cys Thr Ser Thr     450 455 460 Pro Met Ile Asp Ser Phe Val Arg Leu Met Asn Glu Phe Thr Asn Met 465 470 475 480 Pro Val Pro Pro Lys Pro Arg Gln Ala Leu Gly Asp Lys Ile Val Arg                 485 490 495 Asp Ile Arg Pro Gly Ala Ala Phe Glu Pro Thr Tyr Ile Tyr Arg Leu             500 505 510 Leu Thr Val Asn Lys Ser Ser Leu Ser Glu Lys Gly Arg Gln Glu Asp         515 520 525 Ala Glu Glu Tyr Leu Gly Phe Ile Leu Asn Gly Leu His Glu Glu Met     530 535 540 Leu Asn Leu Lys Lys Leu Leu Leu Ser Pro Ser Asn Glu Lys Leu Thr Ile 545 550 555 560 Ser Asn Gly Pro Lys Asn His Ser Val Asn Glu Glu Glu Gln Glu Glu                 565 570 575 Gln Gly Glu Gly Ser Glu Asp Glu Trp Glu Gln Val Gly Pro Arg Asn             580 585 590 Lys Thr Ser Val Thr Arg Gln Ala Asp Phe Val Gln Thr Pro Ile Thr         595 600 605 Gly Ile Phe Gly Gly His Ile Arg Ser Val Val Tyr Gln Gln Ser Ser     610 615 620 Lys Glu Ser Ala Thr Leu Gln Pro Phe Phe Thr Leu Gln Leu Asp Ile 625 630 635 640 Gln Ser Asp Lys Ile Arg Thr Val Gln Asp Ala Leu Glu Ser Leu Val                 645 650 655 Ala Arg Glu Ser Val Gln Gly Tyr Thr Thr Lys Thr Lys Gln Glu Val             660 665 670 Glu Ile Ser Arg Arg Val Thr Leu Glu Lys Leu Pro Pro Val Leu Val         675 680 685 Leu His Leu Lys Arg Phe Val Tyr Glu Lys Thr Gly Gly Cys Gln Lys     690 695 700 Leu Ile Lys Asn Ile Glu Tyr Pro Val Asp Leu Glu Ile Ser Lys Glu 705 710 715 720 Leu Leu Ser Pro Gly Val Lys Asn Lys Asn Phe Lys Cys His Arg Thr                 725 730 735 Tyr Arg Leu Phe Ala Val Val Tyr His His Gly Asn Ser Ala Thr Gly             740 745 750 Gly His Tyr Thr Thr Asp Val Phe Gln Ile Gly Leu Asn Gly Trp Leu         755 760 765 Arg Ile Asp Asp Gln Thr Val Lys Val Ile Asn Gln Tyr Gln Val Val     770 775 780 Lys Pro Thr Ala Glu Arg Thr Ala Tyr Leu Leu Tyr Tyr Arg Arg Val 785 790 795 800 Asp Leu Leu    <210> 14 <211> 3280 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (32) .. (2443)    <400> 14 gcgtgagcag ccggaggatc gcggagtccc a atg aaa cgg gca gcc atg gcc 52                                    Met Lys Arg Ala Ala Met Ala                                      1 5    ctc cac agc ccg cag tat att ttt gga gat ttt agc cct gat gaa ttc 100 Leu His Ser Pro Gln Tyr Ile Phe Gly Asp Phe Ser Pro Asp Glu Phe          10 15 20    aat caa ttc ttt gtg act cct cga tct tca gtt gag ctt cct cca tac 148 Asn Gln Phe Phe Val Thr Pro Arg Ser Ser Val Glu Leu Pro Pro Tyr      25 30 35    agt gga aca gtt ctg tgt ggc aca cag gct gtg gat aaa cta cct gat 196 Ser Gly Thr Val Leu Cys Gly Thr Gln Ala Val Asp Lys Leu Pro Asp  40 45 50 55    gga caa gaa tat cag aga att gag ttt ggt gtc gat gaa gtc att gaa 244 Gly Gln Glu Tyr Gln Arg Ile Glu Phe Gly Val Asp Glu Val Ile Glu                  60 65 70    ccc agt gac act ttg ccg aga acc ccc agc tac agt att tca agc aca 292 Pro Ser Asp Thr Leu Pro Arg Thr Pro Ser Tyr Ser Ile Ser Ser Thr              75 80 85    ctg aac cct cag gcc cct gaa ttt att ctc ggt tgt aca gct tcc aaa 340 Leu Asn Pro Gln Ala Pro Glu Phe Ile Leu Gly Cys Thr Ala Ser Lys          90 95 100    ata acc cct gat ggt atc act aaa gaa gca agc tat ggc tcc atc gac 388 Ile Thr Pro Asp Gly Ile Thr Lys Glu Ala Ser Tyr Gly Ser Ile Asp     105 110 115    tgc cag tac cca ggc tct gcc ctc gct ttg gat gga agt tct aat gtg 436 Cys Gln Tyr Pro Gly Ser Ala Leu Ala Leu Asp Gly Ser Ser Asn Val 120 125 130 135    gag gcg gaa gtt ttg gaa aat gat ggt gtc tca ggt ggt ctt gga caa 484 Glu Ala Glu Val Leu Glu Asn Asp Gly Val Ser Gly Gly Leu Gly Gln                 140 145 150    agg gag cgt aaa aag aag aaa aag cgg cca cct gga tat tac agc tat 532 Arg Glu Arg Lys Lys Lys Lys Lys Arg Pro Pro Gly Tyr Tyr Ser Tyr             155 160 165    ttg aaa gat ggt ggc gat gat agt atc tcc aca gaa gcc ctg gtc aat 580 Leu Lys Asp Gly Gly Asp Asp Ser Ile Ser Thr Glu Ala Leu Val Asn         170 175 180    ggc cat gcc aat tca gca gtc ccg aac agt gtc agt gca gag gat gca 628 Gly His Ala Asn Ser Ala Val Pro Asn Ser Val Ser Ala Glu Asp Ala     185 190 195    gaa ttt atg ggt gac atg ccc ccg tca gtt acg ccc agg act tgt aac 676 Glu Phe Met Gly Asp Met Pro Pro Ser Val Thr Pro Arg Thr Cys Asn 200 205 210 215    agc ccc cag aac tcc aca gac tct gtc agt gac att gtg cct gac agt 724 Ser Pro Gln Asn Ser Thr Asp Ser Val Ser Asp Ile Val Pro Asp Ser                 220 225 230    cct ttc ccc gga gca ctc ggc agt gac acc agg act gca ggg cag cca 772 Pro Phe Pro Gly Ala Leu Gly Ser Asp Thr Arg Thr Ala Gly Gln Pro             235 240 245    gag ggg ggc ccc ggg gct gat ttt ggt cag tcc tgc ttc cct gca gag 820 Glu Gly Gly Pro Gly Ala Asp Phe Gly Gln Ser Cys Phe Pro Ala Glu         250 255 260    gct ggc aga gac acc ctg tca agg aca gct ggg gct cag ccc tgc gtt 868 Ala Gly Arg Asp Thr Leu Ser Arg Thr Ala Gly Ala Gln Pro Cys Val     265 270 275    ggt acc gat act act gaa aac ctt gga gtt gct aat gga caa ata ctt 916 Gly Thr Asp Thr Thr Glu Asn Leu Gly Val Ala Asn Gly Gln Ile Leu 280 285 290 295    gaa tcc tcg ggt gag ggc aca gct acc aac ggg gtg gag ttg cac acc 964 Glu Ser Ser Gly Glu Gly Thr Ala Thr Asn Gly Val Glu Leu His Thr                 300 305 310    acg gaa agc ata gac ttg gac cca acc aaa ccc gag agt gca tca cct 1012 Thr Glu Ser Ile Asp Leu Asp Pro Thr Lys Pro Glu Ser Ala Ser Pro             315 320 325    cct gct gac ggc acg ggc tct gca tca ggc acc ctt cct gtc agc cag 1060 Pro Ala Asp Gly Thr Gly Ser Ala Ser Gly Thr Leu Pro Val Ser Gln         330 335 340    ccc aag tcc tgg gcc agc ctc ttt cat gat tct aag ccc tct tcc tcc 1108 Pro Lys Ser Trp Ala Ser Leu Phe His Asp Ser Lys Pro Ser Ser Ser     345 350 355    tcg ccg gtg gcc tat gtg gaa act aag tat tcc cct ccc gcc ata tct 1156 Ser Pro Val Ala Tyr Val Glu Thr Lys Tyr Ser Pro Pro Ala Ile Ser 360 365 370 375    ccc ctg gtt tct gaa aag cag gtt gaa gtc aaa gaa ggg ctt gtt ccg 1204 Pro Leu Val Ser Glu Lys Gln Val Glu Val Lys Glu Gly Leu Val Pro                 380 385 390    gtt tca gag gat cct gta gcc ata aag att gca gag ttg ctg gag aat 1252 Val Ser Glu Asp Pro Val Ala Ile Lys Ile Ala Glu Leu Leu Glu Asn             395 400 405    gta acc cta atc cat aaa cca gtg tcg ttg caa ccc cgt ggg ctg atc 1300 Val Thr Leu Ile His Lys Pro Val Ser Leu Gln Pro Arg Gly Leu Ile         410 415 420    aat aaa ggg aac tgg tgc tac att aat gct aca ctg cag gca ttg gtt 1348 Asn Lys Gly Asn Trp Cys Tyr Ile Asn Ala Thr Leu Gln Ala Leu Val     425 430 435    gct tgc ccg ccg atg tac cac ctg atg aag ttc att cct ctg tat tcc 1396 Ala Cys Pro Pro Met Tyr His Leu Met Lys Phe Ile Pro Leu Tyr Ser 440 445 450 455    aaa gtg caa agg cct tgt acg tca aca ccc atg ata gac agc ttt gtt 1444 Lys Val Gln Arg Pro Cys Thr Ser Thr Pro Met Ile Asp Ser Phe Val                 460 465 470    cgg cta atg aat gag ttc act aat atg cca gta cct cca aaa ccc cga 1492 Arg Leu Met Asn Glu Phe Thr Asn Met Pro Val Pro Pro Lys Pro Arg             475 480 485    caa gct ctt gga gat aaa atc gtg agg gat att cgc cct gga gct gcc 1540 Gln Ala Leu Gly Asp Lys Ile Val Arg Asp Ile Arg Pro Gly Ala Ala         490 495 500    ttt gag ccc aca tat att tac aga ctc ctg aca gtt aac aag tca agc 1588 Phe Glu Pro Thr Tyr Ile Tyr Arg Leu Leu Thr Val Asn Lys Ser Ser     505 510 515    ctg tct gaa aag ggt cga caa gaa gat gct gag gaa tac tta ggc ttc 1636 Leu Ser Glu Lys Gly Arg Gln Glu Asp Ala Glu Glu Tyr Leu Gly Phe 520 525 530 535    att cta aat gga ctt cat gag gaa atg ttg aac cta aag aag ctt ctc 1684 Ile Leu Asn Gly Leu His Glu Glu Met Leu Asn Leu Lys Lys Leu Leu                 540 545 550    tca cca agt aat gaa aaa ctt acg att tcc aac ggc ccc aaa aac cac 1732 Ser Pro Ser Asn Glu Lys Leu Thr Ile Ser Asn Gly Pro Lys Asn His             555 560 565    tcg gtc aat gaa gaa gag cag gaa gaa caa ggt gaa gga agc gag gat 1780 Ser Val Asn Glu Glu Glu Gln Glu Glu Gln Gly Glu Gly Ser Glu Asp         570 575 580    gaa tgg gaa caa gtg ggc ccc cgg aac aag act tcc gtc acc cgc cag 1828 Glu Trp Glu Gln Val Gly Pro Arg Asn Lys Thr Ser Val Thr Arg Gln     585 590 595    gcg gat ttt gtt cag act cca atc acc ggc att ttt ggt gga cac atc 1876 Ala Asp Phe Val Gln Thr Pro Ile Thr Gly Ile Phe Gly Gly His Ile 600 605 610 615    agg tct gtg gtt tac cag cag agt tca aaa gaa tct gcc act ttg cag 1924 Arg Ser Val Val Tyr Gln Gln Ser Ser Lys Glu Ser Ala Thr Leu Gln                 620 625 630    cca ttt ttc acg ttg cag ttg gat atc cag tca gac aag ata cgc aca 1972 Pro Phe Phe Thr Leu Gln Leu Asp Ile Gln Ser Asp Lys Ile Arg Thr             635 640 645    gtc cag gat gca ctg gag agc ttg gtg gca aga gaa tct gtc caa ggt 2020 Val Gln Asp Ala Leu Glu Ser Leu Val Ala Arg Glu Ser Val Gln Gly         650 655 660    tat acc aca aaa acc aaa caa gag gtt gag ata agt cga aga gtg act 2068 Tyr Thr Thr Lys Thr Lys Gln Glu Val Glu Ile Ser Arg Arg Val Thr     665 670 675    ctg gaa aaa ctc cct cct gtc ctc gtg ctg cac ctg aaa cga ttc gtt 2116 Leu Glu Lys Leu Pro Pro Val Leu Val Leu His Leu Lys Arg Phe Val 680 685 690 695    tat gag aag act ggt ggg tgc cag aag ctt atc aaa aat att gaa tat 2164 Tyr Glu Lys Thr Gly Gly Cys Gln Lys Leu Ile Lys Asn Ile Glu Tyr                 700 705 710    cct gtg gac ttg gaa att agt aaa gaa ctg ctt tct cca ggg gtt aaa 2212 Pro Val Asp Leu Glu Ile Ser Lys Glu Leu Leu Ser Pro Gly Val Lys             715 720 725    aat aag aat ttt aaa tgc cac cga acc tat cgg ctc ttt gca gtg gtc 2260 Asn Lys Asn Phe Lys Cys His Arg Thr Tyr Arg Leu Phe Ala Val Val         730 735 740    tac cat cac ggc aac agt gcg acg ggc ggc cat tac act aca gac gtc 2308 Tyr His His Gly Asn Ser Ala Thr Gly Gly His Tyr Thr Thr Asp Val     745 750 755    ttc cag atc ggt ctg aat ggc tgg ctg cgc atc gat gac cag aca gtc 2356 Phe Gln Ile Gly Leu Asn Gly Trp Leu Arg Ile Asp Asp Gln Thr Val 760 765 770 775    aag gtg atc aac cag tac cag gtg gtg aaa cca act gct gaa cgc aca 2404 Lys Val Ile Asn Gln Tyr Gln Val Val Lys Pro Thr Ala Glu Arg Thr                 780 785 790    gcc tac ctc ctg tat tac cgc cga gtg gac ctg ctg taa accctgtgtg 2453 Ala Tyr Leu Leu Tyr Tyr Arg Arg Val Asp Leu Leu             795 800    cgctgtgtgt gcgcccagtg cccgcttcgt aggacaccac ctcacactca cttcccgcct 2513    ctctttagtg gctctttaga gagaaactct ttctcccttt gcaaaaatgg gctagaatga 2573    aaaggagatg ccttggggtt cgtgcacaac acagcttctg ttgactctaa cttccaaatc 2633    aaaatcattt ggttgaaaca gactgttgct tgattttaga aaatacacaa aaacccatat 2693    ttctgaaata atgctgattc ctgagataag aaagtggatt tgatccccag tctcattgct 2753    tagtagaata aatcctgcac cagcaacaac acttgtaaat ttgtgaaaat gaattttatc 2813    tttccttaaa aaagaaattt tttaatccat cacacttttc ttccctaccc tttagttttt 2873    gataaatgat aaaaatgagc cagttatcaa agaagaacta gttcttactt caaaagaaaa 2933    ataaacataa aaaataagtt gctggttcct aacaggaaaa attttaataa ttgtactgag 2993    agaaactgct tacgtacaca ttgcagatca aatatttgga gttaaaatgt tagtctacat 3053    agatgggtga ttgtaacttt attgccatta aaagatttca aattgcattc atgcttctgt 3113    gtacacataa tgaaaaatgg gcaaataatg aagatctctc cttcagtctg ctctgtttaa 3173    ttctgctgtc tgctcttctc taatgctgcg tccctaattg tacacagttt agtgatatct 3233    aggagtataa agttgtcgcc catcaataaa aatcacaaag ttggttt 3280       <210> 15 <211> 1318 <212> PRT <213> Homo sapiens    <400> 15 Met Ser Gly Gly Ala Ser Ala Thr Gly Pro Arg Arg Gly Pro Pro Gly   1 5 10 15 Leu Glu Asp Thr Thr Ser Lys Lys Lys Gln Lys Asp Arg Ala Asn Gln              20 25 30 Glu Ser Lys Asp Gly Asp Pro Arg Lys Glu Thr Gly Ser Arg Tyr Val          35 40 45 Ala Gln Ala Gly Leu Glu Pro Leu Ala Ser Gly Asp Pro Ser Ala Ser      50 55 60 Ala Ser His Ala Ala Gly Ile Thr Gly Ser Arg His Arg Thr Arg Leu  65 70 75 80 Phe Phe Pro Ser Ser Ser Gly Ser Ala Ser Thr Pro Gln Glu Glu Gln                  85 90 95 Thr Lys Glu Gly Ala Cys Glu Asp Pro His Asp Leu Leu Ala Thr Pro             100 105 110 Thr Pro Glu Leu Leu Leu Asp Trp Arg Gln Ser Ala Glu Glu Val Ile         115 120 125 Val Lys Leu Arg Val Gly Val Gly Pro Leu Gln Leu Glu Asp Val Asp     130 135 140 Ala Ala Phe Thr Asp Thr Asp Cys Val Val Arg Phe Ala Gly Gly Gln 145 150 155 160 Gln Trp Gly Gly Val Phe Tyr Ala Glu Ile Lys Ser Ser Cys Ala Lys                 165 170 175 Val Gln Thr Arg Lys Gly Ser Leu Leu His Leu Thr Leu Pro Lys Lys             180 185 190 Val Pro Met Leu Thr Trp Pro Ser Leu Leu Val Glu Ala Asp Glu Gln         195 200 205 Leu Cys Ile Pro Pro Leu Asn Ser Gln Thr Cys Leu Leu Gly Ser Glu     210 215 220 Glu Asn Leu Ala Pro Leu Ala Gly Glu Lys Ala Val Pro Pro Gly Asn 225 230 235 240 Asp Pro Val Ser Pro Ala Met Val Arg Ser Arg Asn Pro Gly Lys Asp                 245 250 255 Asp Cys Ala Lys Glu Glu Met Ala Val Ala Ala Asp Ala Ala Thr Leu             260 265 270 Val Asp Glu Pro Glu Ser Met Val Asn Leu Ala Phe Val Lys Asn Asp         275 280 285 Ser Tyr Glu Lys Gly Pro Asp Ser Val Val Val His Val Tyr Val Lys     290 295 300 Glu Ile Cys Arg Asp Thr Ser Arg Val Leu Phe Arg Glu Gln Asp Phe 305 310 315 320 Thr Leu Ile Phe Gln Thr Arg Asp Gly Asn Phe Leu Arg Leu His Pro                 325 330 335 Gly Cys Gly Pro His Thr Thr Phe Arg Trp Gln Val Lys Leu Arg Asn             340 345 350 Leu Ile Glu Pro Glu Gln Cys Thr Phe Cys Phe Thr Ala Ser Arg Ile         355 360 365 Asp Ile Cys Leu Arg Lys Arg Gln Ser Gln Arg Trp Gly Gly Leu Glu     370 375 380 Ala Pro Ala Ala Arg Val Gly Gly Ala Lys Val Ala Val Pro Thr Gly 385 390 395 400 Pro Thr Pro Leu Asp Ser Thr Pro Pro Gly Gly Ala Pro His Pro Leu                 405 410 415 Thr Gly Gln Glu Glu Ala Arg Ala Val Glu Lys Asp Lys Ser Lys Ala             420 425 430 Arg Ser Glu Asp Thr Gly Leu Asp Ser Val Ala Thr Arg Thr Pro Pro         435 440 445 Pro Gly Gly Ala Pro His Pro Leu Thr Gly Gln Glu Glu Ala Arg Ala     450 455 460 Val Glu Met Val Pro Pro Met Pro His Ser Pro Val Ser Gly Asp Ser 465 470 475 480 Val Glu Glu Glu Glu Glu Glu Glu Lys Lys Val Cys Leu Pro Gly Phe                 485 490 495 Thr Gly Leu Val Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Val Ile             500 505 510 Gln Ser Leu Ser Asn Thr Arg Glu Leu Arg Asp Phe Phe His Asp Arg         515 520 525 Ser Phe Glu Ala Glu Ile Asn Tyr Asn Asn Pro Leu Gly Thr Gly Gly     530 535 540 Arg Leu Ala Ile Gly Phe Ala Val Leu Leu Arg Ala Leu Trp Lys Gly 545 550 555 560 Thr His His Ala Phe Gln Pro Ser Lys Leu Lys Ala Ile Val Ala Ser                 565 570 575 Lys Ala Ser Gln Phe Thr Gly Tyr Ala Gln His Asp Ala Gln Glu Phe             580 585 590 Met Ala Phe Leu Leu Asp Gly Leu His Glu Asp Leu Asn Arg Ile Gln         595 600 605 Asn Lys Pro Tyr Thr Glu Thr Val Asp Ser Asp Gly Arg Pro Asp Glu     610 615 620 Val Val Ala Glu Glu Ala Trp Gln Arg His Lys Met Arg Asn Asp Ser 625 630 635 640 Phe Ile Val Asp Leu Phe Gln Gly Gln Tyr Lys Ser Lys Leu Val Cys                 645 650 655 Pro Val Cys Ala Lys Val Ser Ile Thr Phe Asp Pro Phe Leu Tyr Leu             660 665 670 Pro Val Pro Leu Pro Gln Lys Gln Lys Val Leu Pro Val Phe Tyr Phe         675 680 685 Ala Arg Glu Pro His Ser Lys Pro Ile Lys Phe Leu Val Ser Val Ser     690 695 700 Lys Glu Asn Ser Thr Ala Ser Glu Val Leu Asp Ser Leu Ser Gln Ser 705 710 715 720 Val His Val Lys Pro Glu Asn Leu Arg Leu Ala Glu Val Ile Lys Asn                 725 730 735 Arg Phe His Arg Val Phe Leu Pro Ser His Ser Leu Asp Thr Val Ser             740 745 750 Pro Ser Asp Thr Leu Leu Cys Phe Glu Leu Leu Ser Ser Glu Leu Ala         755 760 765 Lys Glu Arg Val Val Val Leu Glu Val Gln Gln Arg Pro Gln Val Pro     770 775 780 Ser Val Pro Ile Ser Lys Cys Ala Ala Cys Gln Arg Lys Gln Gln Ser 785 790 795 800 Glu Asp Glu Lys Leu Lys Arg Cys Thr Arg Cys Tyr Arg Val Gly Tyr                 805 810 815 Cys Asn Gln Leu Cys Gln Lys Thr His Trp Pro Asp His Lys Gly Leu             820 825 830 Cys Arg Pro Glu Asn Ile Gly Tyr Pro Phe Leu Val Ser Val Pro Ala         835 840 845 Ser Arg Leu Thr Tyr Ala Arg Leu Ala Gln Leu Leu Glu Gly Tyr Ala     850 855 860 Arg Tyr Ser Val Ser Val Phe Gln Pro Pro Phe Gln Pro Gly Arg Met 865 870 875 880 Ala Leu Glu Ser Gln Ser Pro Gly Cys Thr Thr Leu Leu Ser Thr Gly                 885 890 895 Ser Leu Glu Ala Gly Asp Ser Glu Arg Asp Pro Ile Gln Pro Pro Glu             900 905 910 Leu Gln Leu Val Thr Pro Met Ala Glu Gly Asp Thr Gly Leu Pro Arg         915 920 925 Val Trp Ala Ala Pro Asp Arg Gly Pro Val Pro Ser Thr Ser Gly Ile     930 935 940 Ser Ser Glu Met Leu Ala Ser Gly Pro Ile Glu Val Gly Ser Leu Pro 945 950 955 960 Ala Gly Glu Arg Val Ser Arg Pro Glu Ala Ala Val Pro Gly Tyr Gln                 965 970 975 His Pro Ser Glu Ala Met Asn Ala His Thr Pro Gln Phe Phe Ile Tyr             980 985 990 Lys Ile Asp Ser Ser Asn Arg Glu Gln Arg Leu Glu Asp Lys Gly Asp         995 1000 1005 Thr Pro Leu Glu Leu Gly Asp Asp Cys Ser Leu Ala Leu Val Trp Arg    1010 1015 1020 Asn Asn Glu Arg Leu Gln Glu Phe Val Leu Val Ala Ser Lys Glu Leu 1025 1030 1035 1040 Glu Cys Ala Glu Asp Pro Gly Ser Ala Gly Glu Ala Ala Arg Ala Gly                1045 1050 1055 His Phe Thr Leu Asp Gln Cys Leu Asn Leu Phe Thr Arg Pro Glu Val            1060 1065 1070 Leu Ala Pro Glu Glu Ala Trp Tyr Cys Pro Gln Cys Lys Gln His Arg        1075 1080 1085 Glu Ala Ser Lys Gln Leu Leu Leu Trp Arg Leu Pro Asn Val Leu Ile    1090 1095 1100 Val Gln Leu Lys Arg Phe Ser Phe Arg Ser Phe Ile Trp Arg Asp Lys 1105 1110 1115 1120 Ile Asn Asp Leu Val Glu Phe Pro Val Arg Asn Leu Asp Leu Ser Lys                1125 1130 1135 Phe Cys Ile Gly Gln Lys Glu Glu Gln Leu Pro Ser Tyr Asp Leu Tyr            1140 1145 1150 Ala Val Ile Asn His Tyr Gly Gly Met Ile Gly Gly His Tyr Thr Ala        1155 1160 1165 Cys Ala Arg Leu Pro Asn Asp Arg Ser Ser Gln Arg Ser Asp Val Gly    1170 1175 1180 Trp Arg Leu Phe Asp Asp Ser Thr Val Thr Thr Val Asp Glu Ser Gln 1185 1190 1195 1200 Val Val Thr Arg Tyr Ala Tyr Val Leu Phe Tyr Arg Arg Arg Asn Ser                1205 1210 1215 Pro Val Glu Arg Pro Pro Arg Ala Gly His Ser Glu His His Pro Asp            1220 1225 1230 Leu Gly Pro Ala Ala Glu Ala Ala Ala Ser Gln Ala Ser Arg Ile Trp        1235 1240 1245 Gln Glu Leu Glu Ala Glu Glu Glu Pro Val Pro Glu Gly Ser Gly Pro    1250 1255 1260 Leu Gly Pro Trp Gly Pro Gln Asp Trp Val Gly Pro Leu Pro Arg Gly 1265 1270 1275 1280 Pro Thr Thr Pro Asp Glu Gly Cys Leu Arg Tyr Phe Val Leu Gly Thr                1285 1290 1295 Val Ala Ala Leu Val Ala Leu Val Leu Asn Val Phe Tyr Pro Leu Val            1300 1305 1310 Ser Gln Ser Arg Trp Arg        1315    <210> 16 <211> 4401 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (162) .. (4118)    <400> 16 gggctggctg cggcggtctc gctcggctgt ccgttccttg ctggagaatt tggccacaaa 60    gagctgccaa gatagctggg ccaggaagaa agcgccgcag ccctgaccca gacgctgttg 120    ccgaccccgg ggcactctgg ctgtcgacca agcggctcaa g atg tct ggc ggg gcc 176                                               Met Ser Gly Gly Ala                                                 1 5    agt gcc aca ggc cca agg aga ggg ccc cca gga ctg gag gac acc act 224 Ser Ala Thr Gly Pro Arg Arg Gly Pro Pro Gly Leu Glu Asp Thr Thr                  10 15 20    agt aag aag aag cag aag gat cga gca aac cag gag agc aag gat gga 272 Ser Lys Lys Lys Gln Lys Asp Arg Ala Asn Gln Glu Ser Lys Asp Gly              25 30 35    gat cct agg aaa gag aca ggg tct cga tat gtt gcc cag gct ggt ctt 320 Asp Pro Arg Lys Glu Thr Gly Ser Arg Tyr Val Ala Gln Ala Gly Leu          40 45 50    gaa cct ctg gcc tca ggt gat cct tct gcc tca gcc tcc cat gca gct 368 Glu Pro Leu Ala Ser Gly Asp Pro Ser Ala Ser Ala Ser His Ala Ala      55 60 65    ggg atc aca ggc tca cgc cac cgt acc cgg ctg ttc ttt cct tca tcg 416 Gly Ile Thr Gly Ser Arg His Arg Thr Arg Leu Phe Phe Pro Ser Ser  70 75 80 85    tca ggg tca gca tcc act cct caa gag gag cag acc aaa gag gga gct 464 Ser Gly Ser Ala Ser Thr Pro Gln Glu Glu Gln Thr Lys Glu Gly Ala                  90 95 100    tgt gaa gac cct cat gat ctc ttg gct act ccc act cca gag ttg ttg 512 Cys Glu Asp Pro His Asp Leu Leu Ala Thr Pro Thr Pro Glu Leu Leu             105 110 115    ctc gat tgg agg cag agt gca gaa gag gtg att gtc aag ctt cgt gtg 560 Leu Asp Trp Arg Gln Ser Ala Glu Glu Val Ile Val Lys Leu Arg Val         120 125 130    gga gta ggt ccc ctg cag ctg gag gat gta gat gct gct ttc aca gat 608 Gly Val Gly Pro Leu Gln Leu Glu Asp Val Asp Ala Ala Phe Thr Asp     135 140 145    aca gac tgt gtg gtg cgg ttt gca ggt ggt cag cag tgg ggt ggt gtc 656 Thr Asp Cys Val Val Arg Phe Ala Gly Gly Gln Gln Trp Gly Gly Val 150 155 160 165    ttc tat gct gag ata aaa agc tct tgt gct aaa gtg caa acc cgc aag 704 Phe Tyr Ala Glu Ile Lys Ser Ser Cys Ala Lys Val Gln Thr Arg Lys                 170 175 180    ggc agt ctc ctg cac ctg aca ctg ccc aaa aag gtg cct atg ctc acg 752 Gly Ser Leu Leu His Leu Thr Leu Pro Lys Lys Val Pro Met Leu Thr             185 190 195    tgg ccc tcc ctc ctg gtt gag gct gat gaa cag ctt tgc ata cca ccg 800 Trp Pro Ser Leu Leu Val Glu Ala Asp Glu Gln Leu Cys Ile Pro Pro         200 205 210    ctg aac tcc caa acc tgc ctc ctg ggc tca gag gag aat tta gcc cct 848 Leu Asn Ser Gln Thr Cys Leu Leu Gly Ser Glu Glu Asn Leu Ala Pro     215 220 225    ttg gca gga gag aaa gca gtg cct ccc ggg aat gac cca gtc tct cca 896 Leu Ala Gly Glu Lys Ala Val Pro Pro Gly Asn Asp Pro Val Ser Pro 230 235 240 245    gcc atg gtc cgg agc aga aac cct ggg aaa gat gac tgt gcc aag gag 944 Ala Met Val Arg Ser Arg Asn Pro Gly Lys Asp Asp Cys Ala Lys Glu                 250 255 260    gag atg gca gtg gca gca gat gct gca acc ttg gtg gat gag ccc gag 992 Glu Met Ala Val Ala Ala Asp Ala Ala Thr Leu Val Asp Glu Pro Glu             265 270 275    tcg atg gtg aac ctg gcg ttt gtc aag aat gac tcg tat gag aag ggc 1040 Ser Met Val Asn Leu Ala Phe Val Lys Asn Asp Ser Tyr Glu Lys Gly         280 285 290    ccg gat tca gtg gtg gtg cac gtg tac gtg aag gag atc tgc agg gac 1088 Pro Asp Ser Val Val Val His Val Tyr Val Lys Glu Ile Cys Arg Asp     295 300 305    acc tca aga gta ctt ttc cgt gag cag gac ttc acg ctc atc ttc cag 1136 Thr Ser Arg Val Leu Phe Arg Glu Gln Asp Phe Thr Leu Ile Phe Gln 310 315 320 325    acc agg gat gga aac ttc ctg agg ctg cac ccg ggc tgt ggg ccc cac 1184 Thr Arg Asp Gly Asn Phe Leu Arg Leu His Pro Gly Cys Gly Pro His                 330 335 340    acc acc ttc cgt tgg cag gtg aag ctc agg aat ctg att gag cca gag 1232 Thr Thr Phe Arg Trp Gln Val Lys Leu Arg Asn Leu Ile Glu Pro Glu             345 350 355    cag tgc acc ttc tgt ttc acg gct tct cgc atc gac atc tgc ctt cgt 1280 Gln Cys Thr Phe Cys Phe Thr Ala Ser Arg Ile Asp Ile Cys Leu Arg         360 365 370    aag agg cag agt cag cgc tgg ggg ggc ctg gag gcc ccg gct gca cga 1328 Lys Arg Gln Ser Gln Arg Trp Gly Gly Leu Glu Ala Pro Ala Ala Arg     375 380 385    gtg ggt ggt gca aag gtt gcc gtg ccg aca ggt cca acc cct ctg gat 1376 Val Gly Gly Ala Lys Val Ala Val Pro Thr Gly Pro Thr Pro Leu Asp 390 395 400 405    tca acc cca cca gga ggt gct ccc cac ccc ctg aca ggc cag gag gag 1424 Ser Thr Pro Pro Gly Gly Ala Pro His Pro Leu Thr Gly Gln Glu Glu                 410 415 420    gcc cgg gct gtg gag aag gat aaa tcc aag gca cga tct gag gac aca 1472 Ala Arg Ala Val Glu Lys Asp Lys Ser Lys Ala Arg Ser Glu Asp Thr             425 430 435    ggg cta gac agt gtg gca acc cgc aca ccc cca cca gga ggt gct ccc 1520 Gly Leu Asp Ser Val Ala Thr Arg Thr Pro Pro Pro Gly Gly Ala Pro         440 445 450    cac ccc ctg aca ggc cag gag gag gcc cgg gct gtg gag atg gtg cct 1568 His Pro Leu Thr Gly Gln Glu Glu Ala Arg Ala Val Glu Met Val Pro     455 460 465    ccc atg ccc cac agc cca gtt agt gga gac agc gtg gag gag gag gaa 1616 Pro Met Pro His Ser Pro Val Ser Gly Asp Ser Val Glu Glu Glu Glu 470 475 480 485    gag gaa gag aag aag gtg tgt ctg cca ggc ttc act ggc ctt gtc aat 1664 Glu Glu Glu Lys Lys Val Cys Leu Pro Gly Phe Thr Gly Leu Val Asn                 490 495 500    tta ggc aac acc tgc ttc atg aac agc gtc att cag tct ctg tcc aac 1712 Leu Gly Asn Thr Cys Phe Met Asn Ser Val Ile Gln Ser Leu Ser Asn             505 510 515    act cgg gaa ctc cgg gac ttc ttc cat gac cgc tcc ttt gag gct gag 1760 Thr Arg Glu Leu Arg Asp Phe Phe His Asp Arg Ser Phe Glu Ala Glu         520 525 530    atc aac tac aac aac cca cta ggg act ggt ggg cgt ctg gcc att ggc 1808 Ile Asn Tyr Asn Asn Pro Leu Gly Thr Gly Gly Arg Leu Ala Ile Gly     535 540 545    ttt gcc gtg ctg ctt cgg gcg ctg tgg aag ggc acc cac cat gcc ttc 1856 Phe Ala Val Leu Leu Arg Ala Leu Trp Lys Gly Thr His His Ala Phe 550 555 560 565    cag cct tcc aag ttg aag gcc att gtg gcg agt aag gcc agc cag ttc 1904 Gln Pro Ser Lys Leu Lys Ala Ile Val Ala Ser Lys Ala Ser Gln Phe                 570 575 580    aca ggc tat gca cag cat gat gcc cag gag ttc atg gct ttc ctg ctg 1952 Thr Gly Tyr Ala Gln His Asp Ala Gln Glu Phe Met Ala Phe Leu Leu             585 590 595    gat ggg ctg cac gag gac ctg aat cgc att cag aac aag ccc tac aca 2000 Asp Gly Leu His Glu Asp Leu Asn Arg Ile Gln Asn Lys Pro Tyr Thr         600 605 610    gag acc gtg gat tca gat ggg cgg ccc gat gag gtg gta gct gag gaa 2048 Glu Thr Val Asp Ser Asp Gly Arg Pro Asp Glu Val Val Ala Glu Glu     615 620 625    gca tgg cag cgg cac aag atg agg aat gac tct ttc atc gtg gac cta 2096 Ala Trp Gln Arg His Lys Met Arg Asn Asp Ser Phe Ile Val Asp Leu 630 635 640 645    ttt cag ggg cag tac aag tcg aag ctg gtg tgc cct gtg tgt gcc aag 2144 Phe Gln Gly Gln Tyr Lys Ser Lys Leu Val Cys Pro Val Cys Ala Lys                 650 655 660    gtc tcc atc act ttt gac ccg ttt ctt tat ctg ccg gtg ccc ttg cca 2192 Val Ser Ile Thr Phe Asp Pro Phe Leu Tyr Leu Pro Val Pro Leu Pro             665 670 675    caa aag caa aag gtt ctc cct gtc ttt tat ttt gcc cga gag ccc cac 2240 Gln Lys Gln Lys Val Leu Pro Val Phe Tyr Phe Ala Arg Glu Pro His         680 685 690    agc aag ccc atc aag ttc ctg gtg agc gtc agc aag gag aac tcc act 2288 Ser Lys Pro Ile Lys Phe Leu Val Ser Val Ser Lys Glu Asn Ser Thr     695 700 705    gcg agc gaa gta ttg gac tcc ctc tct cag agt gtt cat gtg aag cct 2336 Ala Ser Glu Val Leu Asp Ser Leu Ser Gln Ser Val His Val Lys Pro 710 715 720 725    gag aac ctg cgt ttg gcg gag gta att aag aat cgt ttt cat cgt gtg 2384 Glu Asn Leu Arg Leu Ala Glu Val Ile Lys Asn Arg Phe His Arg Val                 730 735 740    ttc cta ccc tcc cac tca ctg gac act gtg tcc cca tct gat acg ctc 2432 Phe Leu Pro Ser His Ser Leu Asp Thr Val Ser Pro Ser Asp Thr Leu             745 750 755    ctc tgc ttt gag ctg cta tcc tca gag ttg gct aag gag cgg gta gtg 2480 Leu Cys Phe Glu Leu Leu Ser Ser Glu Leu Ala Lys Glu Arg Val Val         760 765 770    gtg cta gag gtg caa cag cgc ccc cag gtg ccc agc gtc ccc atc tcc 2528 Val Leu Glu Val Gln Gln Arg Pro Gln Val Pro Ser Val Pro Ile Ser     775 780 785    aag tgt gca gcc tgc cag cgg aag caa cag tcg gag gat gaa aag ctg 2576 Lys Cys Ala Ala Cys Gln Arg Lys Gln Gln Ser Glu Asp Glu Lys Leu 790 795 800 805    aag cgc tgt acc cgg tgc tac cgt gtg ggc tac tgc aac cag ctc tgc 2624 Lys Arg Cys Thr Arg Cys Tyr Arg Val Gly Tyr Cys Asn Gln Leu Cys                 810 815 820    cag aaa acc cac tgg cct gac cac aag ggc ctc tgc cga cct gag aac 2672 Gln Lys Thr His Trp Pro Asp His Lys Gly Leu Cys Arg Pro Glu Asn             825 830 835    att ggc tac ccc ttc ctg gtc agt gta cct gcc tca cgc ctc act tat 2720 Ile Gly Tyr Pro Phe Leu Val Ser Val Pro Ala Ser Arg Leu Thr Tyr         840 845 850    gcc cgc ctc gct cag ttg cta gag ggc tat gcc cgg tac tct gtg agt 2768 Ala Arg Leu Ala Gln Leu Leu Glu Gly Tyr Ala Arg Tyr Ser Val Ser     855 860 865    gta ttc cag cca ccc ttt cag cca ggc cgc atg gcc ttg gag tct cag 2816 Val Phe Gln Pro Pro Phe Gln Pro Gly Arg Met Ala Leu Glu Ser Gln 870 875 880 885    agc cct ggc tgc acc aca ctg ctc tcc aca ggt tcc ctg gag gct ggg 2864 Ser Pro Gly Cys Thr Thr Leu Leu Ser Thr Gly Ser Leu Glu Ala Gly                 890 895 900    gac agc gag aga gac ccc att cag cca cct gag ctc cag ctg gtg acc 2912 Asp Ser Glu Arg Asp Pro Ile Gln Pro Pro Glu Leu Gln Leu Val Thr             905 910 915    cct atg gct gag ggg gac aca ggg ctt ccc cgg gtg tgg gca gcc cct 2960 Pro Met Ala Glu Gly Asp Thr Gly Leu Pro Arg Val Trp Ala Ala Pro         920 925 930    gac cgg ggt cct gtg ccc agc acc agt gga att tct tct gag atg ctg 3008 Asp Arg Gly Pro Val Pro Ser Thr Ser Gly Ile Ser Ser Glu Met Leu     935 940 945    gcc agt ggg ccc att gag gtt ggc tcc ttg cca gct ggc gag agg gtg 3056 Ala Ser Gly Pro Ile Glu Val Gly Ser Leu Pro Ala Gly Glu Arg Val 950 955 960 965    tcc cga ccc gaa gct gct gtg cct ggg tac cag cat cca agt gaa gct 3104 Ser Arg Pro Glu Ala Ala Val Pro Gly Tyr Gln His Pro Ser Glu Ala                 970 975 980    atg aat gcc cac aca ccc cag ttc ttc atc tat aaa att gat tca tcc 3152 Met Asn Ala His Thr Pro Gln Phe Phe Ile Tyr Lys Ile Asp Ser Ser             985 990 995    aac cga gag cag cgg cta gag gac aaa gga gac acc cca ctg gag ctg 3200 Asn Arg Glu Gln Arg Leu Glu Asp Lys Gly Asp Thr Pro Leu Glu Leu        1000 1005 1010    ggt gac gac tgt agc ctg gct ctc gtc tgg cgg aac aat gag cgc ttg 3248 Gly Asp Asp Cys Ser Leu Ala Leu Val Trp Arg Asn Asn Glu Arg Leu    1015 1020 1025    cag gag ttt gtg ttg gta gcc tcc aag gag ctg gaa tgt gct gag gat 3296 Gln Glu Phe Val Leu Val Ala Ser Lys Glu Leu Glu Cys Ala Glu Asp 1030 1035 1040 1045    cca ggc tct gcc ggt gag gct gcc cgg gcc ggc cac ttc acc ctg gac 3344 Pro Gly Ser Ala Gly Glu Ala Ala Arg Ala Gly His Phe Thr Leu Asp                1050 1055 1060    cag tgc ctc aac ctc ttc aca cgg cct gag gtg ctg gca ccc gag gag 3392 Gln Cys Leu Asn Leu Phe Thr Arg Pro Glu Val Leu Ala Pro Glu Glu            1065 1070 1075    gcc tgg tac tgc cca cag tgc aaa cag cac cgt gag gcc tcc aag cag 3440 Ala Trp Tyr Cys Pro Gln Cys Lys Gln His Arg Glu Ala Ser Lys Gln        1080 1085 1090    ctg ttg cta tgg cgc ctg cca aat gtt ctc atc gtg cag ctc aag cgc 3488 Leu Leu Leu Trp Arg Leu Pro Asn Val Leu Ile Val Gln Leu Lys Arg    1095 1100 1105    ttc tcc ttt cgt agt ttt atc tgg cgt gac aag atc aat gac ttg gtg 3536 Phe Ser Phe Arg Ser Phe Ile Trp Arg Asp Lys Ile Asn Asp Leu Val 1110 1115 1120 1125    gag ttc cct gtt agg aac ctg gac ctg agc aag ttc tgc att ggt cag 3584 Glu Phe Pro Val Arg Asn Leu Asp Leu Ser Lys Phe Cys Ile Gly Gln                1130 1135 1140    aaa gag gag cag ctg ccc agc tac gat cta tat gct gtc atc aac cac 3632 Lys Glu Glu Gln Leu Pro Ser Tyr Asp Leu Tyr Ala Val Ile Asn His            1145 1150 1155    tat gga ggc atg att ggt ggc cac tac act gcc tgt gca cgc ctg ccc 3680 Tyr Gly Gly Met Ile Gly Gly His Tyr Thr Ala Cys Ala Arg Leu Pro        1160 1165 1170    aat gat cgt agc agt cag cgc agt gac gtg ggc tgg cgc ttg ttt gat 3728 Asn Asp Arg Ser Ser Gln Arg Ser Asp Val Gly Trp Arg Leu Phe Asp    1175 1180 1185    gac agc aca gtg aca acg gta gac gag agc cag gtt gtg acg cgt tat 3776 Asp Ser Thr Val Thr Thr Val Asp Glu Ser Gln Val Val Thr Arg Tyr 1190 1195 1200 1205    gcc tat gta ctc ttc tac cgc cgg cgg aac tct cct gtg gag agg ccc 3824 Ala Tyr Val Leu Phe Tyr Arg Arg Arg Asn Ser Pro Val Glu Arg Pro                1210 1215 1220    ccc agg gca ggt cac tct gag cac cac cca gac cta ggc cct gca gct 3872 Pro Arg Ala Gly His Ser Glu His His Pro Asp Leu Gly Pro Ala Ala            1225 1230 1235    gag gct gct gcc agc cag gct tcc cgg att tgg cag gag ctg gag gct 3920 Glu Ala Ala Ala Ser Gln Ala Ser Arg Ile Trp Gln Glu Leu Glu Ala        1240 1245 1250    gag gag gag ccg gtg cct gag ggg tct ggg ccc ctg ggt ccc tgg ggg 3968 Glu Glu Glu Pro Val Pro Glu Gly Ser Gly Pro Leu Gly Pro Trp Gly    1255 1260 1265    ccc caa gac tgg gtg ggc ccc cta cca cgt ggc cct acc aca cca gat 4016 Pro Gln Asp Trp Val Gly Pro Leu Pro Arg Gly Pro Thr Thr Pro Asp 1270 1275 1280 1285    gag ggc tgc ctc cgg tac ttt gtc ctg ggc acc gtg gcg gct ttg gtg 4064 Glu Gly Cys Leu Arg Tyr Phe Val Leu Gly Thr Val Ala Ala Leu Val                1290 1295 1300    gcc ctc gtg ctc aac gtg ttc tat cct ctg gta tcc cag agt cgc tgg 4112 Ala Leu Val Leu Asn Val Phe Tyr Pro Leu Val Ser Gln Ser Arg Trp            1305 1310 1315    aga tga gct cgcctgcagg cagctgctgt gagctggcct acctgcctgc 4161 Arg                                                         cccaggccat gcctgccttt gttgtgggga acacctctgg gctttgggcc tcagcttatg 4221    catctggtgg gagagggtgg ggaggttgtg gcccctgcag gggcagagta tcctagggtg 4281    tgtatccatc tggctgtctg tccattcatc ctgctgctct gacccttggc ctcaggcttg 4341    gccctgccca agctacttcc tgtacttaaa agtgttaata aaaccagact attcaggccc 4401 <210> 17 <211> 16 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <222> (1) .. (16) <223> Cys box consisting in the polypeptide of SEQ ID No: 1 <400> 17 Gly Leu Lys Asn Ile Gly Asn Thr Cys Tyr Met Asn Ala Ala Leu Gln   1 5 10 15 <210> 18 <211> 18 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <222> (1) .. (18) <223> His box composed in the polypeptide of SEQ ID No: 1 <400> 18 Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Ser Ser Gly   1 5 10 15 His Tyr <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 19 cctaacaaaa tgtcagcttt tcg 23 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 20 ggtttgcgga tcttcttcta c 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 21 gtagaagaag atccgcaacc 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 22 ttgaaaggtc tcgagggtta c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 23 gtaaccctcg agacctttca a 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 24 ccagcttttc ttggaggaac 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 25 gttcctccaa gaaaagctgg 20 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 26 cctcattaga actctctaca tcc 23 <210> 27 <211> 57 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <222> (1) .. (57) <223> Zinc finger domain consist in the polypeptide       of SEQ ID No: 1 <400> 27 Cys Gln Asp Cys Lys Val Gln Gly Pro Asn Leu Trp Ala Cys Leu Glu   1 5 10 15 Asn Arg Cys Ser Tyr Val Gly Cys Gly Glu Ser Gln Val Asp His Ser              20 25 30 Thr Ile His Ser Gln Glu Thr Lys His Tyr Leu Thr Val Asn Leu Thr          35 40 45 Thr Leu Arg Val Trp Cys Tyr Ala Cys      50 55 <210> 28 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 28 gggacaagtt tgtacaaaaa agcaggctac aaaatgtcag cttttcga 48 <210> 29 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 29 ggggaccact ttgtacaaga aagctgggtc tcattagaac tctctacatc c 51 <210> 30 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 30 ggaaatacta gttacatgaa tgcagctttg caggctc 37 <210> 31 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 31 gagcctgcaa agctgcattc atgtaactag tatttcc 37 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 32 aatatgggca ccaatgcctc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 33 ttactctctt gactgataga 20 <210> 34 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 34 gggacaagtt tgtacaaaaa agcaggctat atgggcacca atgcctctgc 50 <210> 35 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 35 ggggaccact ttgtacaaga aagctgggtg ttactctctt gactgataga 50 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a mutator <400> 36 tcaattttgg aaacacatcc tactgtaact ccgtgc 36 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a mutator <400> 37 gcacggagtt acagtaggat gtgtttccaa aattga 36 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 38 gcttcgtgtc acccagaggt g 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 39 cggcacattc tggtagatgg c 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 40 gcaccgtcgt acgtgctcaa g 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 41 tagcccagca gatcacacgg c 21 <210> 42 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 42 gggacaagtt tgtacaaaaa agcaggctta atgggggact ccagggacct t 51 <210> 43 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 43 ggggaccact ttgtacaaga aagctgggtc tagcccagca gatcacacgg c 51 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 44 cctcgggaac tccagctaca tgaacgccg 29 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 45 cggcgttcat gtagctggag ttcccgagg 29 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 46 atgattgact gggtgtcctg g 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 47 ggtgtcctct gtgatgcagt g 21 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 48 ttcacggacc tctctctcgc ct 22 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 49 tcaggttggc agcaggttca c 21 <210> 50 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 50 gggacaagtt tgtacaaaaa agcaggctta atgattgact gggtgtcctg g 51 <210> 51 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 51 ggggaccact ttgtacaaga aagctgggtc tcaggttggc agcaggttca c 51 <210> 52 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 52 caacctgggc aacacaagct atgtcaacag c 31 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 53 gctgttgaca tagcttgtgt tgcccaggtt g 31 <210> 54 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 54 gggacaagtt tgtacaaaaa agcaggctta atgccaatag tggataagtt g 51 <210> 55 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 55 ggggaccact ttgtacaaga aagctgggtc cagtcagcgg cgatagctga g 51 <210> 56 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 56 caaccttggc aacaccagct ttctcaatgc cac 33 <210> 57 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 57 gtggcattga gaaagctggt gttgccaagg ttg 33 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 58 gcggtgcctg ccttgcagcc tcc 23 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 59 cggctggcca ggctggccaa gg 22 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 60 gtcaggtgcc aagtctgcca t 21 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 61 acagtagatg cctccataca tcag 24 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 62 tgcgaaggcg aagcggcaca a 21 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 63 taaggctact cgtattccag g 21 <210> 64 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 64 ccttggccag cctggccagc cg 22 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 65 gagatccaag ctgatgtccc a 21 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 66 ctgggcagct tcaaggtgga ca 22 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 67 tgtccacctt gaagctgccc ag 22 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 68 taccagtgct tcgtgtggag cg 22 <210> 69 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 69 cgctccacaa gccgaactgg ta 22 <210> 70 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 70 gggacaagtt tgtacaaaaa agcaggctcc atggacgccg agctggaggt a 51 <210> 71 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 71 ggggaccact ttgtacaaga aagctgggtc ctactggtat tccaggaact g 51 <210> 72 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 72 gatcaacctt gggaacacag ccttcatgaa ctgcatcgtg c 41 <210> 73 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 73 gcacgatgca gttcatgaag gctgtgttcc caaggttgat c 41 <210> 74 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 74 gggacaagtt tgtacaaaaa agcaggctca atgaaacggg cagccatg 48 <210> 75 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 75 ggggaccact ttgtacaaga aagctgggtt ttacagcagg tccactcg 48 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 76 gggaactgga gctacattaa tgctacactg cag 33 <210> 77 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 77 ctgcagtgta gcattaatgt agctccagtt ccc 33 <210> 78 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 78 gggacaagtt tgtacaaaaa agcaggctta gaaggagata gaaccatgtc tggcggggcc 60 agtgccac 68 <210> 79 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 79 gggaccactt tgtacaagaa agctgggtcc tagtgatggt gatggtgatg tctccagcga 60 ctctggga 68 <210> 80 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 80 tttaggcaac acctccttca tgaacagcg 29 <210> 81 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       polynucleotide for using as a primer <400> 81 cgctgttcat gaaggaggtg ttgcctaaa 29

[Brief description of drawings]

FIG. 1 is an electrophoretogram for confirming the expression of KIAA1097 in Escherichia coli. As a result of detecting KIAA1097 by staining with Coomassie blue, B is anti-His-
It is the result of detection by immunoblotting using a tag antibody. In both figures, lane 1 is a molecular weight marker, lane 2 is a whole cell extract without NaCl induction, and lane 3 is Na.
The total cell extract upon Cl induction, lane 4 is the soluble cell extract upon NaCl induction, and lane 5 is the insoluble cell extract upon NaCl induction.

FIG. 2 is a diagram showing the results of examining the degradation activity of Ub-GST fusion protein for KIAA1097 in a co-expression system. A shows the results using Ub-M-GST as a substrate, lane 1 shows wild type KIAA1097, lane 2 shows mutant KIAA1097 ^C163S , lane 3 shows positive control KIAA0529, and lane 4 shows negative control luciferase. . B is wild type KIAA10
9 shows the results of examining the substrate specificity of 97, lane 6 is Ub-R-GST, lane 7 is Ub-P-GST,
Lane 8 shows Ub-I-GST. C is mutant KIA
A1097 ^C163S , positive control KIAA052
9 shows the results of examining the substrate specificity of luciferase, which is 9 or a negative control. Lane 10 shows Ub-R-
GST / mutant KIAA1097 ^C163S , lane 1
1 is Ub-R-GST / KIAA0529, lane 12
Is Ub-R-GST / luciferase, lane 13 is U
b-I-GST / mutant KIAA1097 ^C163S ,
Lane 14 is Ub-I-GST / KIAA0529, lane 15 is Ub-I-GST / luciferase, lane 16 is Ub-P-GST / KIAA0529, lane 1.
7 shows Ub-P-GST / luciferase. Also,
Lanes 5, 9, and 18 show the results when only Ub-M-GST was expressed.

FIG. 3 is an electrophoretogram for confirming the expression of AK024318 in Escherichia coli.

FIG. 4 Ub-M-GST for AK024318
It is a figure which shows the result of having investigated the degradation activity of a fusion protein in a co-expression system.

FIG. 5: Ub-R-GST for AK024318
It is a figure which shows the result of having investigated the degradation activity of a fusion protein in a co-expression system.

FIG. 6 Ub-I-GST for AK024318
It is a figure which shows the result of having investigated the degradation activity of a fusion protein in a co-expression system.

FIG. 7: Ub-P-GST for AK024318
It is a figure which shows the result of having investigated the degradation activity of a fusion protein in a co-expression system.

FIG. 8 is a diagram showing the results of examining the tissue expression of AK024318 by Northern blotting.

FIG. 9 is an electrophoretogram for confirming the expression of KIAA1003 in E. coli. Lane 1 is a molecular weight marker,
Lane 2 shows the cell extract when NaCl is not induced, and lane 3 shows the cell extract when NaCl is induced.

FIG. 10: KIAA1003 or mutant KIAA1
It is a figure which shows the result of having investigated the degradation activity of Ub-M-GST fusion protein about 003 ^C154S by a co-expression system.
USP15 is a positive control and luciferase is a negative control. A shows the result of degradation activity, and B shows the expression of each protein in the co-expression system.

FIG. 11: Ub-P-GS for KIAA1003
It is a figure which shows the result of having investigated the degradation activity of each fusion protein of T, Ub-R-GST, and Ub-I-GST by the co-expression system. USP15 is a positive control and luciferase is a negative control. A shows the result of degradation activity, and B shows the expression of each protein in the co-expression system.

FIG. 12: Ub for KIAA1003 ^C154S
It is a figure which shows the result of having compared the degradation activity of each fusion protein of -R-GST and Ub-I-GST with the wild-type KIAA1003 by the co-expression system. A is the result of degradation activity,
B shows the expression of each protein in the co-expression system.

FIG. 13 is an electrophoretogram for confirming the expression of KIAA1372 in Escherichia coli. Lane 1 shows the cell extract when NaCl is not induced, and lane 2 shows the cell extract when NaCl is induced.

FIG. 14: Ub-M-GS for KIAA1372
It is a figure which shows the result of having examined the degradation activity of T fusion protein in vitro. Lane 1 is KIAA1372 during non-induction
Lane 2 shows KIAA1372 at the time of induction. Lane 3 is positive control USP15, lane 4 is buffer only. A shows the result of degradation activity, and B shows the expression of each protein in the co-expression system.

FIG. 15: Ub-P-GS for KIAA1372
It is a figure which shows the result of having examined the degradation activity of each fusion protein of T, Ub-I-GST, and Ub-R-GST in vitro. Lanes 1, 4, and 7 are KIAA1372, lanes 2, 5, and 8 are positive controls USP15, lanes 3, 6, 9 are buffer only.

FIG. 16: KIAA1372 and positive control USP1
FIG. 5 shows that the Ub-M-GST degradation activity of 5 is inhibited by the cysteine protease inhibitor NEM.

FIG. 17: Ub for KIAA1372 ^C181S
It is a figure which shows the result of having examined the degradation activity of -M-GST fusion protein in vitro in comparison with wild type KIAA1372. A shows the result of degradation activity, and B shows the result of confirming the expression of each protein. Lanes 1 and 3 show the cell extract at the time of non-induction, and lanes 2 and 4 show the cell extract at the time of induction.

FIG. 18 is an electrophoretogram for confirming the expression of KIAA1453 in Escherichia coli. Lane 1 is whole cell extract without NaCl induction, 2 is soluble extract without NaCl induction, lane 3 is whole cell extract with NaCl induction,
And lane 4 shows the soluble extract upon NaCl induction.

FIG. 19: KIAA1453 or mutant KIAA1
It is a figure which shows the result of having investigated the degradation activity of Ub-M-GST fusion protein about 453 ^C131S by a co-expression system.
USP15 is a positive control and luciferase is a negative control. A shows the result of degradation activity, and B shows the expression of each protein in the co-expression system.

FIG. 20: KIAA1453 or mutant KIAA1
About 453 ^C131S Ub-R-GST, Ub-I
It is a figure which shows the result of having examined the degradation activity of each-GST and Ub-P-GST fusion protein in a co-expression system. USP15 is a positive control and luciferase is a negative control. A shows the result of degradation activity, and B shows the expression of each protein in the co-expression system.

FIG. 21 is an electrophoretogram for confirming the expression of KIAA1063 in Escherichia coli. A shows the expression at 37 ° C, and B shows the expression at 25 ° C. Lanes 1 and 7
Are soluble and insoluble extracts of clone # 1 upon NaCl induction, lanes 2 and 8 are N respectively.
Soluble and insoluble extracts of clone # 1 without aCl induction, lanes 3 and 9 are soluble and insoluble extracts of clone # 2 with NaCl induction, respectively.
And lanes 4 and 10 are soluble and insoluble extracts of clone # 1 without NaCl induction, lanes 5 and 11 are soluble and insoluble extracts of clone # 3 with NaCl induction, lanes 6 and 12, respectively. Indicates a soluble extract and an insoluble extract of clone # 3 when NaCl was not induced.

FIG. 22: KIAA1063 or mutant KIAA1
It is a figure which shows the result of having investigated the degradation activity of Ub-M-GST fusion protein about 453 ^C131S by a co-expression system.
Lane 1 is KIAA1063 when NaCl is not induced, and lane 2 is KIAA106 when it is induced with 0.05 M NaCl.
3, lane 3 is KIAA during induction with 0.1 M NaCl
1063, lane 4 is K when induced with 0.2 M NaCl
IAA1063, lane 3 represents KIAA1063, lane M represents a molecular weight marker upon induction with 0.3 M NaCl.

FIG. 23. Ub-MG for KIAA1063
ST, Ub-R-GST, Ub-I-GST, Ub-P
FIG. 6 is a diagram showing the results of comparative examination of the degradation activity of each GST fusion protein in a co-expression system. Lanes 1 to 4 show KIAA1063 when induced with 0.1 M NaCl, and lanes 5 to 6 show KIAA1063 when not induced. Lanes 1 and 5 are Ub-M-GST, lanes 2 and 6 are Ub-R-GST, lanes 3 and 7 are Ub-IG.
ST, lanes 4 and 8 represent Ub-P-GST.

FIG. 24: Ub for KIAA1063 ^C174S
It is a figure which shows the result of having examined the degradation activity of -M-GST fusion protein in vitro in comparison with wild type KIAA1063. A shows the result of degradation activity, and B shows the result of confirming the expression of each protein. Lanes 1 and 3 show the cell extract at the time of non-induction, and lanes 2 and 4 show the cell extract at the time of induction. Lanes 1 to 5 show each protein when induced with 0.1 M NaCl, and lanes 5 to 6 show each protein when not induced. Lane M represents molecular weight markers. Lane 1
And 6 are KIAA1063 # 1, lanes 2 and 7 are KIAA1063 # 2, and lanes 3 and 8 are KIAA1.
063 ^C174S # 1, ^lanes 4 and 9 are KIAA1
063 ^C174S # 2, lanes 5 and 10 are positive controls, KIAA0529.

FIG. 25 is an electrophoretogram for confirming the expression of KIAA0190 in Escherichia coli. As a result of detecting KIAA0190 by staining with Coomassie blue, A is anti-His-
It is the result of detection by immunoblotting using a tag antibody. In both figures, lane 1 shows the whole cell extract at the time of NaCl induction, lane 2 shows the soluble extract at the time of NaCl induction, and lane 3 shows the whole cell extract at the time of non-induction. Lane M is a molecular weight marker. The examination was conducted on three samples (# 1, # 2, # 3).

FIG. 26: Ub-M-GS for KIAA0190
It is a figure which shows the result of having examined the degradation activity of T fusion protein in vitro. A shows the result of decomposition activity. B is KIA
The result of having examined the influence of the cysteine protease inhibitor NEM on the Ub-M-GST fusion protein degrading activity of A0190 is shown. Lane 1 is KIAA0190 during induction
Lane 2 shows KIAA0190 in the non-induced state. Lane 3 shows the time when NEM was added, and lane 4 shows the time when NEM was not added. The study was conducted on 3 samples (# 1, # 2, #
3). KIAA0529 is a positive control.

FIG. 27: Ub-P-GS for KIAA0190
It is a figure which shows the result of having examined in vitro the degradation activity of each fusion protein of T, Ub-R-GST, and Ub-I-GST. Lane 1 shows KIAA0190 at the time of induction, lane 2
Shows KIAA0190 in the non-induced state. The examination was conducted on two samples (# 1, # 2).

FIG. 28: Ub-M-GS for KIAA0190
T, Ub-P-GST, Ub-R-GST, Ub-I-
It is a figure which shows the result of having investigated the degradation activity of each GST fusion protein in a co-expression system. Lane 1 is KIAA019 during induction
0, lane 2 shows KIAA0190 in the non-induced state.
The study was conducted on 3 samples (# 1, # 2, #
3). Lane 1: NaCl (-) 6 hours, Lane 2: N
aCl (+) 6 hours, lane 3: NaCl (+) 3 hours, lane 4: NaCl (+) 3 hours, lane 5: Na
Cl (+) 6 hours, Lane 6: NaCl (−) 6 hours,
Lane 7: NaCl (+) 3 hours, Lane 8: NaCl
(+) 6 hours, lane 9: NaCl (-), lane 1
0: NaCl (+) 3 hours, lane 11: NaCl
(+) 6 hours, Lane 12: NaCl (-) 6 hours.

FIG. 29. Ub for KIAA0190 ^C429S
-M-GST (A in the figure), Ub-R-GST (B in the figure), Ub-I-GST (C in the figure) The degradation activity of each fusion protein was compared and examined in vitro with wild type KIAA0190. FIG. Lane 1 shows the results after 3 hours of induction with NaCl, lane 2 shows the results of induction with NaCl for 6 hours, and lane 3 shows the results when cultured for 6 hours in the absence of NaCl. The examination was conducted on two samples (# 1, # 2).
KIAA0529 is a positive control and luciferase is a negative control.

FIG. 30. KIAA0891 and mutant KIAA08.
It is the electrophoretogram which confirmed the expression in E. coli of 91 ^C506S . Lane 1 is a molecular weight marker, lane 2 is a negative control luciferase, lane 3 is a positive control USP15, lane 4 is KIAA0891, lane 5.
Indicates a mutant KIAA0891 ^C506S

FIG. 31. KIAA0891 and mutant KIAA0.
About 891 ^C506S , Ub-I-GST (A in the figure), Ub-M-GST (B in the figure), Ub-P-GST
(C in the figure) and Ub-R-GST (D in the figure) are diagrams showing the results of in vitro examination of the degradation activity of each fusion protein. Lane 1 is KIAA upon induction with 0.3 M NaCl
0891, lane 2 shows the result of KIAA0891 when 0.15 M NaCl was induced, and lane 3 shows the result of no induction. Lane 4 is KIAA when induced with 0.3 M NaCl
0891 ^C506S , 0.15M NaC in lane 5
KIAA0891 ^C506S at 1-induction, lane 6 shows the result at non-induction. Lane 7 is USP as a positive control
15, Lane 8 is a negative control luciferase.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61P 21/04 4B065 48/00 25/16 4C084 A61P 21/04 25/28 4C085 25/16 C07K 16 / 40 4C087 25/28 C12N 1/15 4H045 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/64 Z 1/21 C12P 21/02 C 5/10 C12Q 1/02 9/64 1 / 37 C12P 21/02 1/68 A C12Q 1/02 G01N 33/15 Z 1/37 33/50 Z 1/68 C12N 15/00 ZNAA G01N 33/15 5/00 A 33/50 A61K 37/02 ( 72) Inventor Naoya Wada 1-16-13 Kitakasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Co., Ltd. Tokyo Research and Development Center (72) Inventor Makiko Kono 1-1-16 Kitakasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Tokyo Research & Development Center Co., Ltd. (72) Inventor Aya Ito 1-16-13 Kita Kasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Co., Ltd. Tokyo Research & Development Center (72) Inventor Tetsuo Morita 1-16-13 Kitakasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Co., Ltd. Tokyo Research and Development Center (72) Noriko Kobayashi 1-16-1 Kitakasai, Edogawa-ku, Tokyo Pharmaceuticals Co., Ltd. Tokyo Research and Development Center (72) Inventor Hiroshi Yokota 1-16-13 Kita Kasai, Edogawa-ku, Tokyo Dai-ichi Pharmaceutical Co., Ltd. Tokyo Research and Development Center F-term (reference) 2G045 AA25 AA40 BB03 BB20 CA25 CB01 CB03 CB07 CB13 CB21 DA12 DA13 DA20 DA36 DA77 FB01 FB02 FB03 4B024 AA01 AA11 BA14 CA04 CA05 CA06 CA09 CA11 DA02 DA06 EA04 GA11 GA18 GA19 HA03 HA08 HA14 4B050 CC01 CC03 DD11 QR42 QRQQQRQR QRQQQRQQR QRQQQRQQR QRQQRQQR QRQQQR QRQQRQQR QRQQRQQR QRQQRQR QRQQRQR QRQQRQQR QR48 QR55 QR59 QR62 QR67 QR69 QR77 QR80 QR82 QS12 QS24 QS25 QS28 QS34 QS39 QX01 QX07 4B064 AG01 CA02 CA19 CC24 DA01 DA13 4B065 AA26X AA93Y AB01 AC14 BA02 CA33 CA44 4C084 AA02 AA03 AA07 AA13 AA17 BA01 BA02 BA10 BA22 NA14 ZA152 ZA162 ZA942 4C085 AA13 AA14 DD62 4C087 AA01 AA02 BC83 CA12 NA14 ZA15 ZA16 ZA94 4H045 AA10 AA11 AA20 AA30 BA10 CA45 DA75 DA86 DA89 EA21 EA50 FA72 FA74

Claims (22)

[Claims] 1. A polypeptide having deubiquitinating activity selected from the following group; SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 in the sequence listing. Or a polypeptide comprising the amino acid sequence of SEQ ID NO: 15, a polypeptide containing the above polypeptide, having at least about 70% homology in the amino acid sequence with the above polypeptide, and exhibiting a deubiquitinating activity. And a polypeptide having a mutation such as deletion, substitution, addition, or insertion of 1 to several amino acids in the amino acid sequence, and having deubiquitinating activity. 2. A polypeptide selected from the following group, wherein ubiquitin (Ub) is bound to glutathione S.
-Polypeptide having an activity of dissociating Ub from transferase; described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 in the sequence listing. A polypeptide consisting of the amino acid sequence of the above, a polypeptide containing the above polypeptide, a polypeptide having at least about 70% homology in the amino acid sequence with the above polypeptide and having deubiquitinating activity, and the above amino acid A polypeptide having a mutation such as a deletion, substitution, addition, or insertion of one to several amino acids in the sequence and having deubiquitinating activity. 3. A polynucleotide encoding the polypeptide according to claim 1 or 2, or a complementary strand thereof. 4. SEQ ID NO: 2, SEQ ID NO: 4 in the sequence listing, which encodes a polypeptide having a deubiquitinating activity and / or an activity of dissociating Ub from a ubiquitin (Ub) -bound glutathione S-transferase.
Sequence number 6, sequence number 8, sequence number 10, sequence number 1
2, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 16 or a complementary strand thereof. 5. A polynucleotide which hybridizes with the polynucleotide according to claim 3 or 4 or a complementary strand thereof under stringent conditions. 6. A recombinant vector containing the polynucleotide according to any one of claims 3 to 5. 7. The recombinant vector according to claim 6, wherein the recombinant vector is an expression recombinant vector. 8. A transformant introduced with the recombinant vector according to claim 6 or 7. 9. A method for producing the polypeptide according to claim 1 or 2, which comprises culturing a transformant into which the recombinant vector according to claim 7 has been introduced, or claim 6. Alternatively, a method comprising means for synthesizing a cell-free protein using the recombinant vector according to claim 7. 10. An antibody which immunologically recognizes the polypeptide according to claim 1 or 2. 11. The antibody according to claim 10, which suppresses deubiquitination activity. 12. A compound which interacts with the polypeptide according to claim 1 or 2 and inhibits or activates its physiological activity, and / or any one of claims 3 to 5. A method for identifying a compound which interacts with the polynucleotide according to claim 1 to inhibit or promote the expression thereof, wherein the polypeptide according to claim 1 or claim 2 and any one of claims 3 to 5 At least one of the polynucleotide according to claim 6, the recombinant vector according to claim 6 or claim 7, the transformant according to claim 8, and the antibody according to claim 10 or claim 11. A method characterized by using. 13. A compound that interacts with the polypeptide according to claim 1 or claim 2 to inhibit or promote its physiological activity, and / or claim 3 to claim 5.
A method for identifying a compound which interacts with the polynucleotide according to any one of 1 to 4 to inhibit or promote the expression thereof, the conditions allowing interaction between the compound and the polypeptide or the polynucleotide. And contacting the compound with the polypeptide or the polynucleotide,
Then, the compound interacts with the polypeptide or polynucleotide by detecting the presence, absence, or change of a signal generated by the interaction between the compound and the polypeptide or the polynucleotide, thereby allowing the physiology of the polypeptide. A method of determining whether to inhibit or promote activity or expression of said polynucleotide. 14. A compound which interacts with the polypeptide according to claim 1 or claim 2 to inhibit or promote its physiological activity, and / or claim 3 to claim 5.
A method for identifying a compound that interacts with the polynucleotide according to any one of claims 1 to 4 to inhibit or promote the expression thereof, wherein the transformant according to claim 8 is contacted with the compound, and Alternatively, a system using a signal and / or a marker capable of detecting the presence or absence of the expression or physiological activity of the polypeptide according to claim 2 is used to detect the presence, absence, or change of this signal and / or marker. Thereby, the compound is
Alternatively, a method for determining whether to promote or inhibit the expression or physiological activity of the polypeptide according to claim 2. 15. A compound identified by the method according to any one of claims 12 to 14. 16. A compound which interacts with the polypeptide according to claim 1 or 2 to inhibit or activate deubiquitination activity, or 3 to 5.
A compound which interacts with the polynucleotide according to any one of 1. to inhibit or promote its expression. 17. The polypeptide according to claim 1 or 2, the polynucleotide according to any one of claims 3 to 5, the recombinant vector according to claim 6 or 7, The transformant according to claim 8, the antibody according to claim 10 or claim 11, and the antibody according to claim 15.
Alternatively, a pharmaceutical composition containing at least one of the compounds according to claim 16. 18. The polypeptide according to claim 1 or 2, the polynucleotide according to any one of claims 3 to 5, the recombinant vector according to claim 6 or 7, The transformant according to claim 8, the antibody according to claim 10 or claim 11, and the antibody according to claim 15.
Alternatively, an agent for preventing and / or treating a neurodegenerative disease, which comprises at least one of the compounds according to claim 16. 19. The preventive and / or therapeutic agent for a neurodegenerative disease according to claim 18, wherein the neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease. 20. The polypeptide according to claim 1 or 2, the polynucleotide according to any one of claims 3 to 5, the recombinant vector according to claim 6 or 7, The transformant according to claim 8, the antibody according to claim 10 or claim 11, and the antibody according to claim 15.
An agent for preventing and / or treating muscular atrophy, which comprises at least one of the compounds according to claim 16. 21. A method for quantitatively or qualitatively measuring the polypeptide according to claim 1 or 2, or the polynucleotide according to any one of claims 3 to 5. 22. A reagent kit used in the method according to any one of claims 12 to 14 and 21, wherein the polypeptide according to claim 1 or 2. The polynucleotide according to any one of claims 3 to 5, the recombinant vector according to claim 6 or claim 7, the transformant according to claim 8, and the claim 10 or claim 11. At least 1 antibody
A reagent kit comprising one or more.