[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO1999051741A2 - Complexes de proteine lyst et proteines a interaction lyst - Google Patents

Complexes de proteine lyst et proteines a interaction lyst Download PDF

Info

Publication number
WO1999051741A2
WO1999051741A2 PCT/US1999/006831 US9906831W WO9951741A2 WO 1999051741 A2 WO1999051741 A2 WO 1999051741A2 US 9906831 W US9906831 W US 9906831W WO 9951741 A2 WO9951741 A2 WO 9951741A2
Authority
WO
WIPO (PCT)
Prior art keywords
lyst
protein
complex
seq
derivative
Prior art date
Application number
PCT/US1999/006831
Other languages
English (en)
Other versions
WO1999051741A3 (fr
Inventor
Krishnan Nandabalan
Stephen Kingsmore
Original Assignee
Curagen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to AU32140/99A priority Critical patent/AU3214099A/en
Priority to EP99914251A priority patent/EP1066378A2/fr
Priority to JP2000542454A priority patent/JP2002510490A/ja
Priority to CA002325562A priority patent/CA2325562A1/fr
Publication of WO1999051741A2 publication Critical patent/WO1999051741A2/fr
Publication of WO1999051741A3 publication Critical patent/WO1999051741A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to lysosome-associated LYST protein or the novel homolog
  • LYST-2 protein in complex with other proteins.
  • the invention relates to complexes of LYST protein with one or more proteins chosen from the list comprising: 14-3-3 protein, HSl protein, Hrs, BMKl alpha kinase. KB07, Efs, OS9, casein kinase II beta subunit (SU), calmodulin, Troponin, Importin beta, Fte-1, estrogen receptor-related protein (hERRl), Imogen- 38, Atrophin-1, GBDRl, DGS-1, norbin (KIAA0607), OPA containing protein, and M4 protein, and ten novel LYST Interacting Proteins ("LIP-1" through "LIP- 10”) further characterized herein.
  • proteins chosen from the list comprising: 14-3-3 protein, HSl protein, Hrs, BMKl alpha kinase. KB07, Efs, OS9, casein kinase II beta subunit (SU), calmodulin, Trop
  • the invention also relates to complexes of LYST-2 protein with one or more proteins chosen from the list comprising: 14-3-3 protein. XAP-4, and HBF-G2.
  • the invention includes antibodies specific to LYST complexes and LYST-2 complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy.
  • the invention further relates to novel LYST-2, LIPl. LIP2, LIP3, LIP4, LIP5.
  • the lysosomal Chediak-Higashi syndrome protein LYST (GenBank Accession No. U67615). is the human homolog of the mouse lysosomal trafficking regulator beige (BG) (Barbosa et al.. 1996, Nature 382: 262-265: Barbosa et al.. 1997. Human Mol. Genetics 6: 1091 - 1098). Both proteins are crucial for lysosomal. and hence cellular, function. The identification of interactions between LYST and other proteins would be useful in the development of treatments and assays for atopic disorders related to malfunctions or deficiencies in lysosomes and lysosome-related disorders and diseases. Examples of disorders and disease states relevant to this invention are classified below.
  • CH SYNDROME Chediak-Higashi syndrome is an autosomal recessive immune deficiency disease found in humans and modeled in "beige " (bg) mouse strains. Affected individuals have giant, perinuclear lysosomes, and defective granulocytic. natural killer and cytolytic T cell function. The disorder is lethal in that the affected individuals die prematurely of infection or malignancy (Baetz et al.. 1995. J. Immunol. 154: 6122-6131). The disorder is characterized by hypopigmentation. severe immunologic deficiency with neutropenia.
  • Intracellular protein transport to and from lysosomes is disordered in CHS patients; giant vesicles arise from lysosomes and proteins are mis-sorted into cell compartments.
  • Functional defects in the secretory lysosomes of granular cells provide a unifying hypothesis that explains the diverse clinical features of CHS (Griffiths, 1996, Semin. Immunol. 9: 109-1 15).
  • Albinism reflects a deficiency of tyrosinase in melanocytes due to exocytosis instead of delivery to the melanosome. Impaired platelet aggregation and a bleeding tendency are due to absence of dense granules in megakaryocytes and platelets.
  • BG mouse beige
  • LYST human Chediak-Higashi proteins
  • LYST gene predicts a polypeptide of 3,801 amino acids with a molecular mass of 429,153 Da (Nagle et al., 1996, Nature Genetics 14: 307-311). Said homologs have a conserved domain called BEACH (beige and CHS). LYST protein is predicted to form helical domains (Barbosa et al., 1996.
  • the C-terminal region of LYST contains seven consecutive 'WD' repeat motifs (Sondek et al., 1996, Nature 379: 369-374), which form beta sheets arranged in a 7-bladed propeller-like secondary structure. This region is similar to the beta-subunit of heterotrimeric G proteins and is thought to mediate protein-protein interactions.
  • LYST has a region of sequence similar to stathmin (oncoprotein 18), a coiled-coil phosphoprotein that regulates microtubule polymerization and acts as a relay for intracellular signal transduction (Barbosa et al., 1996, Nature 382: 262-265).
  • the LYST transcript predominates in thymus, fetal thymus, spleen and brain. LYST is abundant in brain tissues, peripheral blood leukocytes and bone marrow. The expression distribution of mRNA isoforms in humans is consistent with the pattern of clinical features observed in CHS patients (immune deficiency, platelet storage pool deficiency, neurologic manifestations, albinism) (Barbosa et al., 1997. Human Mol. Genetics 6: 1091-1098).
  • LYST is centrally implicated in many functional activities including, but not limited to. physiological processes such as signal transduction. vesicular transport, the formation and exocytosis of acidic intracellular organelles. and protein trafficking; pathological processes, including but not limited to. atopic diseases, autoimmune diseases, degenerative disorders such as neurodegenerative disease, hyperproliferative disorders such as tumorigenesis and tumor progression, oculocutaneous albinism, hypopigmentation, and platelet dysfunction.
  • physiological processes such as signal transduction. vesicular transport, the formation and exocytosis of acidic intracellular organelles. and protein trafficking
  • pathological processes including but not limited to. atopic diseases, autoimmune diseases, degenerative disorders such as neurodegenerative disease, hyperproliferative disorders such as tumorigenesis and tumor progression, oculocutaneous albinism, hypopigmentation, and platelet dysfunction.
  • Chediak-Higashi Syndrome (CHS) may suggest treatments that are not currently being considered. Patients with CHS still typically die in childhood.
  • novel therapeutic targets may be identified for control of mast cell degranulation and amelioration of consequent damage in diseases such as asthma and urticaria.
  • this invention identifies a rather large number of LYST interacting proteins.
  • the identification of interactions between LYST and other proteins can be useful in the development of treatments and assays for atopic disorders such as asthma, nasal polyps, hay fever rhinitis, and urticaria; autoimmune diseases such as CHS, rheumatoid arthritis, systemic lupus, inflammatory bowel disease, diabetes mellitus. and multiple sclerosis; neurological disorders and diseases; certain forms of cancer: pigmentation defects; and bleeding tendency.
  • a LYST interactant identified in the present invention is human 14-3-3 protein (GenBank
  • 14-3-3 proteins A broad range of organisms and tissues contain a family of highly conserved 14-3-3 proteins, which has been associated with many diverse functions, including signal transduction, exocytosis and cell cycle regulation.
  • 14-3-3 protein is an abundant acidic brain protein of approximately 28 kDa (GenBank Accession No. X56468). This protein activates tyrosine and calcium/calmodulin- dependent protein kinase II.
  • 14-3-3 proteins exist in cells both as membrane-associated and cytoplasmic forms (Martin et al., 1994, J. Neurochem. 63:2259-2265; Roth et al., 1994, Biochem. J. 301 :305-310). They stimulate calcium-dependent exocytosis in several cell types, acting apparently at the priming stage in a manner that is potentiated by protein kinase C (PKC) and associated with actin reorganization (Chamberlain et al., 1995, J. Cell. Biol. 130:1063-1070; Burgoyne et al., 1993, J. Anat. 183:309-314; Roth and Burgoyne, 1995, FEBS Letters 374: 77-81), although, as with LYST protein, the precise mechanism is unknown.
  • PKC protein kinase C
  • 14-3-3 proteins are also important in signal transduction: 14-3-3 protein bind PKC, is phosphorylated by PKC, and inhibits PKC activity. The PKC abnormalities described in CHS are similar to the effects of 14-3-3 on PKC (Ito et al, 1989. Biochem. Biophys. Res. Commun. 160:433-440; Sato et al., 1990, J. Leukocy. Biol. 48:377-381). 14-3-3 protein also binds to serine/threonine protein kinase Raf-1, and is necessary for Raf-1 kinase activity (1 (Zhang et al., 1997, J. Biol. Chem.
  • 14-3-3 also interacts with other kinases, such as Bcr. and can form ternary complexes with Raf and Bcr (Braselman and McCormick, 1995, EMBOJ. 14: 4839-4848). Like the proposed action of LYST, 14-3-3 appears to function as a scaffold protein that facilitates interactions among components of signaling and exocytic pathways (Aitken et al., 1995, Biochem. Soc. Trans. 23:605-61 1).
  • the Raf MECK/ERK/MAP signal transduction pathway and 14-3-3 protein have been suggested to play a role in the initiation of apoptosis.
  • Disorders of the regulation of apoptosis may play an important role in the pathogenesis of autoimmune and atopic diseases, including bronchial asthma. AIDS, and neoplastic diseases (Jagiello & Krasnowska, 1997. Postepy. Hig. Med. Dosw. 51: 385-398).
  • HSl PROTEIN There is a linkage between the induction of apoptosis and signal transduction disorders.
  • HSl protein 80% identical to 14-3-3 protein, is related to the protein kinase C inhibitory protein (KCIP) and is an abundant and ubiquitously expressed acidic protein of approximately 28 kDa. HSl protein is present in purified synaptic membranes.
  • KCIP protein kinase C inhibitory protein
  • HSl protein may influence neurotransmission by regulating exocytosis (Martin et al., 1994, J. Neurochemistry 63: 2259-2265). HSl protein also exists in phosphorylated forms in mammalian and avian brain. The HS 1 protein interacts with and activates Raf-1, a mediator of mitogenesis and differentiation and a key protein involved in transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases (Aitken et al., 1995, J. Biol. Chem. 270: 5706-5709; Fantl et al., 1994, Nature 371: 612-614). HSl protein also interacts with insulin receptor substrate- 1 (Kosaki et al., 1998, J. Biol. Chem. 273: 940-944) (see Imogen 38, infra).
  • Hrs Hepatocyte growth factor-regulated tyrosine kinase substrate
  • Hrs inhibits exocytosis in a dose-dependent manner, probably through binding to SNAP-25. a component of the SNARE protein complex ((Bean et al.. 1997. Nature 385: 826- 829).). While the SNARE complex has a well-defined role in synaptic vesicle docking, there is also evidence for SNAP25 involvement in calcium dependent exocytic membrane fusion reactions (Banerjee et al., 1996. J. Biol. Chem. 271:20227-20230). Calcium and zinc inhibit binding of Hrs to SNAP25 and promote exocytosis (Bean et al.. 1997.
  • Hrs interacts with STAM (signal transducing adaptor molecule), a relay in cytokine-mediated intracellular signal transduction (Asao et al., 1997. J. Biol. Chem. 272:32785-
  • EET RULE 26 32791.
  • Hrs suppresses STAM-mediatd signaling.
  • Cytokine stimulation of cells leads to STAM-mediated cell growth signalling and phosphorylation of Hrs.
  • BMKl ALPHA KINASE Another LYST interactant identified in the present invention is BMKl alpha kinase
  • MAP mitogen-activated protein
  • BMKl The mitogen-activated protein (MAP) kinase BMKl is part of a distinct MAP-kinase signaling pathway that is required for EGF-induced cell proliferation and progression through the cell cycle, see. e.g., Lee et al, 1995, Biochem. Biophys. Res. Commun. 213: 715-724.
  • BMKl plays an important role in shear stress-mediated gene expression in endothelial cells.
  • KB07 kinase Another LYST interactant identified in the present invention is KB07 kinase (GenBank Accession Number AF064606, direct submission). The gene was isolated from human dendritic cells and is similar to tyrosine kinase. Thus, it is likely that KB07 plays a role in the signaling pathway.
  • EFS Another of the LYST interactants identified in this invention is the human phosphoprotein signaling protein Efs (embryonal Fyn-associated substrate) (GenBank Accession No. AB001466; Ishino et al., 1997. Oncogene 15: 1741-1745). Efs has characteristic regions important in intracellular signal transduction. including a Src homology 3 (SH3) domain, a cluster of putative ligands for Src homology 2 (SH2) domains, and proline-rich sequences with SH3-binding consensus (Ishino et al.. 1995, Oncogene 11: 2331-2338).
  • SH3 Src homology 3
  • SH2 Src homology 2
  • Fyn a nonreceptor protein tyrosine kinase that plays a role in signaling processes, including T-cell activation, excitation-contraction coupling, and programmed cell death (apoptosis) (Marks, 1991, Am. J. Physiol. 272: H597-H605).
  • OS9 PRECURSOR Another one of the LYST interactants identified in this invention is OS-9 (GenBank
  • the gene encoding the OS-9 precursor is located within chromosome 12q 13-15, a region frequently amplified in human cancers.
  • the gene is ubiquitously- expressed in human tissues and amplified in sarcomas, frequently coamplified with the CDK4 gene. See. e.g. Su et al. 1996. Mol. Carcinog. 15: 270-275. Thus, it may be involved in cellular growth control.
  • CASEIN-KINASE II BETA SUBUNIT Another LYST interactant identified in the present invention is the ubiquitous serine/threonine protein kinase. casein-kinase II beta subunit (casein kinase II beta SU) (GenBank Accession Number M30448, Jakobi et ai, 1989, Ewr. J. Biochem. 183: 227-233).
  • the highly conserved enzyme is present in the nucleus and cytoplasm, and is another LYST interactant with roles in both signal transduction and the regulation of vesicular trafficking.
  • Casein kinase II beta SU is a regulatory subunit that stabilizes the catalytic alpha-subunit and modulates interactions with substrates (Allende & Allende. 1995, FASEB J. 9: 313-323).
  • Several casein kinase II beta SU substrates are important in vesicular transport, including synaptotagmin. syntaxin (a t-SNAR ⁇ ), synaptobrevin (a v-SNAR ⁇ ), calmodulin, protein kinase C, UNC18 and clathrin (Daveltov et al., 1993, J. Biol. Chem. 268:6816-6822; Nielander et al., 1995, J. Neurochem. 65:1712-20; Sacks & Mazus, 1994, Biochem. Mol. Biol. Int 34:251-259;
  • CALMODULIN Another LYST interactant identified in the present invention is calmodulin (GenBank
  • Calmodulin has a significant role in membrane fusion: it binds to intracellular organelles such as secretory granules, and is required for membrane fusion and exocytosis in several cell types that are aberrant in CHS. including platelets, mast cells and leukocytes. (Sheng et al, 1996. Nature 379: 4541-454) In particular. SNAR ⁇ -mediated docking of acidic organelles has been shown to prompt local calcium release, causing calmodulin to bind tightly to the docked vesicle and promote membrane fusion (Peters & Mayer. 1998. Nature 396:575-580; Schekman. 1998, Nature 396:514-515).
  • calmodulin inhibits interaction of Hrs and SNAP25 (see above).
  • the Ca2+- signaled exocytosis is blocked by SNAP-25.
  • Alpha-SNAP. 14-3-3 proteins, and calmodulin effect regulation of exocytosis in permeabilized adrenal chromaffin cells (Chamberlain et al.. 1995. J. Cell Biol. 130: 1063-1070). All three proteins. alpha-SNAP. 14-3-3 and calmodulin. lead to a Ca2+-dependent increase in catecholamine secretion. These three proteins also have distinct stage-specific actions on exocytosis.
  • TROPONIN I Another LYST interactant identified in the present invention is the troponin I (GenBank
  • Troponin I is a subunit of the thin filament-associated troponin-tropomyosin complex involved in calcium regulation of skeletal and cardiac muscle contraction. Cytoskeletal proteins including Troponin I play a central role in cardiomyopathies. Cardiomyopathies are serious heart muscle disorders in children and adults, which result in morbidity and premature death.
  • Importin beta subunit a soluble protein consisting of a 60 kDa (alpha) and 90 kDa (beta) subunit. is essential for the first step of selective protein import into the cell nucleus.
  • the complex of Importin-beta and Importin-alpha is essential for nuclear protein import.
  • Importin-alpha provides the nuclear localization signal binding site, Importin-beta the site of initial docking to the pore (Gorlich et al, 1995, Nature 377: 246-248). Importin-beta interacts with Rnalp, a GTPase activating protein required for nuclear transport (Koepp et al.. 1996, J. Cell Biol. 133: 1163-1176).
  • the human immunodeficiency virus type 1 (HIV-1) Rev protein nuclear localization signal mediates specific binding to human Importin-beta (Henderson & Percipalle. 1997, J. Mol. Biol. 274: 693-707).
  • the Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localization signal and a distinct nuclear export signal.
  • Rev and Importin-alpha bind (via an arginine-rich sequence) to a similar region on Importin-beta.
  • the Importin-beta subunit is an essential component of the nuclear protein translocation complex.
  • FTE-1 The fte-1 gene, a putative v-fos transformation effector gene (GenBank Accession Number M8471 1 ; Kho & Zarbl. 1992, Proc. Natl. Acad. Sci. USA, 89: 2200-2204), has been shown to encode ribosomal protein S3a.
  • An elevated expression of fte-1 mRNA is frequently associated with cell transformation.
  • the accumulation of ribosomal subunits modulates neoplastic transformation and cell growth.
  • fte-1 might be involved in protein translocation into mitochondria, due to its homology to a yeast gene (MFT1), whose product is involved in protein import.
  • MFT1 yeast gene
  • hERRal The human estrogen receptor-related protein (hERRal) was identified in the present invention as one of the LYST interactants.
  • hERRal (GenBank Accession Number X51416. Yang et al. 1996. J. Biol. Chem. 271: 5795-5804) is an orphan member of the steroid-thyroid hormone receptor superfamily of transcription factors which are expressed in characteristic patterns during organogenesis and postnatal development.
  • hERRal plays a role in bone development and metabolism, and was found expressed in osteoblastic osteosarcoma cell lines (Bonnelye et al, 1997, Mol. Endocrinol. IT. 905-916).
  • hERRal interacts with the human estrogen receptor.
  • Steroid hormone receptors are basically localized in the nucleus, regardless of hormonal status, and considerable amounts of unliganded steroid hormone receptors may be present in the cytoplasm of target cells in exceptional cases (Yamashita. 1998, Histol. Histopathol. 13: 255- 270).
  • Estrogens transiently induce a number of nuclear protooncogenes, such as members of the c-fos and c-jun family of proteins, which act as transcription factors through the estrogen receptor system.
  • the estrogen receptor system appears to participate in the growth response and abnormalities of the epithelium elicited by exogenous estrogen treatment during the neonatal period (Yamashita. 1998, Histol. Histopathol. 13: 255-270).
  • the human Imogen 38 protein (GenBank Accession Number Z68747) was identified in this invention as one of the LYST interactants. and is a novel 38 kDa islet mitochondrial autoantigen (Arden et ai. 1996. J. Clin. Invest. 97: 551-561).
  • Cell-mediated autoimmune attack directed against Imogen 38 is associated with type 1 diabetes. Insulin regulates glucose uptake, mediated by an translocation of glucose transporters from an intercellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells (Cheatham et ai. 1996. Proc. Natl. Acad. Sci. U.S.A. 93: 15169-15173).
  • XAP-4 The protein encoded by the XAP-4 gene was found to be a LYST-2 interacting protein.
  • the XAP-4 mRNA (GenBank Accession Number X79353) encodes for a rap GDP-dissociation inhibitor. See, e.g., Sedlacek et ai. 1994. Mamm. Genome 5: 633-639. Thus, it may play a role in regulating the GDP-GTP exchange reaction, and may therefore be involved in signaling processes.
  • Atrophin I is the protein product of the gene for dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease).
  • DRPLA is a rare, progressive, fatal autosomal dominant neurological disorder characterized by neuronal degeneration, especially in the cerebellar dentate nucleus.
  • Clinical symptoms include variable combinations of myoclonus epilepsy, cerebellar ataxia, choreoathetosis and dementia.
  • DRPLA is known to result from expansion of a CAG trinucleotide repeat encoding glutamine (Margolis et ai. 1996, Brain Res. Mol. Brain Res. 36: 219-226).
  • a further LYST interactant described in this invention is the putative glialblastoma cell differentiation-related protein (GBDRl ) (GenBank Accession Number AF068195. direct submission).
  • GBDRl putative glialblastoma cell differentiation-related protein
  • DGS-I DiGEORGE SYNDROME PROTEIN
  • An additional LYST interactant described in this invention is the DiGeorge syndrome
  • the DGSI gene is located within the minimal DiGeorge syndrome critical region of the chromosome and encodes for a 476 amino acid protein (Gong et al. 1997, Hum. Mol. Genet.
  • the clinical symptoms include cardiac defects, thymic hypoplasia or aplasia and hypocalcemia.
  • NORBIN (KIAA0607 GENE)
  • rat norbin gene GenBank Accession Numbers ABOl 1179 for human KIAA0607 gene and AB006461 for rat norbin; Shinozaki et ai. 1997, Biochem. Biophys. Res. Comm. 240: 766-771
  • Rat norbin is expressed in the brain and is induced by treatment of tetraethylammonium in rat hippocampal slice accompanied with neurite-outgrowth in neuro-2a cells.
  • the neurite-outgrowth-related norbin protein may play a role in neural plasticity because of the formation of new synapses.
  • OPA CONTAINING PROTEIN A LYST interactant was identified as an OPA-containing protein (GenBank Accession No. AF071309. Philbert. et al, 1998, Mol. Psych. 3: 303-309). No function has yet been assigned to this protein.
  • M4 PROTEIN One LYST interactant was found to be identical to the M4 protein (GenBank Accession No. L03532. Datar, et al. 1993, Nucl. Acids Res. 21: 439-446).
  • the M4 protein is a RNA- binding protein.
  • HBF-G2 HUMAN BRAIN FACTOR (HBF)-G2
  • HBF-G2 human brain factor (HBF)-G2 (GenBank Accession Number X78202: Wiese et ai. 1995, Biochim. Biophys. Acta 1262: 105-112).
  • HBF-G2 is a member of the fork head gene family, which expression is restricted to the neurons of the developing telencephalon.
  • HBF-G2 is strongly expressed in embryonic brain and may contribute to early developmental decisions in cell fates during embryogenesis. It should be noted that the citation of a reference in this or in any section of the specification shall not be construed as an admission that such reference is prior art to the present invention.
  • the human Chediak-Higashi syndrome protein LYST plays an important role in regulating lysosomal trafficking and function.
  • the inventors have cloned a large novel human gene fragment, hereforth referred to as LYST-2, whose DNA coding region and translated protein sequence are highly homologous to human LYST.
  • the LYST-2 protein contains a BEACH domain.
  • the present inventors suggest that LYST-2 also plays an important role in lysosomal regulation.
  • the invention herein provides a composition comprising the LYST-2 gene or protein, as well as derivatives, fragments, and analogs thereof.
  • the present invention is based upon the inventors' discovery that certain proteins bind to and form complexes with LYST or LYST-2 proteins, as assayed by a modified yeast two-hybrid system (Chien et al, 1991, Proc. Natl. Acad. Sci. USA 88: 9578-9581). Accordingly, the invention also provides compositions comprising a protein complex of a LYST or LYST-2 protein with a protein that interacts with, i.e., binds to, LYST or LYST-2.
  • a protein shown to bind with LYST or LYST-2 is designated as a "LYST-IP" for LYST-Interacting Protein
  • the complexes of LYST or LYST-2 and a LYST-IP are designated as "LYST:LYST-IP” herein.
  • the present invention is directed to complexes of LYST or LYST-2, and complexes of derivatives, fragments and analogs of LYST or LYST-2, with 14-3-3 protein, with HSl protein, with Hrs, with BMKl, with KB07, with Efs, with XAP-4, with OS9. with casein kinase II beta SU, with calmodulin.
  • LIP-1 with troponin I, with Importin beta, with Fte-1 , with estrogen- receptor related protein, with Imogen 38. with Atrophin-1, with norbin. with HBF-G2, with DGS-I, with GBDR-1 , with OPA containing protein, and with M4 protein and their derivatives, analogs and fragments of these LYST or LYST-2 interacting proteins.
  • Ten genes that interact with LYST or LYST-2 are novel to this invention, and are identified herein as LIP-1, LIP-2, LIP- 3, LIP -4, LIP -5, LIP-6, LIP-7, LIP-8, LIP -9. and LIP-10.
  • the present invention is also directed to complexes of LYST or LYST-2. and complexes of derivatives, fragments and analogs of LYST or LYST-2. with LIPl . with LIP2. with LIP3. with LIP4. with LIP5. with LIP6. with LIP7. with LIP8. with LIP9. and with LIPIO and their derivatives, analogs and fragments of these LYST or LYST-2 interacting proteins.
  • LYST-2 antibodies specific to complexes of derivatives, fragments or analogs of said LYST or LYST-2 and at least one LYST-IP, wherein any one or more of the LYST.
  • LYST-2. and LYST-IP proteins may be a derivative, analog or fragment thereof.
  • the invention further relates to nucleotide and amino acid sequences of LYST-2. LIPl.
  • Nucleic acids able to hybridize to, or complementary to, the foregoing nucleotide sequence, such as the inverse complement of the foregoing sequence, are also provided.
  • the inverse complement is a nucleic acid sequence that has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand.
  • the invention also relates to protein derivatives and analogs of LYST-2, LIPl, LIP2,
  • Such functional activities include, but are not limited to, (i) the ability to bind with or compete for interaction with LYST; (ii) antigenicity, i.e.. the ability to bind or compete with LYST-2, LIPl, LIP2, LIP3, LIP4. LIP5, LIP6. LIP7, LIP8. LIP9, or LIPIO for binding to each LIP-specific antibody, respectively; and (iii) immunogenicity, i.e. the ability to generate an antibody that binds LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5. LIP6, LIP7, LIP8, LIP9, or LIPIO protein, respectively.
  • LIPl LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein, and derivatives, fragments, and analogs of the complexes and/or individual proteins, e.g., by recombinant means, are also provided.
  • Pharmaceutical compositions comprising same are also provided.
  • the present invention further provides methods of modulating (i.e.. inhibiting or enhancing) the activity of LYST:LYST-IP complexes, and methods of modulating a LYST-2, LIPl, LIP2. LIP3. LIP4. LIP5. LIP6, LIP7. LIP8. LIP9. or LIPIO protein.
  • the individual protein components of the above mentioned LYST:LYST-IP complexes have been implicated in cellular functions, including but not limited to, (i) physiological processes including, but not restricted to. signal transduction. vesicular transport, the formation and exocytosis of acidic, intracellular organelles. protein trafficking, pigmentation regulation, platelet function, and viral response; and
  • pathological processes including, but not restricted to. atopic diseases, autoimmune diseases, degenerative disorders including neurodegenerative disease, hyperproliferative disorders including tumorigenesis and tumor progression, oculocutaneous albinism, hypopigmentation. platelet dysfunction and viral infections.
  • the present invention also provides methods for screening a LYST:LYST-IP complex, and screening a LYST-2.
  • LIPl LIP2, LIP3, LIP4.
  • LIPIO protein as well as derivatives and analogs of the LYST:LYST-IP complex; screening a
  • LYST-2 LIP2, LIP3. LIP4, LIP5, LIP6, LIP7, LIP8, LIP9. or LIPIO mRNA; and screening a LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein for the ability to alter cell functions, particularly those cell functions in which LYST, LYST-2, and/or a LYST- IP have been implicated.
  • the present invention also relates to therapeutic and prophylactic, as well as diagnostic, prognostic, and screening methods and compositions based upon LYST:LYST-IP complexes (and the nucleic acid molecules encoding the individual proteins that participate in the complexes), as well as LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9 or LIPIO proteins and encoding nucleic acid molecules.
  • Therapeutic compounds of the invention include, but are not limited to. LYST:LYST-IP complexes and complexes where one or both members of the complex is a derivative or analog of LYST, LYST-2, and/or a LYST-IP.
  • Therapeutic compounds also include LYST-2, LIPl, LIP2, LIP3. LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO proteins and derivatives, fragments or analogs thereof, antibodies to the protein or derivatives, fragments or analogs of the antibody, and nucleic acids encoding the foregoing proteins, derivatives, fragments and analogs.
  • Therapeutic compounds also include antisense nucleic acids to the nucleotide sequences encoding the LYST:LYST-IP complex components, and LYST-2, LIPl. LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO antisense nucleic acids. Diagnostic, prognostic and screening kits are also provided.
  • LYST:LYST-IP complexes are also provided in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 is a depiction of the novel nucleotide sequence (SEQ ID NO: l) and deduced amino acid sequence (SEQ ID NO:2) of LYST-2.
  • the coding sequences beginning at base 3. indicated by an arrow, and ending at base 794. and beginning at base 774, indicated by arrow, and ending at base 1424 were used as "bait" in the assays described in the EXAMPLES, infra.
  • FIGURE 2 is a depiction of the LIP-1 nucleotide sequence (503 nucleotides total,
  • LIP-1 is a TCP10A homolog.
  • the prey sequence identified in the assay described in the EXAMPLES, infra, begins at nucleotide 1 and 5. indicated by arrow.
  • the known sequence does not include the initiation codon, and therefore, the He residue at amino acid position 1 is denoted in bold indicating that the sequence must be extended in the amino-terminal direction.
  • the sequence does not have a STOP codon.
  • the amino acid at position 85 may be encoded by AAC (Asn), ACC (Thr), AGC (Ser) or ATC (He).
  • FIGURE 3 is a depiction of the LIP-2 nucleotide sequence (495 nucleotides total,
  • GenBank Accession No. AAO 10799) (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6).
  • FIGURE 4 is a depiction of the LIP-3 nucleotide sequence (1 198 residues, SEQ ID NO:
  • LIP-3 is a ROAZ homolog.
  • FIGURE 5 is a depiction of the LIP-4 nucleotide sequence (total of 524 nucleotides,
  • LIP-4 is a hnRNP-E2 homolog.
  • the prey sequence identified in the assay described in the EXAMPLES, infra, begins at base 2.
  • ATG initiator methionine codon.
  • FIGURE 6 is a depiction of the LIP-5 nucleotide sequence (total of 517 nucleotides,
  • FIGURE 7 is a depiction of the LIP-6 nucleotide sequence (total of 753 nucleotides,
  • LIP-6 is an ns2-3 homolog.
  • FIGURE 8 is a depiction of the LIP-7 nucleotide sequence (total of 451 nucleotides. SEQ ID NO:15) and amino acid sequence (SEQ ID NO: 16). LIP-7 is a TCP10A homolog. The prey sequence identified in the assay described in the EXAMPLES, infra, begins at base 1. indicated by an arrow.
  • FIGURE 9 is a depiction of the LIP-8 nucleotide sequence (total of 402 nucleotides,
  • LIP-8 is a KAP4L homolog.
  • the prey sequence identified in the assay described in the EXAMPLES, infra, begins at base 1. indicated by an arrow.
  • FIGURE 10 is a depiction of the LIP-9 nucleotide sequence (total of 554 nucleotides,
  • LIP-9 is an etr-1 homolog.
  • the prey sequence identified in the assay described in the EXAMPLES, infra begins at base 138, indicated by an arrow.
  • ATG initiator methionine codon
  • FIGURE 1 1 is a depiction of the LIP-10 nucleotide sequence (total of 373 nucleotides, SEQ ID NO:21 ) and amino acid sequence (SEQ ID NO:22).
  • LIP-10 is an chicken tyrosine kinase homolog. The prey sequence identified in the assay described in EXAMPLES, infra, begins at base 1.
  • FIGURE 12 demonstrates the specificity of LYST interactions. Shown is the matrix of results of the yeast two hybrid system assays. The results of assays using the bait proteins LYST and LYST-2 are indicated above the columns. LYST was used in two screens, a forward (LYST
  • BMKl KB07.
  • CDK2. retinoblastoma. p27(Kipl ), RGL-2 and vector control are indicated to the left of the columns.
  • a positive interaction between the indicated bait and prey proteins is indicated as "+" in the box forming the intersection between the particular bait and prey proteins, a lack of interaction is designated by "-".
  • N.D. not determined in this test.
  • FIGURE 13 is a depiction of the corrected nucleotide sequence (SEQ ID NO:23) and deduced amino acid sequence (SEQ ID NO:24) of LYST-2.
  • the coding sequences beginning at base 3. indicated by an arrow, and ending at base 794, and beginning at base 774. indicated by arrow, and ending at base 1424 were used as "bait" in the assays described in the EXAMPLES. infra.
  • the present invention is based upon two proteins important in regulating lysosomal trafficking and function, namely, LYST and a novel LYST homolog described herein and termed LYST-2.
  • LYST-2 has a 74% amino acid similarity to LYST, and its protein contains the conserved BEACH domain described in the Chediak-Higashi LYST protein and the murine beige protein.
  • one embodiment of the present invention provides a composition comprising the LYST-2 gene or LYST-2 protein [SEQ ID NO:l and SEQ ID NO:2, respectively (FIG. 1 ) and the more complete SEQ ID NO:24 and SEQ ID NO:24 (FIG. 13)], and derivatives, fragments, and analogs thereof.
  • the invention is directed toward the inventors' discovery that certain proteins bind to and form complexes with LYST or LYST-2 proteins (a said LYST or LYST-2 interacting protein being designated herein as a "LYST-IP" for LYST-Interacting Protein).
  • Said LYST-IPs were identified using an improved, modified form of the yeast two hybrid system. See e.g. Chien, et al. Proc Natl Acad Sci USA. 88: 9578-9581, 1991.
  • LYST-interacting protein- 1 (LIP-1. SEQ ID NO:3), LYST- interacting protein-2 (LIP-2, SEQ ID NO:5), LYST-interacting protein-3 (LIP-3, SEQ ID NO:7), LYST-interacting protein-4 (LIP-4. SEQ ID NO:9), LYST-interacting protein-5 (LIP-5. SEQ ID NO: l 1 ).
  • LYST-interacting protein-6 (LIP-6.
  • LYST:LYST-IP complexes by virtue of the interaction, are implicated in modulating the functional activities of LYST or LYST-2 and its binding partners.
  • Such functional activities include, but are not limited to. the physiological processes and the pathological processes that occur within individual cells, or that occur throughout the tissues and organs of an organism in question.
  • Hepatocyte growth factor-regualted tyrosine kinase substrate, Hrs was found to interact with LYST fragement 6586-7449. This sequence was isolated once from a screen of a fetal brain cDNA library. The 5 ' end of the Hrs clone corresponds to amino acid 84.
  • the N-terminal region of LYST interacted with 14-3-3 protein.
  • Four colonies, representing two independent isolates of 14-3-3 protein were identified in reverse two-hybrid screens of the fetal brain cDNA library (see Table lb).
  • Another LYST fragment (bp 4009-4821) interacted with the same region of 14-3-3 protein. This interaction was found once in a reverse screen of a fetal brain library (see Table lb).
  • eight colonies representing seven independent isolates of 14-3-3 protein were identified in forward two-hybrid screens of fetal liver, fetal brain, and adult heart libraries (see Table la).
  • the LYST-interacting domain of 14-3-3 protein did not contain the dimerization domain, membrane-binding domain, or PKC-inhibition domain, but did include the annexin-like domain that is important in promoting Calcium-regulated exocytosis.
  • 14-3-3 beta also called HSl protein
  • 14-3-3 beta also called HSl protein
  • the HSl interaction was identified twice in screens of a fetal brain library (Table lb).
  • the LYST-interacting region of HSl protein (amino acids 31-246) was the same as the LYST-interacting region of 14-3-3 protein.
  • Neither of the 14-3-3 interacting domains of LYST contained the recently described consensus 14-3-3 binding motif (Yaffe et al., 1997, Cell 91 :961-71).
  • 14-3-3 protein was also found to interact with LYST-2 fragment 774-1424 (Table Ic).
  • the LYST N-terminus also interacted with the C-terminal twelve amino acids of calmodulin. an interaction identified once in a screen of a fetal brain library (Table lb).
  • the LYST N-terminus also interacted with the C-terminal half of a steroid hormone receptor. estrogen receptor-related protein (hERRl). an interaction detected three times in a screen of a fetal brain library (Table lb).
  • the LYST fragment 2347-3213 was found to interact witlrtrifc ' C-terminus of BMKl alpha kinase in an interaction detected three times representing two different isolates in a reverse screen of a fetal brain library (Table lb).
  • the LYST fragment 3190-4032 was found to interact with the carboxy-terminal half of OS-9 precursor in an interaction detected three times in a forward screen of a keratinocyte library (Table la). The same LYST fragment interacted with the
  • the LYST fragment 4819-5700 was found to interact with the fos transformation effector gene Fte- 1 in an interaction detected twice in a forward screen of a fetal brain library (Table la).
  • the LYST fragment 6586-7449 interacted with twelve proteins: the c-terminal halves of the importin beta-subunit, imogen 38 and norbin; DGS-I; and seven novel genes (referred to as LIPl, LIP2, LIP4, LIP5, LIP6. LIP7, LIP8) (see Table lb).
  • LIP6 interacted with the LYST domain containing WD40 repeats (bp 10576-1 161 1). Two independent isolates of LIP6 were identified among interactions with the WD40 repeats. Casein kinase II beta subunit which interacted with LYST fragment 3190-4032 in forward screens, also interacted with the LYST WD40 repeat domain in reverse screens (see Table lb).
  • the BEACH domain of LYST (bp 9502-10590) interacted with six proteins: the amino terminal third of atrophin-1 and embryonic Fyn-substrate 1 (Efsl), the amino terminus of OPA- containing protein, heterogeneous nuclear riboprotein M4, LIP3 and LIP9 (see Table lb).
  • LIP3 also interacted with LYST 9037-9585.
  • LIP3 interacted as well with LYST domain 9037-9585 which includes part of the BEACH domain.
  • the LYST fragment 9037-9585 which includes the 5' part of the BEACH domain was found to interact with LIP10, as detected in seven identical colonies in a forward screen of a heart library (Table la); with Glialblastoma cell differentiation-related protein (four identical isolates of a keratinocyte library); and with KB07 (five isolates representing two different clones of a heart library) (Table la).
  • LIPl Tcp-10 homolog
  • LIP2 LIP2 (LI 7 homolog).
  • LIP3 Roaz protein homolog
  • LIP4 hnRNP-e2 homolog
  • LIP6 Ns2-3 homolog
  • LIP7 TCP10A homolog
  • LIP8 KAP4L homolog
  • LIP9 etr-1 homolog
  • LIP10 chicken tyrosine kinase homolog
  • the invention provides for a LYST-2. LIP-1. LIP-2. LIP-3. LIP-4, LIP-5, LIP-6. LIP-7. LIP-8. LIP-9. or LIP-10 protein and gene encoding the protein from many different species, particularly vertebrates, and more particularly mammals.
  • the LYST-IP subgroup comprising the LIP-1.
  • LIP-9, and LIP- 10 proteins or genes encoding said proteins are designated herein as the ten
  • novel LIP proteins and genes are of human origin. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is also provided in the present invention.
  • the present invention further relates to a LYST-IP protein derivative or analog that is functionally active, i.e.. capable of displaying one or more known functional activities associated with a full length (wild-type) LYST-IP protein. Functional activities include, but are not limited to, immunogenicity, antigenicity, and the ability to form a complex with LYST or LYST-2.
  • Derivatives, fragments, and analogs provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively. Fragments are, at most, one nucleic acid-less or one amino acid-less than the wild type full length sequence.
  • Derivatives and analogs may be full length if said full length derivative or analog contains a modified nucleic acid or amino acid, as described infra.
  • Derivatives or analogs of LYST or LYST-2 and LYST-IPs include, but are not limited to.
  • molecules comprising regions that are substantially homologous to LYST or LYST-2 or LYST- IPs, in various embodiments, by at least about 30% through about 95% identity (with a preferred identity of 90-95%) over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art. or whose encoding nucleic acid is capable of hybridizing to the complement (e.g., the inverse complement) of a sequence encoding LYST or LYST-2 or a LYST-IP under stringent (the preferred embodiment), moderately stringent, or low stringent conditions. See e.g. Ausubel. et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons. New York. NY, 1993, and infra.
  • fragments of LYST or LYST-2 and LYST-IPs include, but are not limited to. molecules comprising regions that are substantially homologous to LYST or LYST-2 or LYST- IPs, in various embodiments, by at least about 30% through acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art. or whose encoding nucleic acid is capable of hybridizing to the complement (e.g., the inverse complement) of a sequence encoding LYST or LYST-2 or a LYST-IP under stringent (the preferred embodiment), moderately stringent, or low stringent conditions.
  • complement e.g., the inverse complement
  • the aforementioned fragments possess nucleotide or amino acid sequences which are comprised of at least 80%) of the wild type, full length LYST or LYST-2 or LYST-IP proteins or 80% of the nucleic acid sequences encoding these proteins, with an overall 90% nucleic acid or amino acid sequence identity.
  • LYST:LYST-IP complexes by virtue of the interaction, are implicated in modulating the functional activities of LYST or LYST-2 and its binding partners.
  • Such functional activities include, but are not limited to, (i) physiological processes including, but not restricted to. signal transduction, vesicular transport, the formation and exocytosis of acidic intracellular organelles. and protein trafficking; and (ii) pathological processes including, but not restricted to. atopic diseases, autoimmune diseases, degenerative disorders including neurodegenerative disease, hyperproliferative disorders including tumorigenesis and tumor progression, oculocutaneous albinism, hypopigmentation and platelet dysfunction.
  • LYST-2 protein or said ten novel LIP proteins identified supra, and fragments, derivative, and analogs thereof, contain determinants important for antigenicity (defined herein as the ability to bind with an antibody specific to a designated protein or protein complex, or the ability to compete for binding with same), and immunogenicity (defined herein as the ability to generate an antibody that binds to a designated protein or protein complex).
  • Derivatives or analogs of the LYST:LYST-IP complex that have the desired immunogenicity or antigenicity can be used in immunoassays, for immunization, for inhibition of LYST:LYST-IP complex activity, etc.
  • Derivatives or analogs of the LYST:LYST- IP complex that retain or enhance, or alternatively lack or inhibit, a property of interest (e.g.. participation in a LYST:LYST-IP complex) can be used as inducers. or inhibitors, respectively, of such a property and its physiological correlates.
  • a specific embodiment relates to a LYST:LYST-IP complex of a fragment of a LYST or LYST-2 protein and/or a fragment of a LYST-IP protein that can be bound by an anti-LYST and or anti-LYST-IP antibody or by an antibody specific for a LYST:LYST-IP complex, when such fragment is included in a LYST:LYST-IP complex.
  • the present invention is directed to methods of screening for proteins ' that nt'Sattf fW
  • the invention further relates to LYST or LYST-2 complexes, in particular LYST or LYST-2 complexes with one or more of the above said LYST-IP proteins.
  • the invention further relates to complexes of derivatives, analogs and fragments of LYST or LYST-2, with derivatives, analogs and fragments thereof of said LYST-IPs.
  • LYST:LYST-IP complexes may be comprised of a derivative of LYST or LYST-2 with a wild type LYST-IP, a wild type LYST or LYST-2 with a derivative LYST-IP, or a derivatives LYST or LYST-2 with a derivative LYST-IP, wherein said derivative has wild type function.
  • such complexes bind an anti-LYST:LYST-IP complex antibody.
  • complexes of human LYST or LYST-2 with human LYST-IP protein are provided.
  • the invention also provides methods of producing and/or isolating LYST:LYST-IP complexes.
  • the invention provides methods of using recombinant DNA techniques to express both LYST or LYST-2 and its binding partner (or fragments, derivatives or homologs of one or both members of the complex) either where both binding partners are under the control of one heterologous promoter (i.e., a promoter not naturally associated with the native gene encoding the particular complex component) or where each is under the control of a separate heterologous promoter.
  • the present invention provides for methods of diagnosis, prognosis, and screening for diseases and disorders associated with aberrant levels of one or more components of a group of proteins and protein complexes comprising: LYST:LYST-IP complexes and LYST-IP proteins.
  • the invention also provides methods of treating or preventing such diseases or disorders, comprising administration of the LYST:LYST-IP complex or of the LYST-IP proteins, or of modulators of LYST:LYST-IP complex formation or activity (e.g., antibodies that bind the LYST:LYST-IP complex, or non-complexed LYST or its binding partner or a fragment thereof-- preferably the fragment containing the portion of LYST or the LYST-IP that is directly involved in complex formation).
  • modulators of LYST:LYST-IP complex formation or activity e.g., antibodies that bind the LYST:LYST-IP complex, or non-complexed LYST or its binding partner or a fragment thereof-- preferably the fragment
  • These methods also include administering mutants of LYST or the LYST-IP that increase or decrease binding affinity, administering small molecule inhibitors/enhancers of LYST:LYST-IP complex formation, or administering antibodies that either stabilize or neutralize the LYST:LYST-IP complex.
  • Methods of assaying LYST:LYST-IP complexes or of assaying LYST-IP proteins for activity as therapeutics or diagnostics as well as methods of screening for LYST:LYST-IP complex or LYST-IP protein modulators i.e.. inhibitors, agonists and antagonists
  • LYST-IP protein modulators i.e.. inhibitors, agonists and antagonists
  • all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector.
  • the necessary transcriptional and translational signals may be provided with the expression vector or may be supplied by the native promoter, and/or their flanking regions.
  • the LYST. LYST-2, or LYST-IP proteins either alone or in a complex, can be obtained by methods well known in the art for protein purification and recombinant protein expression.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector. /. e. , a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence.
  • the necessary transcriptional and translational signals can also be supplied by the native promoter for LYST or LYST-2 or any LYST-IP genes, and/or their flanking regions.
  • a LYST:LYST-IP complex is obtained by expressing the entire LYST or LYST-2 sequence and a LYST-IP coding sequence in the same cell, either under the control of the same promoter or under two separate promoters.
  • a derivative, fragment or homolog of LYST or LYST-2 and/or a derivative, fragment or homolog of a LYST-IP are recombinantly expressed.
  • the derivative, fragment or homolog of LYST or of LYST-2 and or of the LYST-IP protein forms a complex with a binding partner identified by a binding assay, such as the modified yeast two hybrid system (see e.g. Chien. et al, 1991, Proc Natl Acad Sci USA. 88: 9578-9581), and more preferably forms a complex that binds to an anti-LYST:LYST-IP complex antibody.
  • a vector is used that comprises a promoter operably linked to nucleotide sequences encoding LYST or LYST-2 and/or a LYST-IP, or a fragment, derivative or homolog thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g.. an antibiotic resistance gene).
  • a vector is used that comprises a promoter operably linked to nucleotide sequences encoding both LYST or LYST-2 and a LYST-IP. one or more origins of replication, and optionally, one or more selectable markers.
  • Expression vectors or derivatives which can be used include, but are not limited to. human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus: yeast vectors: bacteriophage vectors such as lambda phage; plasmids such as pBR322. pUC plasmid derivatives, or the pBlueScript vector (Stratagene. La Jolla. CA); and cosmid vectors.
  • Methods used may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Any suitable transcription and translation elements may be used, and expression may be regulated by any promoter and or enhancer known in the art. Promoters sources which may be used include, but are not limited to, viral, mammalian. prokaryotic. and plant promoters, and promoter element from yeast and other fungi.
  • Transcriptional control regions may exhibits tissue specificity or ubiquitous expression. Positive clones may be identified by any method known in art. See e.g. Ausubel, et al. CURRENT
  • vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the pBlueScript vector (Stratagene, La Jolla. CA).
  • Insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically "polished” to ensure compatibility.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and the LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO gene may be modified by homopolymeric tailing.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • Promoters which may be used include but are not limited to the SV40 early promoter (Bernoist and Chambon. 1981. Nature 290: 304-310); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980. Cell 22: 787-797); the Herpes thymidine kinase promoter (Wagner et ai. 1981 , Proc. Natl. Acad. Sci. USA 78: 1441-1445); the regulatory sequences of the metallothionein gene (Brinster et ai. 1982.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff et ai. 1978. Proc. Natl. Acad. Sci. USA 75: 3727-3731) or the tac promoter (DeBoer et al. 1983. Proc. Natl. Acad. Sci. USA 80: 21-25. see also "Useful Proteins from Recombinant Bacteria". In: Scientific American 1980. 242: 79-94); plant expression vectors comprising the nopaline synthetase promoter (Herrar-Estrella et ai. 1984. Nature 303: 209-213) or the cauliflower mosaic virus 35S
  • RNA promoter (Garder et ai. 1981. Nucleic Acids Res. 9: 2871), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et ai. 1984. Nature 310: 1 15-120); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter; and the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et ai, 1984.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et ai, 1986, Cell 45: 485-495), albumin gene control region which is active in liver (Pinckert et ai, 1987, Genes and Devel 1: 268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et ai.
  • alpha-1 antitrypsin gene control region which is active in liver (Kelsey et ai, 1987, Genes and Devel 1: 161-171), beta globin gene control region which is active in myeloid cells (Mogram et ai, 1985. Nature 315: 338-340; Kollias et al. 1986. Cell 46: 89-94).
  • myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et ai, 1987, Cell 48: 703-712), myosin light chain- 2 gene control region which is active in skeletal muscle (Sani 1985. Nature 314: 283-286), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al. 1986, Science 234: 1372-1378).
  • an expression vector containing the coding sequence, or a portion thereof, of LYST or LYST-2 and a LYST-IP either together or separately is made by subcloning the gene sequences into the EcoRI restriction site of one of the three pGEX vectors (glutathione S-transferase expression vectors: Smith and Johnson, 1988, Gene 7: 31-40; Promega Corp., Madison. WL). This allows for the expression of products in the correct reading frame.
  • Expression vectors containing the sequences of interest can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of marker gene function, and (c) expression of the inserted sequences. In the first approach. LYST. LYST-2.
  • GBDRl, OPA containing protein M4 protein.
  • LIP9. or LIPIO. or other LYST-IP sequences can be detected by nucleic acid hybridization to probes comprising sequences homologous and complementary to the inserted sequences.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain marker functions (e.g., binding to an anti-LYST, anti-LYST-
  • IP or anti-LYST:LYST-IP complex antibody, resistance to antibiotics, occlusion body formation in baculovirus, etc.
  • a LYST or LYST-2 or LYST-IP gene, or portion thereof is inserted within the marker gene sequence of the vector, recombinants containing the LYST or LYST-2 or LYST-IP fragment will be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying for LYST, LYST-2, 14-3-3 protein, HSl protein, Hrs, BMKl, KB07, Efs, XAP-4, OS9, casein kinase II beta SU, calmodulin, troponin I, importin beta, Fte-1, hERRal, imogen 38, atrophin-1, norbin, HBF-G2, DGS-I, GBDRl, OPA containing protein, M4 protein, LIPl, LIP2, LIP3, LIP4, LIP5.
  • LIP6, LIP7, LIP8, LIP9, or LIPIO or other LYST-IP products expressed by the recombinant vector.
  • assays can be based, for example, on the physical or functional properties of the interacting species in in vitro assay systems, e.g., formation of a LYST:LYST-IP complex or immunoreactivity to antibodies specific for the protein.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.
  • the individual gene product or complex can be isolated and analyzed. This is achieved by assays based on the physical and/or functional properties of the protein or complex, including, but not limited to. radioactive labeling of the product followed by analysis by gel electrophoresis. immunoassay, cross-linking to marker- labeled product, etc.
  • assays based on the physical and/or functional properties of the protein or complex, including, but not limited to. radioactive labeling of the product followed by analysis by gel electrophoresis. immunoassay, cross-linking to marker- labeled product, etc.
  • the expression vectors or derivatives which can be used include, but are not limited to. human or animal viruses such as vaccinia virus or adenovirus: insect viruses such as baculovirus; yeast vectors; bacteriophage vectors such as lambda phage; and plasmid and cosmid vectors.
  • a host cell strain may be chosen that modulates the expression of the inserted sequence, or modifies or processes the expressed protein in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically-engineered LYST or LYST-2 and/or LYST-IP may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation. phosphorylation, etc.) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein is achieved.
  • expression in a bacterial system can be used to produce an unglycosylated core protein
  • expression in mammalian cells can ensure native glycosylation of a heterologous mammalian protein.
  • different vector/host expression systems may effect processing reactions to different extents.
  • the LYST or LYST-2 and/or LYST-IP encoding nucleotide sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis and in vitro site-directed mutagenesis (Hutchinson et ai, 1978, J. Biol. Chem 253: 6551-6558), use of TABTM linkers (Pharmacia), etc.
  • host- vector systems may be utilized to express the protein coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g.. baculovirus); microorganisms such as yeast containing yeast vectors or bacteria transformed with bacteriophage DNA, plasmid DNA. or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g.. baculovirus
  • microorganisms such as yeast containing yeast vectors or bacteria transformed with bacteriophage DNA, plasmid DNA. or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • Different host cells have characteristic and specific mechanisms for the translational and post-translation
  • acetylation. phosphorylation of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the introduced protein is achieved.
  • any one of a number of suitable transcription and translation elements may be used, see, e.g., supra.
  • Manipulations of LYST or LYST-2 and or LYST-IP sequences may be made at the protein level (e.g. post-translational). Included within the scope of the invention are derivatives of complexes of LYST or LYST-IP fragments, derivatives or analogs thereof that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation. amidation, prenylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4. acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.; acetylation; formylation; oxidation; reduction; or modification by metabolic processes.
  • nucleotide coding sequences that encode substantially the same amino acid sequence as a LYST or LYST-2 or LYST-IP gene can be used in the practice of the present invention.
  • Said nucleotide sequences may be altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • Substitute codons for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic), polar neutral, positively charged (basic), and negatively charged (acidic) amino acids.
  • LYST or LYST-2 and LYST-IP derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of LYST or LYST-2 or a LYST-IP. including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine.
  • the polar neutral amino acids include glycine. serine. threonine. cysteine. tyrosine, asparagine. and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the LYST or LYST-2 and or LYST-IP protein sequences are modified to include a fluorescent label or to have a heterofunctional reagent; such heterofunctional reagents may be used to crosslink the protein to other members of the complex or to other LYST-IPs, or if desired, contain non-classical amino acids or chemical amino acid analogs introduced as a substitution or addition into the LYST or LYST-2 and/or a LYST-IP.
  • Non-classical amino acids include but are not limited to amino isobutyric acid, 3-amino propionoic acid, ornithine, norleucine. norvaline, hydroxyproline, sarcosine.
  • citrulline cysteic acid, t-butylglycine, t-butylalanine.
  • phenylglycine cyclohexylalanine, fluoro-amino acids, C- methyl amino acids, N-methyl amino acids, designer amino acids and amino acid analogs in general.
  • classical or non-classical amino acids can be D (dextrorotary) or L (levorotary).
  • analogs and derivatives of LYST or LYST-2 and/or a LYST-IP can be chemically synthesized.
  • a peptide corresponding to a portion of LYST or LYST-2 and or a LYST-IP, which comprises the desired domain or mediates the desired activity in vitro e.g., LYST:LYST-IP complex formation
  • LYST:LYST-IP complex formation can be synthesized by use of a peptide synthesizer.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the LYST or LYST-2 and or a LYST-IP.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, amino isobutyric acid, 4-aminobutyric acid (4-Abu), 2-aminobutyric acid (2-Abu), 6- amino hexanoic acid (e-Ahx), 2-amino isobutyric acid (Aib), 3-amino propionoic acid, ornithine, norleucine. norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine.
  • t- butylalanine phenylglycine, cyclohexylalanine. ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids. C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general.
  • classical or non-classical amino acids can be D (dextrorotary) or L (levorotary).
  • any of the methods described infra for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleotide sequences encoding LYST or LYST-2 and a LYST-IP. or a derivative, fragment or homolog thereof), may be regulated by a second nucleotide sequence so that the gene or gene fragment thereof is expressed in a host transformed with the recombinant
  • DNA molecule(s) DNA molecule(s).
  • expression of the proteins may be controlled by any promoter/enhancer known in the art.
  • the promoter is not native to the gene for LYST, LYST-2 or for LYST-IP.
  • Chimeric genes comprising portions of LYST or LYST-2 and/or a LYST-IP, or LIP-1, LIP-2, LIP-3. LIP-4. LIP-5. LIP-6, LIP-7, LIP-8, LIP-9, or LIP-10, fused to any heterologous protein-encoding sequences may be constructed.
  • a specific embodiment relates to a chimeric protein comprising a fragment of LYST and or a LYST-IP. or a fragment of LIP-1. LIP-2, LIP-3, LIP-4, LIP-5, LIP-6, LIP-7, LIP-8, LIP-9, or LIP- 10 protein, of at least six amino acids.
  • DNA fragments may be used to construct a chimeric gene in an expression vector using recombinant methodologies well known in the art.
  • the LYST or LYST-2 and or LYST-IP, or fragment, homolog or derivative thereof may be expressed as a fusion or chimeric protein product comprising the protein, fragment, homolog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein.
  • chimeric products can be made by ligating the appropriate nucleic acids encoding the desired amino acids to each other in the proper coding frame by methods known in the art, and expressing the chimeric products in a suitable host by methods commonly known in the art.
  • a chimeric product can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • the amino acid sequence of LYST or LYST-2. or a LYST-IP isolated from the natural source, as well as those expressed in vitro can be determined from analysis of the DNA sequence, or alternatively, by direct sequencing of the isolated protein. Such analysis may be performed by manual sequencing or through use of an automated amino acid sequencer.
  • the LYST. LYST-2, or LYST-IP proteins can be isolated and purified by standard methods known in the art. including but not restricted to column chromatography (e.g.. ion exchange, affinity, gel exclusion, reversed-phase high pressure, fast protein liquid, etc.). differential centrifugation. differential solubility, or by any other standard technique used for the purification of proteins.
  • Starting material may be either from natural sources or recombinant host cells expressing the complexes or proteins.
  • compositions comprising a LYST protein, a LYST-2 protein, a LYST-IP protein, or a LYST:LYST-IP complex may be purified to as little as 5% homogeneity, from 5% to 90% homogeneity, or from
  • a protein or its derivative can be synthesized by standard chemical methods known in the art (see. e.g., Hunkapiller et al, 1984, Nature 310: 105-
  • the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene from which it was encoded.
  • the protein or its derivative can be synthesized by standard chemical methods known in the art (see, e.g., Hunkapiller et al, 1984, Nature 310: 105-1 11).
  • the LYST:LYST-IP complex or LIPl, LIP2, LIP3, LIP4.
  • LIP5, LIP6, LIP7, LIP8, LIP9. or LIP10 protein may also be analyzed by hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828).
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the proteins, and help predict their orientation to aid in the design of substrates for experimental manipulation, such as in binding experiments, antibody synthesis, etc.
  • Secondary structural analysis can also be done to identify regions of the LYST or LYST-2 and/or a LYST-IP that assume specific structures (Chou and Fasman, 1974, Biochemistry 13: 222-223). Manipulation, translation, secondary structure prediction, hydrophilicity and hydrophobicity profiles, open reading frame prediction and plotting, and determination of sequence homologies. can be accomplished using computer software programs available in the art.
  • the present invention relates to LYST-2.
  • the fragment, derivative or analog is functionally active, i.e.. capable of exhibiting one or more functional activity associated with wild type LIPl.
  • BMKl BMKl , KB07.
  • Homologs or paralogs of species other than human can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning, see, e.g., as described infra. Such hybridization conditions and techniques are well known in the art.
  • Nucleic acids encoding LYST, LYST-2, and LYST-IPs disclosed herein can be obtained by any method known in the art, e.g., by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide specific for the gene sequence, see e.g., as described infra.
  • LYST or LYST-2 or LYST-IP gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 1989, MOLECULAR CLONING, A
  • sequences can be cleaved at appropriate sites with restriction endonuclease(s), modified further by enzymatic manipulation if desired, isolated, and ligated in vitro.
  • LYST or LYST-2 and or LYST-IP can be made by altering their respective sequence by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as a LYST or LYST-2 or LYST-IP gene can be used in the practice of the present invention. Said nucleotide sequences may be altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Substitute codons for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic). polar neutral, positively charged (basic), and negatively charged (acidic) amino acids.
  • the LYST. LYST-2. or LYST-IP derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned LYST or LYST-2 or
  • LYST-IP gene sequence can be modified by any of numerous strategies known in the art
  • Chimeric genes comprising portions of LYST or LYST-2 and/or a LYST-IP. or LIPl. LIP2, LIP3, LIP4. LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO, fused to any heterologous protein- encoding sequences may be constructed.
  • a specific embodiment relates to a chimeric protein comprising a fragment of LYST and/or a LYST-IP, or a fragment of LIPl, LIP2, LIP3.
  • fusion proteins are provided that contain the interacting domains of the LYST or LYST-2 protein and a LYST-IP and/or, optionally, a hetero-functional reagent, such as a peptide linker between the two domains, where such a reagent promotes the interaction of the LYST or LYST-2 and LYST-IP binding domains.
  • a hetero-functional reagent such as a peptide linker between the two domains, where such a reagent promotes the interaction of the LYST or LYST-2 and LYST-IP binding domains.
  • fusion proteins may be particularly useful where the stability of the interaction is desirable (due to the formation of the complex as an intra-molecular reaction), for example in production of antibodies specific to the LYST:LYST-IP complex.
  • LIP6 LIP6
  • LIP7, LIP8, LIP9. or LIPIO derivatives can be made by altering their respective sequence by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • other DNA sequences that encode substantially the same amino acid sequence as a LYST or LYST-2 or LYST-IP gene can be used in the practice of the present invention. These include but are not limited, to a nucleotide sequence comprising all or a portion of LYST, LYST-2. or a LYST-IP gene that is altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • LYST or LYST-2 or a LYST-IP are provided.
  • the fragment consists of at least about 10, 20, 30, 40, or 50 amino acids of LYST or LYST-2 or a LYST-IP. In specific embodiments, such fragments are not larger than about 35. 100 or 200 amino acids.
  • Derivatives or analogs of LYST or LYST-2 and LYST-IPs include, but are not limited to.
  • molecules comprising regions that are substantially homologous to LYST or LYST-2 or LYST-IPs, in various embodiments, by at least about 30%, and ranging up to 99% identity (with a preferred identity of 90-95% ) over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art.
  • derivatives or analogs of LYST or LYST-2 and LYST-IPs may differ by as little as one chemical moiety or side group.
  • Derivatives or analogs of LYST or LYST-2 and LYST-IPs whose encoding nucleic acid is capable of hybridizing to the complement (e.g., the inverse complement) of a sequence encoding LYST or LYST-2 or a LYST-IP under stringent, moderately stringent, or nonstringent conditions, as described supra, are also provided.
  • such LYST:LYST-IP complex or LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 protein, whether produced by recombinant DNA techniques, chemical synthesis methods, or by purification from native sources, include but are not limited to those containing as a primary amino acid sequence all or part of the amino acid sequences substantially, as well as fragments and other analogs and derivatives thereof, including proteins homologous thereto.
  • the amino acid sequence of LYST or LYST-2, or a LYST-IP isolated from the natural source, as well as those expressed in vitro, or from synthesized expression vectors in vivo or in vitro can be determined from analysis of the DNA sequence, or alternatively, by direct sequencing of the isolated protein. Such analysis may be performed by manual sequencing or through use of an automated amino acid sequenator.
  • the present invention relates to the nucleotide sequences encoding a LIPl .
  • the LIPl, LIP2, LIP3. LIP4. LIP5. LIP6. LIP7. LIP8. LIP9. or LIP10 nucleic acid sequence comprises the sequence of SEQ ID NOS: 3. 5. 7. 9. 1 1. 13. 15, 17. 19, and 21. respectively, or a portion thereof. or a nucleotide sequence encoding, in whole or in part, a LIPl. LIP2, LIP3, LIP4. LIP5, LIP6.
  • LIP7, LIP8. LIP9. or LIPIO protein e.g., a protein comprising the amino acid sequence of SEQ
  • the invention provides purified nucleic acids consisting of at least 6 nucleotides (i.e., a hybridizable portion) of a LYST-2. LIPl . LIP2, LIP3, LIP4. LIP5, LIP6. LIP7. LIP8. LIP9, or LIPIO sequence. In other embodiments, the nucleic acids consist of at least about 25 (continuous) nucleotides. 50 nucleotides. 100 nucleotides, 150 nucleotides, or 200 nucleotides of a LIPl, LIP2. LIP3, LIP4,
  • nucleic acids are smaller than about 35, 200 or 500 nucleotides in length. Nucleic acids can be single or double stranded.
  • the invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences, in particular the invention provides the inverse complement to nucleic acids hybridizable to the foregoing sequences (i.e., the inverse complement of a nucleic acid strand has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize with little or no mismatches to the nucleic acid strand; thus, for example, where the coding strand is hybridizable to a nucleic acid with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding strand).
  • nucleic acid molecules comprise a sequence complementary to (specifically are the inverse complement of) at least about 10. 25. 50. 100, or 200 nucleotides or the entire coding region of a LIPl. LIP2. LIP3, LIP4, LIP5. LIP6. LIP7, LIP8, LIP9, or LIP10 gene.
  • LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 nucleic acid sequence e.g., having sequence SEQ ID NOS: 3, 5, 7. 9, 1 1, 13, 15, 17, 19, and 21, respectively
  • LIP10 protein derivative e.g.
  • a nucleic acid sequence which is hybridizable to a LIPl. LIP2, LIP3, LIP4. LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 nucleic acid sequence (or a complement of the foregoing), or to a nucleic acid sequence encoding a derivative of the same, under conditions of high stringency is provided. It should be noted that the most preferred embodiment of the present invention utilizes high stringency hybridization conditions.
  • a nucleic acid sequence which is hybridizable to a LIPl. LIP2. LIP3, LIP4, LIP5. LIP6. LIP7. LIP8. LIP9, or LIP10 nucleic acid sequence or to a nucleic acid sequence encoding a LIPl . LIP2. LIP3. LIP4, LIP5, LIP6. LIP7. LIP8. LIP9. or LIP10 derivative (or a complement of the foregoing) under conditions of moderate stringency is provided. For example, but not limited to. procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhardt's solution.
  • LIP5, LIP6, LIP7. LIP8, LIP9. or LIP10 proteins (supra), or antisense nucleic acids to the same (see, e.g., infra) are additionally provided.
  • a "nucleic acid encoding a fragment or portion of a LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9. or LIP10 protein, and not the other contiguous portions of the LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 as a continuous sequence.
  • potential open reading frames can be identified using the NCBI BLAST program ORF Finder available to the public. Because all known protein translation products are at least 60 amino acids or longer (Creighton, 1992, PROTEINS, 2nd Ed., W.H. Freeman and Co.. New York), only those ORFs potentially encoding a protein of 60 amino acids or more are considered. If an initiation methionine codon (ATG) and a translational stop codon (TGA, TAA, or TAG) are identified, then the boundaries of the protein are defined. Other potential proteins include any open reading frames that extend to the 5' end of the nucleotide sequence, in which case the open reading frame predicts the C-terminal or core portion of a longer protein.
  • any open reading frame that extends to the 3' end of the nucleotide sequence predicts the N-terminal portion of a longer protein.
  • This methodology was used to identify open reading frames encoding the interactant LIPl, LIP2. LIP3, LIP4, LIP5. LIP6, LIP7, LIP8. LIP9. or LIP10 Any method available in the art can be used to obtain a full length (i.e.. encompassing the entire coding region) cDNA clone encoding a LIPl , LIP2, LIP3. LIP4, LIP5. LIP6. LIP7, LIP8, LIP9, or LIP10 protein.
  • PCR polymerase chain reaction
  • Oligonucleotide primers that hybridize to sequences at the 3 " and 5 " termini of the identified sequences can be used as primers to amplify by PCR sequences from a nucleic acid sample (cDNA or DNA).
  • a cDNA library from an appropriate source (e.g., the sample from which the initial cDNA library for the modified yeast two hybrid assay fusion population was derived, or. e.g. the genomic DNA of the same).
  • PCR can be carried out. e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase.
  • the DNA being amplified can include genomic DNA or cDNA sequences from any eukaryotic species.
  • It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions to amplify nucleic acid homologs e.g., to obtain LIPl, LIP2.
  • LIP3, LIP4. LIP5, LIP6, LIP7. LIP8. LIP9, or LIPIO by allowing for greater or lesser degrees of nucleotide sequence similarity between the known nucleotide sequence and the nucleic acid homolog being isolated.
  • LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO sequence that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In this fashion, the nucleotide sequence of the entire LIPl, LIP2, LIP3, LIP4. LIP5. LIP6.
  • LIP7, LIP8, LIP9, or LIPIO gene as well as additional genes encoding a LIPl, LIP2, LIP3, LIP4.
  • LIP5, LIP6, LIP7, LIP8, LIP9. or LIPIO protein or analog, may be identified.
  • Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of a LIPl.
  • the nucleic acids can be isolated from vertebrates, including human and other primate sources, porcine, bovine, feline, avian. equine, canine, as well as additional mammalian sources, and insects, plants, etc.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA.
  • Clones derived from genomic DNA may contain regulatory and intronic DNA regions in addition to coding regions: clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNase in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and or polyacrylamide gel electrophoresis, and column chromatography. Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, a portion of the LIPl, LIP2, LIP3, LIP4, LIP5.
  • LIP6, LIP7, LIP8, LIP9, or LIPIO gene (of any species) (e.g., a PCR amplification product obtained as described above, or an oligonucleotide having a sequence of a portion of the known nucleotide sequence) or its specific RNA. or a fragment thereof, may be purified and labeled, and the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196: 180-182; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72: 3961-3964). Those DNA fragments with substantial homology to the probe will hybridize.
  • LIPl LIP2, LIP3, LIP4, LIP5. LIP6, LIP7, LIP8, LIP9, or LIPIO. If an anti-LIPl, anti-LIP2, anti-LIP3, anti-LIP4, anti-LIP5. anti-LIP6. anti-LIP7, anti-LIP8, anti-LIP9 or anti-LIPIO antibody is available, the protein may be identified by binding of labeled antibody to the putative LIPl. LIP2, LIP3, LIP4, LIP5. LIP6. LIP7.
  • LIP8. LIP9, or LIP10 synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay )-type procedure.
  • LIPIO cDNA includes, but is not limited to, chemically synthesizing the gene sequence itself from a known sequence. Other methods are possible and within the scope of the invention. The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated LIPl, LIP2, LIP3, LIP4. LIP5, LIP6, LIP7, LIP8. LIP9. or LIPIO gene, cDNA. or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO nuclear acid sequence includes those nucleotide sequences encoding substantially the same amino acid sequence as found in the respective native protein, those encoded amino acid sequences with functionally equivalent amino acids, and those sequences encoding other LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO derivatives or analogs, as described supra.
  • LIP3, LIP4, LIP5, LIP6. LIP7, LIP8, LIP9, or LIPIO protein or fragments, homologs and derivatives thereof may be used as immunogens to generate antibodies which immunospecifically bind such immunogens.
  • Said antibodies include but are not limited to, polyclonal.
  • antibodies to complexes of human LYST or LYST-2 and a human LYST-IP are produced.
  • complexes formed from fragments of a LYST or LYST-2 and a LYST-IP. where the fragments contain the protein domain that interacts with the other member of the complex are used as immunogens for antibody production.
  • LIP6. LIP7, LIP8, LIP9, or LIPIO proteins or fragments, derivatives, or homologs thereof are used as immunogens.
  • LIPl LIP2. LIP3. LIP4. LIP5. LIP6. LIP7. LIP8, LIP9, or LIPIO protein, or derivative, fragment or analog thereof.
  • various host animals can be immunized by injection with the native LYST:LYST-IP complex, or LYST-2, LIPl, LIP2, LIP3. LIP4. LIP5. LIP6, LIP7, LIP8, LIP9, or LIPIO protein, or a synthetic version, or a derivative of the foregoing, such as a cross-linked LYST:LYST-IP.
  • host animals include but are not limited to rabbits, mice, rats. etc.
  • adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin (BCG) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • LIPl For preparation of monoclonal antibodies directed towards a LYST:LYST-IP complex or to a LYST-2, LIPl, LIP2, LIP3, LIP4.
  • LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein, or derivatives, fragments or analogs thereof any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • Such techniques include but are not restricted to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497), the trioma technique (Rosen et al, 1977, Cell 11: 139-147), the human B- cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4: 72), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al, 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • monoclonal antibodies can be produced in germ-free animals (See International Application No. PCT/US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (Cole et al, 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030), or by transforming human B cells with EBV virus in vitro (Cole et al.. 1985, In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77- 96).
  • techniques developed for the production of chimeric antibodies (Morrison et al.. 1984. Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al. , 1984.
  • LIP6 LIP7. LIP8. LIP9, or LIPIO protein-specific single chain antibody.
  • An additional embodiment of the invention utilizes techniques described for the construction of Fab expression libraries (Huse et al. 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the LYST:LYST-IP complex, or an individual LYST-2, LIPl, LIP2, LIP3, LIP4. LIP5. LIP6,
  • Non-human antibodies can be humanized by known methods (see, e.g., U.S. Patent No. 5,225,539).
  • LIPl, LIP2, LIP3, LIP4, LIP5, LIP6. LIP7, LIP8, LIP9, or LIPIO protein can be generated by techniques known in the art.
  • fragments include but are not limited to: the F(ab) 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of the F(ab) 2 fragment; the Fab fragments that can be generated by treating the antibody molecular with papain and a reducing agent; and Fv fragments.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies specific to a domain of the LYST:LYST-IP complex are also provided, as are antibodies to specific domains of the LYST-2.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and/or quantitation of a LYST:LYST-IP complex or of a LYST-2.
  • LIPl . LIP2, LIP3. LIP4. LIP5. LIP6. LIP7. LIP8. LIP9. or LIPIO protein of the invention e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods. etc.
  • anti-LYST LYST-IP complex antibodies and fragments thereof, or anti-LYST-2.
  • anti-LIPl anti-LIP2.
  • ant-LIP5. anti- LIP6, anti-LIP7, anti-LIP8. or anti-LIP9, or anti-LIPIO antibodies or fragments thereof. containing the binding domain, are therapeutics, as described infra.
  • LYST:LYST-IP complexes may be markers of normal physiological processes including, but not limited to, the physiological processes including signal transduction, vesicular transport, the formation and exocytosis of acidic intracellular organelles, and protein trafficking; and pathological processes including but not restricted to, atopic diseases, autoimmune diseases, degenerative disorders including neurodegenerative disease, hyperproliferative disorders including tumorigenesis and tumor progression, oculocutaneous albinism and hypopigmentation and platelet dysfunction, and thus have diagnostic utility. Further, definition of particular groups of patients with elevations or deficiencies of a LYST:LYST-IP complex, or a LYST-2. LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein, can lead to new classifications of diseases, furthering diagnostic ability.
  • Detecting levels of LYST:LYST-IP complexes or individual proteins that have been shown to form complexes with LYST or LYST-2, or a LYST-2.
  • LIP5, LIP6, LIP7, LIP8, or LIP9 protein may be used in prognosis, to follow the course of disease state, to follow therapeutic response, etc.
  • Such molecules can be used in assays, such as immunoassays. to detect, prognose. diagnose, or monitor various conditions, diseases, and disorders, and treatment thereof, characterized by aberrant levels of LYST:LYST-IP complexes, or by aberrant levels of
  • LIPl LIP2, LIP3, LIP4, LIP5, LIP6. LIP7. LIP8. LIP9, or LIPIO protein.
  • an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-LYST:LYST-IP complex antibody, or an antibody specific to LYST-2, LIPl, LIP2. LIP3. LIP4. LIP5, LIP6, LIP7, LIP8, LIP9. or LIPIO, under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding that occurs by such an antibody.
  • such binding of antibody for example in tissue sections, can be used to detect aberrant LYST:LYST-
  • IP complex formation or aberrant LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein localization, or aberrant (e.g., high, low or absent) levels of LYST:LYST- IP complex or complexes, or aberrant levels of LYST-2, LIPl, LIP2, LIP3. LIP4. LIP5, LIP6, LIP7. LIP8. LIP9, or LIPIO protein.
  • an antibody against a LYST:LYST-IP complex can be used to assay a patient tissue or serum sample for the presence of the LYST:LYST-IP complex, where an aberrant level of the LYST:LYST-IP complex is an indication of a disease condition.
  • an antibody against LYST-2, LIP 1 , LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO can be used to assay a patient tissue or serum sample for the presence of said protein, where an aberrant level of LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO is an indication of a disease condition.
  • aberrant levels is meant increased or decreased levels relative to levels previously found present, or to a standard level in an analogous sample either from another portion of a body or from a subject not having the disorder. Immunoassay s that can be used are described infra.
  • Nucleic acids encoding the components of the LYST:LYST-IP complexes and nucleic acids encoding a LYST-2, LIPl, LIP2. LIP3, LIP4, LIP5, LIP6, LIP7, LIP8. LIP9, or LIPIO protein, and related nucleotide sequences and subsequences, including complementary sequences, can also be used in hybridization assays.
  • the LYST or LYST-2 and or LYST-IP nucleotide sequence, or a subsequence thereof, comprising about at least 6 nucleotides, can be used as hybridization probes. Hybridization assays can be used to detect, prognose.
  • such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to LYST or LYST-2 or a LYST-IP DNA or RNA. under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization. See supra for hybridization conditions.
  • the hybridization assay is carried out using nucleic acid probes capable of hybridizing to LYST or
  • LYST-2 and to a binding partner of LYST or LYST-2 to measure concurrently the expression of both members of a LYST:LYST-IP complex.
  • the expression of mRNAs encoding LYST-2. LIPl, LIP2, LIP3. LIP4. LIP5. LIP6, LIP7. LIP8. LIP9. or LIPIO is measured.
  • diseases and disorders involving or characterized by aberrant levels of LYST:LYST-IP complexes can be diagnosed, or their suspected presence can be screened for. or a predisposition to develop such disorders can be detected, by detecting aberrant levels of a LYST:LYST-IP complex, or un-complexed LYST or LYST-2 and/or a LYST-IP protein or nucleic acids or functional activity, including but not restricted to, binding to an interacting partner, or detecting mutations in LYST or LYST-2 and or in a LYST-IP RNA.
  • DNA or protein e.g., translocations, truncations, changes in nucleotide or amino acid sequence relative to wild-type LYST and/or LYST-IP
  • DNA or protein e.g., translocations, truncations, changes in nucleotide or amino acid sequence relative to wild-type LYST and/or LYST-IP
  • LYST:L YST-IP complex and or LYST or LYST-2 and/or protein that binds to LYST or L YST- 2.
  • diseases and disorders include but are not limited to those described in later subsections, infra.
  • levels of a LYST:L YST-IP complex or the individual components of a LYST:L YST-IP complex can be detected by immunoassay; levels of LYST and or of LYST-IP mRNA can be detected by hybridization assays (e.g.. Northern blots, dot blots); binding of LYST or to a LYST-IP can be measured by binding assays commonly known in the art.
  • translocations and point mutations in LYST or LYST-2 and/or in genes encoding a LYST-IP can be detected by Southern blotting, RFLP analysis, PCR using primers that preferably generate a fragment spanning at least most of the LYST or LYST-2 and or LYST-IP gene, sequencing of the LYST or LYST-2 and/or LYST-IP genomic DNA or cDNA obtained from the patient, etc.
  • Assays well known in the art can be used to determine whether one or more particular LYST:LYST-IP complexes are present at either increased or decreased levels, or are absent, in samples from patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder as compared to the levels in samples from subjects not having such a disease or disorder.
  • these assays can be used to determine whether the ratio of the LYST:LYST- IP complex to the un-complexed components of the LYST:L YST-IP complex, i.e.. LYST or LYST-2 and/or the specific LYST-IP in the complex of interest, is increased or decreased in samples from patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, as compared to the ratio in samples from subjects not having such a disease or disorder.
  • LYST:LYST-IP complexes are determined to be altered in patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, then the particular disease or disorder or predisposition for a disease or disorder can be diagnosed, have prognosis defined for, be screened for, or be monitored by detecting altered levels of the one or more LYST:LYST-IP protein complexes, the mRNA that encodes the members of the one or more particular LYST:L YST-IP complexes, or LYST:LYST-IP complex functional activity.
  • diseases and disorders involving increased levels of one or more LYST:L YST-IP complexes can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting increased levels of the one or more LYST:L YST-IP complexes, the mRNA encoding both members of the complex, or complex functional activity, or by detecting mutations in LYST or LYST-2 or the LYST-IP (e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type LYST or LYST-2 or LYST-IP) that stabilize or increase LYST:LYST-IP complex formation.
  • mutations in LYST or LYST-2 or the LYST-IP e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type LYST
  • diseases and disorders involving decreased levels of one or more LYST:L YST-IP complexes can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting decreased levels of the one or more LYST:L YST-IP complexes, the mRNA encoding the members of the one or more complexes, or complex functional activity, or by detecting mutations in LYST or LYST-2 or the LYST-IP (e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild- type LYST or LYST-2 or the LYST-IP) that inhibit or reduce LYST:LYST-IP complex formation.
  • mutations in LYST or LYST-2 or the LYST-IP e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to
  • diseases and disorders involving aberrant expression of a LYST-2, LIPl, LIP2. LIP3. LIP4. LIP5. LIP6, LIP7, LIP8, LIP9. or LIPIO protein are diagnosed, or their suspected presence can be screened for. or a predisposition to develop such disorders can be detected, by detecting aberrant levels of LYST-2. LIPl . LIP2. LIP3. LIP4. LIP5. LIP6. LIP7. LIP8, LIP9, or LIPIO proteins, or mRNA, or functional activity, or by detecting mutations in LYST-2. LIPl. LIP2. LIP3. LIP4. LIP5. LIP6. LIP7, LIP8, LIP9.
  • LIPIO protein or mRNA or DNA e.g., translocations in nucleic acids, truncations in the gene or protein. changes in nucleotide or amino acid sequence relative to wild-type LYST-2 or a wild-type novel
  • LIP gene or protein that cause aberrant expression or activity of said LYST-2 or said novel LIP protein.
  • diseases and disorders include but are not limited to those described infra.
  • LIP6, LIP7, LIP8. LIP9. or LIPIO mRNA or protein, LYST or LYST-2 binding activity, or the presence of translocations or point mutations, can be determined as de ribed above.
  • Assays well known in the art can be used to determine whether LYST- 2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO are present at either increased or decreased levels, or are absent, in samples from patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, as compared to the levels in samples from subjects not having such a disease or disorder.
  • LIP9, or LIPIO are determined to be altered in patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, then the particular disease or disorder or predisposition for a disease or disorder can be diagnosed, have its prognosis determined, be screened for, or be monitored by detecting altered levels of protein, mRNA, or functional activity (e.g., binding to LYST) of said LYST-2 or novel LIP.
  • diseases and disorders involving increased levels of a LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting increased levels of a LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP 10 protein, encoding nucleic acids, or functional activity, or by detecting mutations in LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5. LIP6.
  • LIP7, LIP8, LIP9, or LIPIO e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type LYST-2 or novel LIP
  • diseases and disorders involving decreased levels of LYST-2, LIPl . LIP2. LIP3. LIP4, LIP5, LIP6, LIP7. LIP8. LIP9. or LIPIO can be diagnosed, or their suspected presence can be screened for. or a predisposition to develop such disorders can be detected, by detecting decreased levels of LIPl. LIP2. LIP3. LIP4. LIP5.
  • LIP6 LIP7. LIP8. LIP9. or LIPIO protein, nucleic acids, or functional activity, or by detecting mutations in LYST-2, LIPl. LIP2. LIP3. LIP4. LIP5. LIP6. LIP7. LIP8. LIP9, or
  • LIPIO e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type LYST-2 or novel LIP
  • LIPl LIP2.
  • LIP9, or LIPIO functional activity LIPIO (e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type LYST-2 or novel LIP) that destabilize or reduce LYST-2, LIPl. LIP2.
  • LIP6. LIP7, LIP8. LIP9, or LIPIO functional activity LIPIO
  • LYST:L YST-IP complexes or against a LYST-2, LIPl, LIP2, LIP3, LIP4.
  • LIP8, LIP9, or LIPIO protein provides a method of detecting specific cells that express the complex or protein.
  • specific cell types can be defined in which one or more particular LYST:LYST-IP complex, or said individual LYST-2 or novel LIP protein, is expressed, and the presence of the complex or protein can be correlated with cell viability.
  • This embodiment includes cell sorting of prokaryotes such as, but not restricted to, bacteria (Davey and Kell, 1996, Microbiol. Rev.
  • tissue specimens from eukaryotes. including mammalian species such as human (Steele et al, 1996, Clin. Obstet. Gynecol 39: 801-813), and continuous cell cultures (Orfao and Ruiz-Arguelles, 1996, Clin. Biochem. 29: 5-9). Such isolations can also be used as methods of diagnosis, described supra.
  • Kits for diagnostic use comprise in one or more containers an anti- LYST:LYST-IP complex antibody or an antibody specific to LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO, and, optionally, a labeled binding partner to the antibody.
  • the anti-LYST:LYST-IP complex antibody, or antibody specific to LYST-2, LIPl , LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8. LIP9, or LIPIO can be labeled with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
  • kits comprises in one or more containers a nucleic acid probe capable of hybridizing to LYST or LYST-2 and or a LYST-IP mRNA.
  • a kit can comprise in one or more containers a pair of primers (e.g., each in the size range of about 6-40 nucleotides) that are capable of priming amplification [e.g , by polymerase chain reaction (see e.g., Innis et al. 1990, PCR PROTOCOLS, Academic Press. Inc.. San Diego. CA).
  • ligase chain reaction see EP 320,308). use of Q ⁇ replicase.
  • kits can optionally further comprise in a container a predetermined amount of a purified LYST:LYST-IP complex. LYST or LYST-2 and/or a LYST-IP. or said individual
  • LYST-2 or novel LIP protein or an encoding nucleic acid molecule thereof, e.g., for use as a standard or control.
  • the invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic”).
  • “Therapeutics” include but are not limited to: LYST:LYST-IP complexes, LYST and the individual LYST-IP proteins and analogs and derivatives (including fragments) of the foregoing (e.g., as described herein above); antibodies there to (as described herein above); nucleic acids encoding LYST or LYST-2 and or a LYST-IP, and analogs or derivatives thereof (e.g., as described herein above); LYST or LYST-2 and/or LYST-IP antisense nucleic acids, and LYST:LYST-IP complex and LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO modulators (i.e., inhibitors, agonists and antagonists).
  • LYST and LYST-2 are centrally implicated in physiological processes including but not limited to signal transduction, vesicle morphology, the formation and exocytosis of acidic intracellular organelles. and sorting of proteins which is necessary for function of intracellular organelles.
  • LYST has been strongly implicated in pathological conditions, including but not limited to, atopic diseases (including bronchial asthma), severe immunologic deficiency with deficient functions of granulocytes. lymphocytes and natural killer cells, oculocutaneous albinism, hypopigmentation, platelet dysfunction (bleeding diathesis), neurodegeneration such as peripheral neuropathy and ataxia. and cancer.
  • LYST has been strongly implicated in protection from pathological conditions including but not limited to autoimmune disorders (including systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease).
  • LYST or LYST-2 interactants described in the present invention are likewise involved in signal transduction and secretion processes including calcium-dependent exocytosis, see BACKGROUND Section, supra. This relates to particularly 1 -3-3 protein. HSl protein. Hrs. BMKl . KB07. Efs. XAP-4. OS9, HRS. calmodulin. Troponin I. Fte-1. casein kinase II subunit beta, and norbin. A linkage between the signal transduction disorders and induction of apoptosis exists, which may relate to LYST. LYST-2. and particularly the interactants 14-3-3 protein, HSl protein, Efs (Fyn-associated substrate), HRS. calmodulin. casein kinase II subunit beta, and norbin.
  • LYST or LYST-2 interactants found in this invention with a role in autoimmune-diseases and inflammation are particularly the estrogen-receptor related protein, 14-3-3 proteins, and
  • Imogen 38 The estrogen-receptor related protein is expressed in human arthritic synovium. and estrogen modulates local inflammation in various diseases. 14-3-3 proteins play a role in autoimmune-diseases, for example, by interacting with insulin receptor substrate 1.
  • Imogen 38 is an mitochondrial autoantigen which is a target for autoimmune attack in diabetes.
  • LYST and LYST-2 and most of its binding partners are significantly implicated in disorders of neurodegeneration, see BACKGROUND Section, supra. 14-3-3 is present in Alzheimer's Disease neurofibrillary tangles and in the cerebrospinal fluid of patients with Creutzfeldt- Jakob disease. The ATPase Hrs is implicated in calcium-regulated secretion. Calmodulin is involved in neurotransmitter release and activated exocytosis in neuroendocrine cells.
  • the phosphoprotein Efs is associated with the tyrosine- kinase Fyn, which is involved in many signal transduction processes, including those in neuronal cells.
  • Importin beta is a nuclear protein involved in nuclear protein translocation.
  • Atrophin-I is the protein product of the gene implicated in dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease) and is ubiquitously expressed in neuronal tissues.
  • the LYST-2 interactant HBF-G3 is a brain factor expressed in fetal tissues and is likely to be involved in development and embryogenesis.
  • DGS-I is the protein associated with DiGeorge syndrome, a developmental defect which is characterized by hypoplasia or aplasia of the thymus and parathyroids, facial dysmorphism and conotruncal cardiac malformations.
  • LYST LYST-2.
  • HSl protein and estrogen-receptor related protein.
  • Chediak-Higashi patients have an increased tumor growth rate and higher metastatic frequency.
  • non-Hodgkin's lymphoma occurred in over one third of CHS patients (Hayakawa et al. 1986. Jpn. J. Cancer Res. 77: 74-79).
  • the effect of the LYST protein on tumorigenesis may be due to the involvement of natural killer cells in cancer.
  • Norbin may be accompanied with neurite-outgrowth in neuro-2a cells and may play a role in the formation of new synapses. Thus, it may be involved in disorders of neurodegeneration.
  • Importin beta subunit is implicated in cellular response to viral infection, including HIV- 1 (AIDS).
  • LIP2 Most of the novel LYST interactants encoded by the genes for LIPl. LIP2. LIP3, LIP4, LIP5. LIP6, LIP7, LIP8, LIP9, or LIPIO can also be related to the implicated functions of LYST and LYST-2 and the interactants described above.
  • Tcp-10 human T-complex protein
  • cytoskeletal and vesicular proteins Two interactants, LIPl and LIP7, have homologies to human T-complex protein (Tcp)-lO and also to cytoskeletal and vesicular proteins.
  • Tcp-10 genes are expressed exclusively in male germ cells during spermatogenesis.
  • Tcp-10 has a conserved C-terminal region with unique nonapeptide repeat and a conserved N-terminal region containing a pair of leucine zippers within a region that shows additional similarity to the coiled-coil regions of various cytosolic polypeptides (Islam et al, Human Mol. Genet. 2: 2075-2079).
  • LIP2 and LIP9 may have nuclear function.
  • LIP2 is homologous to ribosomal protein LI 7.
  • LIP9 is homolog to the Xenopus laevis elav-type ribonucleoprotein Etr-1, a putative RNA-binding protein found in neural tissue, and thus, LIP9 might be involved in neurodegenerative diseases.
  • LIP3 Another interactant, LIP3, may be involved in disorders of neurodegeneration.
  • LIP3 is homolog to rat O/E-l -associated zinc finger protein (Roaz), that plays a role in regulating the temporal and spatial pattern of olfactory neuronal-specific gene expression.
  • Roaz protein functions by interacting with the olfactory factor O/E- 1 and modulating its transcriptional activity.
  • the Roaz mRNA was found in brain, eye, olfactory epithelium, spleen, and heart and was expressed in the basal layer, consisting of neural precursor cells and immature sensory neurons of the olfactory epithelium, but not in the mature receptor cells (Tsai & Reed, 1997, J. Neurosci.
  • LIP-8 is the human homolog of the rat norbin gene.
  • Rat norbin is expressed in the brain and is induced by treatment of tetraethylammonium in rat hippocampal slice accompanied with neurite-outgrowth in neuro-2a cells.
  • the neurite-outgrowth-related norbin protein may play a role in neural plasticity because of the formation of new synapses.
  • LYST:LYST-IP complex activity A wide range of cell diseases affected by intracellular signal transduction. vesicle transport and protein trafficking can be treated or prevented by administration of a Therapeutic that modulates (i.e.. inhibits, antagonizes, enhances or promotes) LYST:LYST-IP complex activity. All of these disorders can be treated or prevented by administration of a Therapeutic that modulates (i.e.. inhibits, antagonizes, enhances or promotes) LYST:L YST-IP complex activity, or modulates LYST-2. LIPl. LIP2, LIP3. LIP4. LIP5. LIP6. LIP7. LIP8. LIP9, or LIPIO activity.
  • LIP5 LIP6. LIP7, LIP8, LIP9, or LIPIO.
  • LIPIO LIP5
  • LIP6. LIP7, LIP8, LIP9, or LIPIO may be treated by administration of a Therapeutic that modulates LYST:L YST-IP complex formation or activity, or LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9. or LIPIO activity.
  • the activity or level of LYST is modulated by administration of a LYST-IP.
  • the activity or level of a LYST-IP is modulated by administration of LYST or LYST-2.
  • LYST:LYST-IP complex Diseases and disorders associated with underexpression of a LYST:LYST-IP complex, or LYST or LYST-2 or a particular LYST-IP, are treated or prevented by administration of a Therapeutic that promotes (i.e., increases or supplies) LYST:L YST-IP complexes or function.
  • Examples of such a Therapeutic include but are not limited to LYST:L YST-IP complexes and derivatives, analogs and fragments thereof that are functionally active (e.g., active to form LYST:LYST-IP complexes), un-complexed LYST or LYST-2 and LYST-IP proteins, and derivatives, analogs, and fragments thereof, and nucleic acids encoding the members of a LYST:L YST-IP complex, or functionally active derivatives or fragments thereof (e.g., for use in gene therapy).
  • LYST:L YST-IP complex or functionally active derivatives or fragments thereof (e.g., for use in gene therapy).
  • derivatives, homologs or fragments of LYST or LYST-2 and or a LYST-IP that increase and/or stabilize LYST:LYST-IP complex formation.
  • Examples of other agonists can be identified using in vitro assays or animal models, examples of which are described herein.
  • LYST:LYST-IP levels or activity can be treated with Therapeutics that antagonize (i.e.. reduce or inhibit) LYST:LYST-IP complex formation or activity, or LYST-2.
  • LIPl . LIP2. LIP3. LIP4.
  • Therapeutics that can be used include but are not limited to.
  • LYST or LYST-2 or a LYST-IP. or analogs, derivatives or fragments thereof; anti-LYST:LYST-IP complex antibodies e.g., antibodies specific for LYST: 14-3-3 protein.
  • LYST.BMK1 LYST:KB07, LYST:Efs, LYST:OS9. LYSTxasein kinase II beta SU.
  • LYST-2 14-3-3
  • LYST-2 XAP-4 complexes
  • antibodies specific to LYST-2, LIPl antibodies specific to LYST-2, LIPl
  • LYST-2 or individual novel LIP antisense nucleic acids or of LYST or LYST-2 and LYST-IP nucleic acids or of individual LYST-2 or novel LIP nucleic acids that are dysfunctional (e.g., due to a heterologous (non-LYST or LYST-2 and/or non-LYST-IP) insertion within the coding sequences of the LYST or LYST-IP coding sequences)) that are used to "knockout" endogenous LYST or LYST-2 and/or LYST-IP function by homologous recombination (see, e.g., Capecchi, 1989, Science 244: 1288-1292).
  • a nucleic acid containing a portion of a LYST or LYST-2 and/or a LYST-IP gene in which the LYST or LYST-2 and or LYST-IP sequences flank (are both 5' and 3' to) a different gene sequence is used as a LYST or LYST-2 and/or a LYST-IP antagonist, or to promote LYST or LYST-2 and/or LYST-IP inactivation by homologous recombination (see also Koller and Smithies. 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935. Zijlstra et al, 1989, Nature 342: 435-438).
  • mutants or derivatives of a first LYST-IP protein that have greater affinity for LYST than a second LYST-IP may be administered to compete with the second LYST-IP protein for LYST binding, thereby reducing the levels of LYST complexes with the second LYST-IP.
  • LIP3, LIP4, LIP5, LIP6, LIP7. LIP8, LIP9, or LIPIO function can be identified by use of known convenient in vitro assays, e.g.. based on their ability to inhibit LYST:LYST-IP binding or as described infra.
  • Therapeutics that antagonize LYST:LYST-IP complex formation or activity, or a LYST-2, LIPl, LIP2. LIP3. LIP4, LIP5. LIP6, LIP7. LIP8. LIP9. or LIPIO activity are administered therapeutically (including prophylactically): ( 1 ) in diseases or disorders involving an increased (relative to normal or desired) level of LYST:LYST-IP complex, or a LYST-2. LIPl. LIP2. LIP3. LIP4. LIP5. LIP6. LIP7. LIP8. LIP9. or LIPIO protein. for example, in patients where a LYST:LYST-IP complex or said individual LYST-2 or novel
  • LIP protein is overactive or overexpressed; (2) in diseases or disorders involving an absence or decreased (relative to normal or desired) level of said LYST:L YST-IP complex, or said individual LYST-2 or novel LIP protein, for example, in patients where LYST:LYST-IP complexes (or the individual components necessary to form the complexes), or where said individual LYST-2 or novel LIP protein is lacking, genetically defective, biologically inactive or underactive. or under-expressed; (3) in diseases or disorders wherein in vitro (or in vivo) assays
  • Increased, decreased, or absent levels of LYST:L YST-IP complexes or of LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO protein levels can be readily detected, e.g., by quantifying protein and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or protein levels, structure and/or activity of the expressed LYST:LYST-IP complex (or for the concurrent expression of mRNA encoding the two components of the LYST:L YST-IP complex), or said individual LYST-2 or novel LIP protein or mRNA levels.
  • a more specific embodiment includes methods of reducing L YST:LYST-IP complex expression (i.e.. the expression of the two components of the LYST:L YST-IP complex and/or formation of the complex), or reducing LYST-2, LIPl. LIP2. LIP3. LIP4, LIP5. LIP6, LIP7. LIP8, LIP9, or LIPIO expression, by targeting mRNAs that express the protein moieties.
  • RNA therapeutics currently fall within three classes, antisense species, ribozymes. or RNA aptamers (Good et al. 1997, Gene Therapy 4: 45-54). Antisense oligonucleotides have been the most widely used.
  • Ribozyme therapy involves the administration, induced expression, etc.. of small RNA molecules with enzymatic ability to cleave, bind, or otherwise inactivate specific RNAs to reduce or eliminate expression of particular proteins (Grassi and
  • RNA aptamers are specific RNA ligands for proteins, such as for Tat and Rev RNA (Good et al, 1997, Gene Therapy 4: 45-54) that can specifically inhibit their translation.
  • Aptamers specific for LYST or LYST-2 or a LYST-IP can be identified by many methods well known in the art, for example but not limited to the protein-protein interaction assay described in Section 5.7.1 infra.
  • the activity or level of LYST or LYST-2 is reduced by administration of a LYST-IP, or a nucleic acid that encodes a LYST-IP, or antibody that immunospecifically binds to a LYST-IP, or a fragment or a derivative of the antibody containing the binding domain thereof.
  • the level or activity of a LYST-IP may be reduced by administration of a LYST or LYST-2 or a LYST-IP nucleic acid, or an antibody that immunospecifically binds LYST or LYST-2, or a fragment or derivative of the antibody containing the binding domain thereof.
  • diseases or disorders associated with increased levels of LYST or LYST-2 or a particular LYST-IP may be treated or prevented by administration of a Therapeutic that increases LYST:L YST-IP complex formation, if the complex formation acts to reduce or inactivate LYST or LYST-2 or the particular LYST-IP through LYST:LYST-IP complex formation.
  • Such diseases or disorders can be treated or prevented by administration of one member of the LYST:LYST-IP complex, including mutants of a member of the LYST:L YST-IP that have increased affinity for the other member of the LYST:LYST-IP complex (to cause increased complex formation), administration of antibodies or other molecules that stabilize the LYST:LYST-IP complex, etc.
  • the activity or level of LYST or LYST-2 is increased by administration of a LYST-IP, or derivative or analog thereof, a nucleic acid encoding a LYST-IP, or an antibody that immunospecifically binds a LYST-IP. or a fragment or derivative of the antibody contains the binding domain thereof.
  • the activity or levels of a LYST-IP are increased by administration of LYST or LYST-2. or derivative or analog thereof, a nucleic acid encoding LYST or LYST-2. or an antibody that immunospecifically binds
  • LYST or LYST-2 or a fragment or derivative of the antibody contains the binding domain thereof.
  • a human LYST:LYST-IP complex or LIPl .
  • suitable in vitro or in vivo assays are utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of an affected tissue.
  • in vitro assays can be carried out with representative cells or cell types involved in a patient's disorder to determine if a Therapeutic has a desired effect upon such cell types.
  • Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats. mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used. Additional descriptions and sources of Therapeutics that can be used according to the invention are found herein.
  • LYST has been implicated in atopic disorders.
  • Atopy is defined as high immunoglobulin E (IgE) responsiveness (Kawakami et al . 1997. Respirology 2: 7-15). since high serum IgE concentrations are found in individuals with atopic disorders.
  • IgE plays a central role in mediating the pathology of allergic disease. In IgE allergic diseases both mast cells and T lymphocytes play an important role. Whereas mast cells have been implicated in immediate allergic responses, T-lymphocytes mediate subsequent late phase responses and chronic inflammation. Cellular mechanisms that mediate the sensitization, immediate and late-phase reactions that follow encounters with allergen, as well as cell surface and signaling events that result in mediator release from inflammatory cells have been discussed.
  • LYST The interaction of LYST with the IgE receptor may be one cause for immunological disorders such as bronchial asthma, nasal polyps, hay fever (allergic) rhinitis, urticaria, atopic dermatitis, food allergies, nonhereditary angioedema, systemic anaphylaxis, and allergic conjunctivitis. All these atopic disorders are associated with a positive family history, and 20-30% of the population has a strong genetic predisposition for this condition (Casolaro et al , 1996, Cwrr. Opin. Immunol. 8: 796- 803).
  • Atopic dermatitis is the most common chronic skin disease of young children and is frequently associated with asthma and allergies (Boguniewicz, 1997, Cwrr. Opin. Pediatr. 9: 577-581).
  • Therapeutics of this invention may be effective in treating or preventing atopic diseases or disorders.
  • Therapeutics of the invention can be assayed by any method known in the art for efficacy in treating or preventing such atopic diseases and disorders.
  • Such assays include in vivo assays using animal models of atopic diseases or disorders, or in vitro assays as described infra.
  • Potentially effective Therapeutics may act by decreasing the abnormal degranulation of basophilic granulocytes.
  • atopic disease or disorder has been shown to be amenable to treatment by modulation of LYST:LYST-IP complex activity, that atopic disease or disorder can be treated or prevented by administration of a Therapeutic that modulates the LYST:L YST-IP complex formation (including supplying LYST:LYST-IP complexes or function).
  • a Therapeutic that modulates the LYST:L YST-IP complex formation (including supplying LYST:LYST-IP complexes or function).
  • LYST has been implicated in autoimmune disorders. Mice with LYST mutations (beige mice) do not develop renal failure when affected by systemic lupus erythematosus. LYST mutation appears to be protective from development of end-organ damage as a consequence of auto-immune disease (Clark et al , 1982, In: NK CELLS AND OTHER NATURAL EFFECTOR CELLS.
  • the LYST protein protects cells against autoimmune disease, including, but not limited to. arthritis (Barthold, 1995. J. Infect. Dis. 172:
  • systemic lupus systemic lupus
  • systemic autoimmune diseases systemic autoimmune diseases
  • IBD inflammatory bowel disease
  • LYST interactant Imogen 38 is a target for autoimmune attack in diabetes mellitus.
  • Therapeutics of the invention particularly those that mimic, modulate (or supply) LYST:LYST-IP complex activity may be effective in treating or preventing autoimmune diseases or disorders.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of LYST:L YST-IP) can be assayed by any method known in the art for efficacy in treating or preventing such autoimmune diseases and disorders.
  • Such assays include in vitro assays using cell culture models as described infra, or in vivo assays using animal models of autoimmune diseases or disorders, also described infra.
  • Potentially effective Therapeutics for example but not by way of limitation, reduce autoimmune responses in animal models in comparison to controls.
  • Autoimmune disorders that can be treated or prevented include but are not limited to those listed in Isslebacher et al, 1997 (In: HARRISON'S PRINCIPALS OF INTERNAL MEDICINE, 13 th Ed., McGraw Hill, New York, pp. 1543-1559, incorporated herein by reference).
  • autoimmune disease or disorder can be treated or prevented by administration of a Therapeutic that modulates the LYST:LYST-IP complex formation (including supplying LYST:LYST-IP complexes) or function.
  • HSl protein, norbin, HBF-G2 have been implicated in neurodegenerative disease.
  • the Chediak-Higashi syndrome is characterized by neurologic disorders such as peripheral neuropathy and ataxia. Beige mice show a progressive neurological disorder accompanied by a complete loss of cerebellar Purkinje cells (Murphy and Roths. 1978. Jackson
  • Atrophin-I has been associated with the neurodegenerative disease dentatorubralpallidoluysian atrophy (DRPLA. Smith's disease).
  • DGS-I is associated with a developmental disease DiGeorge syndrome. 14-3-3 protein which interacts with both LYST and
  • LYST-2 is present in Alzheimer ' s disease neurofibrillary tangles and is present in cerebrospinal fluid of patients with Creutzfeldt- Jakob disease. Furthermore, the LYST-2 interactant HBF-G3 is a human brain factor expressed in fetal tissues. LIP3, a homolog to rat Roaz protein, may have a role in regulation neuronal-specific gene expression. Further, LYST and certain binding partners of LYST (e.g., 14-3-3 protein. HSl protein, calmodulin. casein kinase subunit II, Hrs. Efs. estrogen receptor related protein.
  • norbinLIPl Tcp-10 homolog
  • LIP7 Tcp-10 homolog
  • LIP5 LIP5
  • LIP6 Ns2-3 protease homolog
  • LIP4 hnRNP-E2 homolog
  • LIP8 KAP4L
  • Therapeutics of the invention may be effective in treating or preventing neurodegenerative disease.
  • Therapeutics of the invention that modulate LYST:LYST-IP complexes involved in neurodegenerative disorders can be assayed by any method known in the art for efficacy in treating or preventing such neurodegenerative diseases and disorders.
  • Such assays include in vitro assays for regulated cell secretion, protein trafficking, and/or folding or inhibition of apoptosis or in vivo assays using animal models of neurodegenerative and/or developmental diseases or disorders, or any of the assays described infra.
  • Potentially effective Therapeutics for example but not by way of limitation, promote regulated cell maturation and prevent cell apoptosis in culture, or reduce neurodegeneration in animal models in comparison to controls.
  • Such diseases include all degenerative disorders involved with aging, especially osteoarthritis and neurodegenerative disorders.
  • Neurodegenerative disorders that can be treated or prevented include but are not limited to those listed in Isslebacher et al. 1997 (In: HARRISON'S PRINCIPALS OF INTERNAL MEDICINE. 13 ,h Ed.. McGraw Hill. New York, incorporated herein by reference).
  • neurodegenerative disease or disorder has been shown to be amenable to treatment by modulation of LYST:LYST-IP complex activity, that neurodegenerative disease or disorder can be treated or prevented by administration of a Therapeutic that modulates
  • LYST.LYST-IP complex formation including supplying LYST:LYST-IP complexes.
  • LYST LYST-IP complexes
  • LYST and LYST-2 Components of LYST:LYST-IP complexes
  • estrogen-receptor related protein casein kinase II beta SU, 14-3-3 protein, Ftel. BMKl , HSl protein, troponin
  • the estrogen-receptor related protein has been found expressed in several cancers, including breast cancer, prostate cancer, pancreatic cancer, osteoblastic osteosarcoma, gastric cancer, laryngeal carcinoma, papillary thyroid carcinoma, intracranial tumors, colorectal cancer and colon cancer.
  • 14-3-3 protein has been found in lung cancer cells, but also in other cancers, including colorectal adenocarcinoma, lymphoblastoma, salivary gland tumors. Chediak-Higashi patients have an increased tumor growth rate and higher metastatic frequency, and non-Hodgkin's lymphoma occurred in over one third of CHS patients (Hayakawa et al, 1986, Jpn. J. Cancer Res. 77: 74-79). This may be due to the involvement of natural killer cells in cancer.
  • Therapeutics of the invention may be useful in treating or preventing diseases or disorders associated with cell hyperproliferation or loss of control of cell proliferation, particularly cancers, malignancies and tumors.
  • Therapeutics of the invention can be assayed by any method known in the art for efficacy in treating or preventing malignancies and related disorders.
  • Such assays include in vitro assays using transformed cells or cells derived from the tumor of a patient, or in vivo assays using animal models of cancer or malignancies, or any of the assays described infra.
  • Potentially effective Therapeutics for example but not by way of limitation, inhibit proliferation of tumors or transformed cells in culture, or cause regression of tumors in animal models in comparison to controls, e.g., as described infra.
  • malignancy or dysproliferative changes are treated or prevented in the bladder, breast, colon, lung, prostate, pancreas, or uterus.
  • Such cancers and malignancies include but are not limited to those listed in Fishman el al, 1985 MEDICINE. 2d Ed.. J.B. Lippincott Co., Philadelphia, a review of such disorders (incorporated herein by reference). Accordingly, once a malignancy or cancer has been shown to be amenable to treatment by modulating (;.e..
  • LYST inhibiting, antagonizing, enhancing or agonizing
  • LYST LYST-IP complex activity, or modulating LYST-2, LIPl, LIP2.
  • LIPIO activity, that cancer or malignancy can be treated or prevented by administration of a Therapeutic that modulates LYST:LYST-IP complex formation and function, or LYST-2. LIPl.
  • LYST:LYST-IP complexes and the individual binding partners of a LYST:LYST-IP complex.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Metaplasia is a form of controlled cell growth in which one type of adult cell or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, skin, oral cavity, and gall bladder.
  • LYST-IP complex activity Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia. metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype. or of a malignant phenotype. displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic of the invention that modulates LYST:LYST-IP complex activity, or that modulates LYST-2.
  • Such characteristics of a transformed phenotype include morphological changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release. increased sugar transport, decreased serum requirement, expression of fetal antigens. disappearance of the 250 kDa cell surface protein, etc. (see also Id., pp. 84-90 for characteristics associated with a transformed or malignant phenotype).
  • leukoplakia a benign-appearing hype ⁇ lastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ
  • fibrocystic disease cystic hyperplasia. mammary dysplasia. particularly adenosis (benign epithelial hype ⁇ lasia)
  • a Therapeutic of the invention is administered to a human patient to prevent progression to breast, colon, lung, pancreatic, prostate or uterine cancer, or melanoma or sarcoma.
  • a patient that exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma.
  • a chromosomal translocation associated with a malignancy e.g., the Philadelphia chromosome for chronic my
  • retinoblastoma cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell. 1976. BASIC PATHOLOGY. 2nd Ed.. W.B. Saunders Co., Philadelphia, pp. 112-113. etc.. inco ⁇ orated herein by reference).
  • a Therapeutic is administered to treat or prevent hype ⁇ roliferative or benign dysproliferative disorders.
  • Therapeutics of the invention can be assayed by any method known in the art for efficacy in treating or preventing hype ⁇ roliferative diseases or disorders, such assays include in vitro cell proliferation assays, in vitro or in vivo assays using animal models of hype ⁇ roliferative diseases or disorders, or any of the assays described infra.
  • Potentially effective Therapeutics include but are not limited to. Therapeutics that reduce cell proliferation in culture or inhibit growth or cell proliferation in animal models in comparison to controls.
  • LIPl LIP2, LIP3.
  • LIP9, or LIPIO protein activity, that hype ⁇ roliferative disease or disorder can be treated or prevented by administration of a
  • LIPl LIP2, LIP3.
  • LIP9. or LIPIO activity including supplying a LYST:LYST-IP complex and or the individual binding partners of a LYST:LYST-IP complex).
  • Specific embodiments are directed to treatment or prevention of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hype ⁇ lasia).
  • LYST is strongly implicated in pigmentation disorders, such as oculocutaneous albinism and hypopigmentation.
  • Albinism connotes a large group of genetic disorders that are characterized by diminished ocular and often cutaneous pigmentation.
  • Genetically based syndromes such as Chediak-Higashi syndrome (CHS), phenotypically resembling albinism, in which the synthesis of pigmented melanosomes, as well as specialized organelles of other cell types, is compromised.
  • Oculocutaneous albinism is characterized by a congenital reduction or absence of melanin pigment in the skin, hair and eyes. The reduction in the hair and skin results in a change in color but no change in the development or function of these tissues, while the absence of melanin pigment in the eye leads to abnormal development and function.
  • Therapeutics of the invention particularly those that modulate (or supply) LYST:L YST- IP complex activity may be effective in treating or preventing pigmentation diseases or disorders.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of LYST:LYST-IP complexes) can be assayed by any method known in the art for efficacy in treating or preventing such viral diseases and disorders.
  • Such assays include widely used in vitro assays using cell culture models, and in vivo assays using animal models of pigmentation diseases or disorders (see McGeoch et ai. 1986. J. Gen. Virol. 67: 813-825 for review).
  • Potentially effective Therapeutics for example but not by way of limitation, reduce pigmentation diseases or disorders in animal models in comparison to controls.
  • LYST:LYST-IP complex formation including supplying a LYST:LYST-IP complex, or LYST-2 or at least one of the ten novel LIP genes or proteins.
  • LYST is strongly implicated in platelet abnormality that can lead to increased bleeding tendency in Chediak-Higashi syndrome.
  • azurophilic granules of neutrophils and melanosomes of melanocytes are generally larger in size and irregular in mo ⁇ hology in CHS patients, indicating that a common pathway in storage organellogenesis is affected in those patients.
  • Platlet dysfunction that may be treated by modulation of LYST:L YST-IP complex activity is of two general classes: (1) diseases associated with platelet storage pool deficiency (e.g., Hermansky-Pudlak Syndrome, Chediak-Higashi Syndrome, gray platelet syndrome) or thrombocytopenia; and (2) diseases associated with thrombocytosis or increased clotting tendency (e.g., myocardial infarction, deep venous thrombosis, cardiovascular accident, transient ischemic attack).
  • the platelet storage pool deficiency in CHS is associated with prolonged bleeding time and low dense granule content (Novak et al, 1984, Blood 63: 536-544).
  • Therapeutics of the invention particularly those that modulate (or supply) LYST:L YST- IP complex activity may be effective in treating or preventing platelet dysfunction diseases or disorders.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of LYST:LYST-IP complexes) can be assayed by any method known in the art for efficacy in treating or preventing such platelet dysfunction diseases and disorders.
  • Such assays include widely used in vitro assays using cell culture models, and in vivo assays using animal models of platelet dysfunction diseases or disorders (see McGeoch et al. 1986. J. Gen. Virol. 67: 813-825 for review).
  • Potentially effective Therapeutics for example but not by way of limitation, reduce platelet dysfunction diseases or disorders in animal models in comparison to controls. Accordingly, once a platelet. dysfunction disease or disorder has been shown to be amenable to treatment by modulation of LYST:L YST-IP complex activity, that platelet dysfunction disease or disorder can be treated or prevented by administration of a Therapeutic that modulates LYST:LYST-IP complex formation, including supplying a LYST:LYST-IP complex.
  • LYST and the LYST interactant Importin beta subunit are strongly implicated in viral infection mechanisms, including that for the AIDS virus, HIV-1.
  • An enormous number of human diseases result from virulent and opportunistic viral infection see, e.g., Belshe (Ed.) 1984 TEXTBOOK OF HUMAN VIROLOGY, PSG Publishing, Littleton, MA).
  • Viral diseases of a wide array of tissues including the respiratory tract, the CNS, the skin, the genitourinary tract, the eyes and ears, the immune system, the gastrointestinal tract, and the musculoskeletal system, affect a vast number of humans, of all ages (see Table 328-2, In: Wyngaarden and Smith, 1988, CECIL TEXTBOOK OF MEDICINE, 18th Ed., W.B. Saunders Co., Philadelphia, pp.1750- 1753).
  • diseases and disorders including, but not limited to, e.g.
  • Therapeutics of the invention particularly those that modulate (or supply) LYST:LYST-IP complex (particularly LYST:importin beta subunit complex) activity may be effective in treating or preventing viral diseases or disorders.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of LYST:importin beta subunit complex) can be assayed by any method known in the art for efficacy in treating or preventing such viral diseases and disorders.
  • Such assays include widely used in vitro assays using cell culture models, and in vivo assays using animal models of viral diseases or disorders (see McGeoch et al, 1986, J. Gen. Virol. 67: 813-825 for review).
  • Potentially effective Therapeutics for example but not by way of limitation, reduce viral responses in animal models in comparison to controls.
  • a viral disease or disorder has been shown to be amenable to treatment by modulation of Importin beta subunit complex activity
  • that viral disease or disorder can be treated or prevented by administration of a Therapeutic that modulates LYST:L YST-IP complex (particularly LYST:Importin beta subunit complex) formation, including supplying a LYST:LYST-IP complex, particularly a LYST:Importin beta subunit complex).
  • a Therapeutic that modulates LYST:L YST-IP complex (particularly LYST:Importin beta subunit complex) formation, including supplying a LYST:LYST-IP complex, particularly a LYST:Importin beta subunit complex).
  • nucleic acid molecule comprising a sequence encoding
  • LYST or LYST-2 and/or a LYST-IP are administered to modulate LYST:LYST-IP complexes, or to modulate LYST-2.
  • LIP7. LIP8. LIP9. or LIPIO function, by way of gene therapy.
  • at least one nucleic acid encoding either or both LYST or LYST-2 and a LYST-IP, or functional derivatives thereof are administered by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid molecule to a subject.
  • the nucleic acid molecule produces its encoded protein(s) that mediates a therapeutic effect by modulating the
  • LYST LYST-IP complex, or by modulating LYST-2, LIPl. LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO function. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • the Therapeutic comprises a LYST or LYST-2 and/or a LYST-IP nucleic acid, or a LYST-2.
  • LIP7, LIP8, LIP9, or LIPIO nucleic acid that is part of an expression vector that expresses the LYST or LYST-IP protein(s), or expresses a LYST-2.
  • such a nucleic acid has a promoter(s) operably linked to the LYST and or the LYST-IP coding region(s), or linked to the LYST-2, LIPl , LIP2. LIP3. LIP4, LIP5, LIP6, LIP7. LIP8, LIP9. or LIPIO coding region, said promoter(s) being inducible or constitutive, and optionally, tissue-specific. See, e.g., as discussed, supra, in RECOMBINANT METHODOLOGIES.
  • a nucleic acid molecule is used in which the LYST or LYST-2 and/or LYST-IP coding sequence, or the individual LYST-2 or novel LIP coding sequences, and any other desired sequences, are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intra-chromosomal expression of the LYST and the LYST-IP nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935. Zijlstra et ai. 1989. Nature 342:
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case. cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo and ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular. e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Patent No. 4.980.286). or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by encapsulation in liposomes.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide that disrupts endosomes, preventing lysosomal degradation of the nucleic acid.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression by targeting a specific receptor (see. e.g., International Patent Publications WO 92/06180, WO 92/22635, WO 92/20316. WO 93/14188, and WO 93/20221).
  • the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935, Zijlstra et ai. 1989. N ⁇ twre 342: 435-438).
  • a viral vector that contains the LYST or LYST-2 and/or the LYST-IP encoding nucleic acid sequence, or the individual LYST-2 or novel LIP encoding nucleic acid sequence is used.
  • a retroviral vector can be used (see Miller et al, 1993. Meth. Enzymol 217: 581 -599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell D ⁇ A.
  • the LYST or LYST-2 and/or LYST-IP (preferably both LYST and LYST-IP) encoding nucleic acids, or individual LYST-2 or novel LIP encoding nucleic acids, to be used in gene therapy is/are cloned into the vector, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors and the use of retroviral vectors in gene therapy can be found in Boesen et al. 1994. Biotherapy 6: 291-302, Clowes et ai. 1994, J. Clin. Invest. 93: 644-651,
  • Adeno viruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300).
  • Another approach to gene therapy involves transferring a gene into cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that took up and are expressing the transferred gene. Those cells are then delivered to a patient by various means available in the art.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art. including but not limited to transfection. electroporation, micro injection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see. e.g., Loeffler and Behr. 1993. Meth. Enzymoi 217: 599-618, Cohen et al, 1993, Meth. Enzymol.
  • Ther. 29 may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell, and is heritable and expressible by its cell progeny.
  • recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected, e.g.. subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • Recombinant secretory cells e.g., cells modified to express dopamine
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, nucleated blood cells, various stem or progenitor cells, in particular embryonic heart muscle cells, liver stem cells (International Patent Publication WO 94/08598), neural stem cells (Stemple and Anderson. 1992. Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures (Rheinwald, 1980. Meth. Cell Bio. 21: 229). ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture (Rheinwald. 1980. Meth. Cell Bio. 21a: 229; Pittelkow and Scott, 1986, Mayo Clinic Proc. 61: 111). If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.
  • a method for suppression of host versus graft reactivity e.g., irradiation, drug or antibody administration to promote moderate immunosuppression
  • HSC hematopoietic stem cells
  • any technique which provides for the isolation, propagation, and maintenance in vitro of HSCs can be used in this embodiment of the invention.
  • Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic.
  • Non-autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient.
  • human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see. e.g., Kodo et al. 1984. J. Clin. Invest. 73: 1377-
  • the HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-term culturing. and can be done by any technique known in the art. Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified
  • the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Additional methods can be adapted for use to deliver a nucleic acid molecule encoding the LYST or LYST-2 and or LYST-IP proteins, or functional derivatives thereof, e.g., as described supra.
  • LYST:LYST-IP complex function or individual LYST-2 or novel LIP protein function is inhibited by use of antisense nucleic acids for LYST or LYST-2 and or a LYST-IP, (preferably both LYST and the LYST-IP), or individual antisense nucleic acids for LYST-2.
  • LIPl LIP2. LIP3, LIP4. LIP5. LIP6. LIP7. LIP8, LIP9, or LIPIO.
  • the present invention provides the therapeutic or prophylactic use of nucleic acids of at least 6 nucleotides that are antisense to a gene or cDNA encoding LYST or LYST-2 and/or a LYST-IP, or encoding LYST-2, LIPl, LIP2, LIP3, LIP4. LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO. or a portion thereof.
  • a LYST or a LYST-IP "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of LYST or a LYST-IP nucleic acid (preferably mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acid may be complementary to a coding and/or noncoding region of a LYST or LYST-IP mRNA.
  • Such antisense nucleic acids have utility as Therapeutics that inhibit LYST:LYST-IP complex formation or activity, or individual LYST-2 or novel LIP protein function or activity, and can be used in the treatment or prevention of disorders as described, supra.
  • the antisense nucleic acids of the invention can be oligonucleotides that are double- stranded or single-stranded.
  • RNA or DNA or a modification or derivative thereof which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • the invention is directed to methods for inhibiting the expression of LYST and or a LYST-IP nucleotide sequence, or individual LIPl. LIP2. LIP3. LIP4. LIP5. LIP6, LIP7. LIP8. LIP9. or LIPIO nucleotide sequences, in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense nucleic acid of LYST or LYST-2 and LYST-IP, or an antisense nucleic acid of LIPl, LIP2, LIP3,
  • the LYST or LYST-2 and or LYST-IP antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 200 oligonucleotides). In specific aspects, the oligonucleotide is at least about 10 nucleotides. at least about 15 nucleotides, at least about 100 nucleotides. or at least about 200 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556, Lemaitre et ai, 1987, Proc. Natl. Acad. Sci. 84: 648-652, PCT Publication No.
  • WO 88/09810 transport across the blood- brain barrier (see, e.g., PCT Publication No. WO 89/10134), hybridization-triggered cleavage agents (see. e.g., Krol et al, 1988. BioTechniques 6: 958-976). or intercalation with other agents (see, e.g., Zon. 1988, Pharm. Res. 5: 539-549).
  • a LYST or LYST-2 and/or LYST-IP antisense oligonucleotide is provided, preferably as single-stranded DNA.
  • the oligonucleotide may be modified at any position on its structure with constituents generally known in the art.
  • the LYST or LYST-2 and/or LYST-IP antisense oligonucleotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5- fluorouracil. 5-bromouracil. 5-chlorouracil, 5-iodouracil. hypoxanthine. xanthine. 4- acetylcytosine. 5-(carboxyhydroxylmethyl) uracil. 5-carboxymethylaminomethyl-2-thiouridine. 5-carboxymethylaminomethyluracil. dihydrouracil, beta-D-galactosylqueosine. inosine, N6- isopentenyladenine. 1-methylguanine. 1-methylinosine.
  • SUBST ⁇ UTE SHEET (RULE 26) methoxycarboxymethyluracil. 5-methoxyuracil. 2-methylthio-N6-isopentenyladenine. uracil-5- oxyacetic acid (v), wybutoxosine, pseudouracil, queosine. 2-thiocytosine. 5-methyl-2-thiouracil.
  • 2-thiouracil 4-thiouracil. 5-methyluracil. uracil-5-oxyacetic acid methylester. uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil. 3-(3-amino-3-N-2-carboxypropyl) uracil. (acp3)w. and 2,6- diaminopurine.
  • the oligonucleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose. 2-fluoroarabinose, xylulose. and hexose.
  • the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.
  • the oligonucleotide is a 2-anomeric oligonucleotide.
  • An anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15: 6625- 6641).
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al, 1988, Nucl. Acids Res. 16: 3209
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, 1988. Proc. Natl Acad. Sci. U.S.A. 85: 7448-7451), etc.
  • the LYST or LYST-2 and/or LYST-IP antisense oligonucleotides comprise catalytic RNAs, or ribozymes (see, e.g., PCT International Publication WO 90/1 1364, published October 4, 1990, Sarver et al. 1990, Science 247: 1222-1225).
  • the oligonucleotide is a 2'-0-methyIribonucleotide (Inoue et al, 1987,
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the LYST and/or LYST-IP antisense nucleic acids of the invention are produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding LYST or LYST-2 and/or a LYST- IP (preferably, both a LYST or LYST-2 and a LYST-IP antisense nucleic acid) antisense nucleic acid(s), or individual LYST-2.
  • LIPl LIP2. LIP3. LIP4. LIP5. LIP6. LIP7, LIP8. LIP9. or LIPIO antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated. as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid. viral, or others known in the art to be capable of replication and expression in mammalian cells.
  • Expression of the sequences encoding the LYST or LYST-2 and/or LYST-IP antisense RNAs can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. See description in RECOMBINANT TECHNOLOGIES section, supra.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a LYST or LYST-2 or a LYST-IP gene, preferably a human LYST or LYST-2 or LYST-IP gene.
  • a LYST or LYST-2 or a LYST-IP gene preferably a human LYST or LYST-2 or LYST-IP gene.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded LYST or LYST-2 or LYST-IP antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex nucleic acid formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Cell types that express or overexpress LYST or LYST-2 and/or LYST-IP mRNA. or individual LYST-2 or novel LIP RNA can be identified by various methods known in the art. Such methods include, but are not limited to. those methods detailed in the RECOMBINANT METHODOLOGIES section, supra, or the various ASSAYS sections, infra.
  • primary tissue from a patient can be assayed for LYST or LYST-2 and/or LYST-IP expression prior to treatment, e.g., by immunocytochemistry or in situ hybridization.
  • compositions of the invention comprising an effective amount of a LYST or LYST-2 and/or a LYST-IP antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a disease or disorder that is of a type that expresses or overexpresses LYST:L YST-IP complexes, LYST and/or LYST-IP mRNA, or LIPl,
  • LIP2 LIP3. LIP4, LIP5, LIP6, LIP7. LIP8, LIP9, or LIPIO mRNA or protein.
  • LYST or LYST-2 and or LYST-IP antisense nucleic acid that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity in vitro, and then in useful animal model systems prior to testing and use in humans.
  • compositions comprising LYST or LYST-2 or
  • LYST-IP antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the LYST or LYST-2 and/or LYST-IP antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable central nervous system cell types (Leonetti et ai, 1990, Proc.
  • LIP2 LIP3, LIP4, LIP5, LIP6, LIP7. LIP8, LIP9, or LIPIO protein, and derivatives, fragments and analogs thereof, can be assayed by various methods known in the art.
  • Potential modulators LIP3, LIP4, LIP5, LIP6, LIP7. LIP8, LIP9, or LIPIO protein, and derivatives, fragments and analogs thereof, can be assayed by various methods known in the art.
  • LYST LYST-IP complex activity
  • LIPl LIP2
  • LIP4, LIP5, LIP6, LIP7, LIP8. LIP9, or LIPIO activity can be assayed for the ability to modulate LYST:LYST-IP complex formation and/or activity, and for the ability to modulate LYST-2 or individual novel LIP activity.
  • Immunoassays which can be used include but are not limited to competitive and non- competitive assay systems. For example, in one embodiment, where one is assaying for the ability to bind or compete with wild-type LYST:L YST-IP complexes, or LYST-2. LIPl . LIP2. LIP3. LIP4. LIP5. LIP6.
  • LIP7, LIP8, LIP9, or LIPIO protein for binding to anti-LYST:LYST-IP antibodies, or antibodies specific to LYST-2 or any one of the ten novel LIP proteins
  • various immunoassays known in the art can be used, including but not limited to competitive and non- competitive assay systems using techniques such as radioimmunoassays, enzyme linked immunosorbent assay (ELISA), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunohistochemistrv assays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, immunoprecipitation assays, precipitin reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A immunoassays, immunoelectrophoresis assay
  • antibody binding is assayed by detecting a label on the primary antibody.
  • the primary antibody is assayed by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. See, e.g., Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, R. Coico (Ed.), John Wiley & Sons. New York, 1998.
  • Derivatives e.g., fragments
  • analogs of LYST-IPs can be assayed for binding to
  • LYST or LYST-2 by any method known in the art, for example the modified yeast two hybrid assay system described infra, immunoprecipitation with an antibody that binds to LYST or LYST-2 in a complex followed by analysis by size fractionation of the immunoprecipitated proteins (e.g., by denaturing or nondenaturing polyacrylamide gel electrophoresis), Western analysis, non-denaturing gel electrophoresis. etc.
  • LYST or LYST-2 and or LYST-IP genes both endogenous genes and those expressed from recombinant DNA containing these genes
  • LYST or LYST-2 and or LYST-IP genes can be detected using techniques known in the art, including but not limited to Southern hybridization, Northern hybridization, restriction endonuclease mapping, DNA sequence analysis, polymerase chain reaction amplification (PCR), or RNase protection with probes specific for LYST or LYST-2 or LYST-IP genes, in various cell types.
  • LYST or LYST-2 or LYST-IP gene mutations to physiological or pathological states can be characterized for their expression of LYST or LYST-2 and or a LYST-IP (particularly expression of LYST or LYST-2 and/or a LYST-IP at the same time and in the same cells), or LYST-2, LIPl, LIP2. LIP3, LIP4, LIP5, LIP6. LIP7. LIP8, LIP9, or LIPIO protein expression.
  • the stringency of the hybridization conditions for northern or Southern blot analysis can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific probes used, as described supra. Modifications to these methods and other methods commonlv known in the art can be used.
  • One embodiment of the invention provides a method for screening a derivative or analog of LYST or LYST-2 for biological activity comprising contacting said derivative or analog of LYST or LYST-2 with a protein selected from the group consisting of 14-3-3 protein.
  • LIP9, or LIPIO and detecting the formation of a complex between said derivative or analog of LYST or LYST-2 and said LYST-IP protein; wherein detecting formation of said complex indicates that said derivative or analog of LYST or LYST-2 has biological (e.g., binding) activity.
  • another embodiment of the invention relates to a method for screening a derivative or analog of a protein selected from the group consisting of 14-3-3 protein, HSl protein, Hrs, BMKl, KB07, Efs, XAP-4, OS9, casein kinase II beta SU. calmodulin, troponin I, importin beta, Fte-1, ERRal, imogen 38, atrophin-1.
  • norbin HBF-G2, DGS-I, GBDRl. OPA containing protein, M4 protein, LIPl. LIP2, LIP3. LIP4, LIP5. LIP6, LIP7, LIP8, LIP9, or LIPIO for biological activity comprising contacting said derivative or analog of said protein with LYST or LYST-2; and detecting the formation of a complex between said derivative or analog of said protein and LYST or LYST-2; wherein detecting the formation of said complex indicates that said derivative or analog of said protein has biological activity.
  • the present invention also provides methods of modulating the activity of a protein that can participate in a LYST:LYST-IP complex by administration of a binding partner of that protein, or derivative or analog thereof.
  • LYST or LYST-2, and derivatives and analogs thereof can be assayed for the ability to modulate the activity or level of a LYST-IP by contacting a cell, or administering to an animal, expressing a LYST-IP gene with a LYST or LYST-2 protein, or a nucleic acid encoding a LYST or LYST-2 protein, or an antibody that immunospecifically binds the LYST or LYST-2 protein, or a fragment or derivative of said antibody containing the binding domain thereof, and measuring a change in LYST-IP levels or activity, wherein a change in LYST-IP levels or activity indicates that LYST or LYST-2 can modulate LYST-IP levels or activity.
  • a LYST-IP can be assayed for the ability to modulate the activity or levels of a LYST or LYST-2 protein by contacting a cell, or administering to an animal.
  • LIP8. LIP9, or LIPIO protein, or derivative, analog, or fragment thereof can also be screened for activity in modulating (i.e.. increasing or decreasing) the activity of LYST and the LYST binding partners (i.e.. the LYST-IPs involved in particular LYST:L YST-IP complexes).
  • Specific LYST:LYST-IP complexes may be associated with specific diseases or tissue types. The relevance of different LYST-IP proteins in specific diseases or disorders are set forth in the background section.
  • LYST protein has been implicated in atopic disorders (see supra), including asthma and allergic diseases. Accordingly, LYST:LYST-IP complexes, and derivatives, analogs, and fragments thereof, nucleic acids encoding the LYST genes. anti-LYST:LYST-IP antibodies and antisense nucleic acids, and other modulators of the LYST:LYST-IP complex, can be tested for activity in treating or preventing atopic disorders in in vitro and in vivo assays.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing atopic disorders by contacting cultured cells that exhibit an indicator of an atopic reaction, in vitro with the Therapeutic, and comparing the level of indicator in the cells contacted with the Therapeutic with said level of the indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing atopic disorders.
  • Cell models that can be used for such assays include, but are not limited to a model of EBV-B cells for serum-IgE-facilitated allergen presentation in atopic disease (van der Heijden et al, 1993, J.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing atopic disease by administering said Therapeutic to a test animal exhibiting an atopic reaction or which test animal does not exhibit an atopic reaction and is subsequently challenged with an agent that elicits an atopic reaction: and measuring the change in the atopic reaction after the administration of said Therapeutic, wherein a reduction in said atopic reaction or a prevention of said atopic reaction indicates that the Therapeutic has activity in treating or preventing an atopic disease.
  • a number of animal models of atopic disorders are known in the art. These models accurately mimic natural human atopic diseases. Examples of specific models include, but are not limited to. a mouse model of atopic asthma where anti-interleukin-4 inhibits IgE production (Zhou et ai. 1997. J. Asthma 34: 195-201 ); a primate model of asthma using anti-allergy/anti- asthma drugs (Turner et ai, 1996, Inflamm. Res. 45: 239-245); a mouse model of allergen- induced airway inflammation and epithelial changes (Blyth et al. 1996. Am. J. Respir. Cell Mol. Biol.
  • LYST:LYST-IP complexes particularly LYST:Imogen 38 complexes, and derivatives, analogs, and fragments thereof, nucleic acids encoding the LYST and Imogen 38 genes, anti-LYST:L YST-IP and particularly anti-LYST:Imogen 38 antibodies and antisense nucleic acids, and other modulators of the LYST:L YST-IP and particularly LYST:Imogen 38 complex activity, can be tested for activity in treating or preventing autoimmune disease in in vitro and in vivo assays.
  • a Therapeutic of the present invention can be assayed for activity in treating or preventing autoimmune disease by contacting cultured cells that exhibit an indicator of an autoimmune reaction, e.g., chemokine secretion, in vitro with the Therapeutic, and comparing the level of said indicator in the cells contacted with the Therapeutic with said level of said indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing autoimmune disease.
  • Cell models that can be used for such assays include, but are not limited to, leukocyte and other synovial cells that secrete chemokines mediating inflammation (Kunkel et ai, 1996. J. Leukoc. Biol.
  • a Therapeutic of the present invention can be assayed for activity in treating or preventing autoimmune disease by administering said Therapeutic to a test animal exhibiting an autoimmune reaction or which test animal does not exhibit an autoimmune reaction and is subsequently challenged with an agent that elicits an autoimmune reaction; and measuring the change in the autoimmune reaction after the administration of said Therapeutic, wherein a reduction in said autoimmune reaction or a prevention of said autoimmune reaction indicates that the Therapeutic has activity in treating or preventing an autoimmune disease.
  • a number of animal models of autoimmune disease are known in the art. These models, including those for arthritis, systemic lupus erythematosus. diabetes, etc., accurately mimic natural human autoimmune diseases (Farine, 97, Toxicol. 119: 29-35) and include, but are not limited to, experimental allergic encephalomyelitis for multiple sclerosis (Brabb et al. , 1997, J. Immunol.
  • ihyroglobulin-induced experimental thyroiditis multiple organ- localized autoimmune disease, e.g., thyroiditis and gastritis in BALB/c nu/nu mice receiving rat thymus grafts under their renal capsules (Taguchi and Takahashi, 1996, Immunology 89: 13-19), virus-induced autoimmune diseases such as insulin-dependent diabetes mellitus (Oldstone and von Herath, 1996 APM1S. 104: 689-97. Review), experimental autoimmune encephalomyelitis (Encinas et al. 1996, J. Neurosci. Res. 45: 655-669), experimental autoimmune labyrinthitis
  • LYST and LYST-2 and the LYST and LYST-2 binding partners, such as 14-3-3 protein.
  • HBF-G2 is expressed in the neurons of the developing telencephalon, and the DGS-I gene is involved in the DiGeorge syndrome.
  • the human homolog to rat norbin is likely to be involved in neural plasticity.
  • LYST:LIP3, LYST orbin LYST-2- :HBF-G2, LYST-2: 14-3-3. complexes), derivatives, analogs and fragments thereof, nucleic acids encoding the LYST and LYST-IP genes, anti-LYST:LYST-
  • IP antibodies, and other modulators of LYST:LYST-IP complex activity can be tested for activity in treating or preventing neurodegenerative disease in in vitro and in vivo assays.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing neurodegenerative disease by contacting cultured cells that exhibit an indicator of a neurodegenerative disease, such as overexpression of the ⁇ -A4 peptide. in vitro with the Therapeutic, and comparing the level of said indicator in the cells contacted with the Therapeutic with said level of said indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing neurodegenerative disease.
  • Specific examples of cell culture models for neurodegenerative disease include, but are not limited to, cultured rat endothelial cells from affected and nonaffected individuals (Maneiro et ai, 1997, Methods Find. Exp. Clin. Pharmacol.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing neurodegenerative disease by administering the Therapeutic to a test animal that exhibits symptoms of a neurodegenerative disease, such as premature development of cognitive deficiencies in transgenic animals expressing ⁇ -APP, or that is predisposed to develop symptoms of a neurodegenerative disease: and measuring the change in said symptoms of the neurodegenerative disease after administration of said Therapeutic, wherein a reduction in the severity of the symptoms of the neurodegenerative disease or prevention of the symptoms of the neurodegenerative disease, indicates that the Therapeutic has activity in treating or preventing neurodegenerative disease.
  • a test animal can be any one of a number of animal models know ⁇ in the art for neurodegenerative disease. These models, including those for Alzheimer's Disease and mental retardation of trisomy 21. which accurately mimic the natural human neurodegenerative disease (Campbell, et al. 1997. Mol. Psychiatry 2: 125-129; Schultz et al.
  • Alzheimer's disease Johnson- Wood et al, 1997. Proc. Natl. Acad. Sci. USA 94: 1550-1555
  • experimental autoimmune dementia Oron et al. 1997. J. Neural Transm. Suppl 49: 77-84.
  • LYST and LYST-2 and several of the identified binding partners of LYST (e.g., 14-3-3 protein. HS-1 protein, estrogen-receptor related protein. Ftel, BMKl, and casein kinase II SU) have roles in the control of cell proliferation and, therefore, cell-transformation and tumorigenesis. Accordingly, methods of the invention are provided for screening LYST:LYST- IP complexes, proteins, and fragments, derivatives and analogs of the foregoing, for activity in altering cell proliferation, cell transformation and/or tumorigenesis in vitro and in vivo.
  • the LYST:LYST-IP complexes or LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIPIO proteins, derivatives, fragments, and analogs thereof, can be assayed for activity to alter (i.e., increase or decrease) cell proliferation in cultured cells in vitro using methods which are well known in the art for measuring cell proliferation.
  • Specific examples of cell culture models include, but are not limited to. lung cancer, primary rat lung tumor cells (Swafford et al, 1997, Mol. Cell.
  • cell proliferation can be assayed by measuring
  • one embodiment of the present invention provides a method of screening LYST:LYST-IP complexes, or an individual LYST-2, LIPl, LIP2, LIP3. LIP4, LIP5,
  • LIP6, LIP7, LIP8, LIP9, or LIPIO protein for activity in altering (i.e., increasing or decreasing) proliferation of cells in vitro, comprising contacting the cells with a LYST:LYST-IP complex, or an individual LYST-2 or novel LIP protein, or a derivative, analog, or fragment thereof, measuring the proliferation of cells that have been so contacted, and comparing said proliferation with the proliferation of cells not so contacted, wherein a change in the level of proliferation in said contacted cells indicates that the complex or protein of the invention has activity to alter cell proliferation.
  • LIP8, LIP9, or LIPIO protein, derivative, fragment or analog thereof can also be screened for activity in inducing or inhibiting cell transformation (or progression to malignant phenotype) in vitro.
  • the complexes and proteins of the invention can be screened by contacting either: (i) cells with a normal phenotype (for assaying for cell transformation) or (ii) a transformed cell phenotype (for assaying for inhibition of cell transformation), with the complex or protein of the invention, and examining such cells for acquisition or loss of characteristics associated with a transformed phenotype (i.e., a set of in vitro characteristics associated with a tumorigenic ability in vivo), including, but not limited to, colony formation in soft agar.
  • the LYST:LYST-IP complexes or a LYST-2.
  • LIPl LIP2.
  • LIP6, LIP7, LIP8, LIP9, or LIP10 protein, derivative, fragment, or analog thereof, can also be screened for activity to promote or inhibit tumor formation in vivo in a non-human test animal.
  • a vast number of animal models of hype ⁇ roliferative disorders, including tumorigenesis and metastatic spread, are known in the art (see Table 317-1. Chapter 317. "Principals of Neoplasia.” in HARRISON'S PRINCIPALS OF INTERNAL MEDICINE. 13th Edition. Isselbacher et al. eds., McGraw- Hill.
  • the complexes and proteins of the present invention can be administered to non-human test animals (preferably test animals predisposed to develop a type of tumor) and the non-human test animal subsequently examined for an increased incidence of tumor formation in comparison with controls not administered the complex or protein of the invention.
  • the complexes and proteins of the present invention can be administered to non- human test animals having tumors (e.g.. animals in which tumors have been induced by introduction of malignant, neoplastic. or transformed cells, or by administration of a carcinogen) and subsequently examining the tumors in the test animals for tumor regression in comparison to controls not administered the complex a protein of the present invention.
  • tumors e.g.. animals in which tumors have been induced by introduction of malignant, neoplastic. or transformed cells, or by administration of a carcinogen
  • LYST protein has been implicated in pigmentation disorders (see supra), including oculocutaneous albinism and hypopigmentation. Accordingly, LYST:LYST-IP complexes, and derivatives, analogs, and fragments thereof, nucleic acids encoding the LYST or LYST-2 genes. anti-LYST:LYST-IP antibodies or nucleic acids, and other modulators of the LYST:L YST-IP complex, can be tested for activity in treating or preventing pigmentation disorders in in vitro and in vivo assays.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing pigmentation disorders by contacting cultured cells that exhibit an indicator of a pigmentation reaction, in vitro with the Therapeutic, and comparing the level of indicator in the cells so contacted with said level of the indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing pigmentation disorders.
  • Cell models that can be used for such assays include, but are not limited to, in vitro studies using cultured vitiligo melanocytes and keratinocytes (Bessou et al, 1997, Br. J. Dermatol.
  • mice 137: 890-897 lines of immortal, severely hypopigmented melanocytes and melanoblasts from mice of the null genotype p(cp)/p(25H) (Sviderskaya et al, 1997, J. Invest. Dermatol. 108: 30-34); and organotypic culture of human skin to study melanocyte migration (Le Poole et al, 1994, Pigment. Cell Res. 7: 33-43).
  • a Therapeutic of the invention can be assayed for activity in treating or preventing pigmentation disease by administering said Therapeutic to a test animal exhibiting a pigmentation reaction or which test animal does not exhibit a pigmentation reaction and is subsequently challenged with an agent that elicits a pigmentation reaction; and measuring the change in the pigmentation reaction after the administration of said Therapeutic, wherein a reduction in said pigmentation reaction or a prevention of said pigmentation reaction indicates that the Therapeutic has activity in treating or preventing an pigmentation disease.
  • a number of animal models of pigmentation disorders are known in the art. These models accurately mimic natural human pigmentation diseases. Examples of specific models include, but are not limited to, smyth line (SL) chickens that develop a spontaneous, autoimmune loss of melanocytes (vitiligo) from the feather (Erf et al. 1997. Vet. Immunol. Immunopathol. 58: 335-343); and mouse pale ear (ep) mutation (Gardner et ai. Proc. Natl Acad. Sci. U.S.A. 94: 9238-9243).
  • SL smyth line
  • ep mouse pale ear
  • LYST protein has been implicated in platelet dysfunction (see supra). Accordingly,
  • LYST:LYST-IP complexes, and derivatives, analogs, and fragments thereof, nucleic acids encoding the LYST genes, anti-LYST:LYST-IP antibodies or nucleic acids, and other modulators of the LYST:LYST-IP. can be tested for activity in treating or preventing platelet dysfunction in in vitro and in vivo assays.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing platelet dysfunction by contacting cultured cells in vitro that exhibit an indicator of a platelet reaction with the Therapeutic, and comparing the level of indicator in the cells so contacted with said level of the indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing platelet dysfunction.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing platelet dysfunction by administering said Therapeutic to a test animal exhibiting a platelet reaction or which test animal does not exhibit a platelet reaction and is subsequently challenged with an agent that elicits a platelet reaction; and measuring the change in the platelet reaction after the administration of said Therapeutic, wherein a reduction in said pigmentation reaction or a prevention of said platelet reaction indicates that the Therapeutic has activity in treating or preventing an platelet dysfunction.
  • a number of animal models of platelet dysfunction are known in the art. These models accurately mimic natural human platelet dysfunction. Examples of specific models include, but are not limited to, a skin bleeding time test in a rat model (MacDonald et ai.
  • the LYST interactant Importin beta subunit protein is strongly implicated in viral infection mechanisms, including that for the AIDS virus, HIV-1.
  • the nuclear localization signal of the HIV-1 Rev protein mediates specific binding to human Importin-beta.
  • An enormous number of human diseases result from virulent and opportunistic viral infection (see Belshe (Ed.) 1984 TEXTBOOK OF HUMAN VIROLOGY. PSG Publishing, Littleton. MA).
  • Diseases of a wide array of tissues including the respiratory tract, the CNS, skin, the genitourinary tract, eyes. ears.
  • LYST:LYST-IP complexes particularly the LYST:Importin beta complex, and derivatives.
  • nucleic acids containing the LYST or LYST-IP gene in particular the Importin beta gene
  • anti-LYST:LYST-IP antibodies or nucleic acids in particular anti-LYST:Importin beta antibodies or nucleic acids, and other modulators of the LYST:L YST- IP complex activity, can be tested for activity in treating or preventing viral diseases in in vitro and in vivo assays.
  • a Therapeutic of the invention can be assayed for activity in treating or preventing viral disease by contacting cultured cells that exhibit an indicator of a viral reaction, e.g., formation of inclusion bodies, in vitro with the Therapeutic, and comparing the level of said indicator in the cells contacted with the Therapeutic with said level of said indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing viral disease.
  • Cell models that can be used for such assays include, but are not limited to, viral infection of T lymphocytes (Selin et al, 1996, J. Exp. Med.
  • hepatitis B infection of dedifferentiated hepatoma cells (Raney et al, 991, J. Virol. 71: 1058-1071); viral infection of cultured salivary gland epithelial cells (Clark et al, 1994, Autoimmunity 18: 7-14); synchronous HIV-1 infection of CD4+ lymphocytic cell lines (Wainberg et al, 1997, Virology 233: 364-373); viral infection of respiratory epithelial cells (Stark et al. 1996, Human Gene Ther. 7: 1669-1681); and amphotrophic retroviral infection ofNIH-3T3 cells (Morgan et al, 1995, J. Virol. 69: 6994-7000).
  • a Therapeutic of the invention can be assayed for activity in treating or preventing viral disease by administering said Therapeutic to a test animal having symptoms of a viral infection, such as characteristic respiratory symptoms in animal models of virally-induced asthma, or which test animal does not exhibit a viral reaction and is subsequently challenged with an agent that elicits an viral reaction; and measuring the change in the viral reaction after the administration of said Therapeutic, wherein a reduction in said viral reaction or a prevention of said viral reaction indicates that the Therapeutic has activity in treating or preventing viral disease.
  • Animal models that can be used for such assays include, but are not limited to, respiratory viral infections in guinea pigs (Kudlacz and Knippenberg, 1995. Inflamm. Res.
  • mice 1 16); encephalomyocarditis infection of mice (Hirasawa et al, 1997, J. Virol. 1: 4024-4031); and cytomegalovirus infection of mice (Orange and Biron, 1996. J. Immunol. 156: 1 138-1 142).
  • LYST:LYST-IP complexes or individual LYST-2 or novel LIP proteins, and derivatives, fragments and analogs thereof, as well as nucleic acids encoding LYST, LYST-2, LYST-IPs.
  • LIPl, LIP2, LIP3, LIP4, LIP5, LIP6. LIP7, LIP8, LIP9, or LIPIO. as well as derivatives, fragments and analogs thereof, can be used to screen for compounds that bind to said nucleic acids, proteins, or derivatives, and thus have potential uses as agonists or antagonists of LYST:L YST-IP complexes, or as antagonists or agonists of an individual LYST-2 or novel LIP protein function.
  • the present invention thus provides assays to detect molecules that specifically bind to LYST:LYST-IP complexes, and LYST, LYST-2, LYST-IP, nucleic acids, proteins or derivatives.
  • recombinant cells expressing both LYST or LYST-2 and/or LYST-IP nucleic acids, or expressing individual LYST-2 or novel LIP nucleic acids can be used to recombinantly produce the complexes or proteins used in these assays, to screen for molecules that bind or interfere with LYST:LYST-IP complexes, or that interfere with an individual LYST- 2 or novel LIP function.
  • peptide analogs that have superior stabilities yet retain the ability to form LYST:LYST-IP complexes are used to screen for modulators which are, e.g., molecules generated by substitution of amino acids at proteolytic cleavage sites, such as the use of chemically derivatized amino acids at proteolytic susceptible sites, and replacement of amino acid residues subject to oxidation, i.e.. methionine and cysteine.
  • Molecules e.g., putative binding partners of a LYST:LYST-IP complex, or of an individual LYST-2 or novel LIP protein
  • Molecules are contacted with the LYST:LYST-IP complex or with an individual LYST-2 or novel LIP protein, or fragment thereof, under conditions conducive to binding, and then molecules that specifically bind to a LYST:LYST-IP complex, or to an individual LYST-2 or novel LIP protein, are identified. Similar methods can be used to screen for molecules that bind to LYST or LYST-2 and/or LYST-IP nucleic acids or derivatives.
  • a particular aspect of the invention relates to identifying molecules that inhibit or promote formation or dissolution or degradation of a LYST:L YST-IP complex, e.g., using the method described for screening inhibitors using the modified yeast two hybrid assay described infra.
  • a molecule that modulates activity of LYST or LYST-2 or LYST-IP. or a complex of LYST or LYST-2 and said protein is identified by contacting one or more candidate molecules with LYST or LYST-2 in the presence of said protein; and measuring the amount of complex that forms between LYST or LYST-2 and said protein; wherein an increase or decrease in the amount of complex that forms relative to the amount that forms in the absence of the candidate molecules indicates that the molecules modulate the activity of LYST or LYST-2 or said protein or said complex of LYST or LYST-2 and said protein.
  • modulators are identified by administering a candidate molecule to a transgenic non-human animal expressing both LYST or LYST-2 and a LYST-IP from promoters that are not the native LYST or LYST-2 or the native LYST-IP promoters, more preferably where the candidate molecule is also recombinantly expressed in the transgenic non-human animal.
  • the method for identifying such modulators can be carried out in vitro, preferably with purified LYST or LYST-2, purified LYST-IP, and a purified candidate molecule.
  • Agents to be screened can be provided as mixtures of a limited number of specified compounds, or as compound libraries, peptide libraries and the like. Agents to be screened may also include all forms of antisera, antisense nucleic acids, etc., that can modulate LYST:L YST-IP complex activity, or modulate an individual LYST-2 or novel LIP activity.
  • diversity libraries such as random or combinatorial peptide or non- peptide libraries can be screened for molecules that specifically bind to a LYST:LYST-IP complex, or to an individual LYST-2 or novel LIP protein.
  • Many libraries are known in the art that can be used, e.g.. chemically synthesized libraries, recombinant libraries (e.g., phage display libraries), and in vitro translation-based libraries.
  • In vitro translation-based libraries include but are not limited to those described in International Patent Publication WO 91/05058; and Mattheakis et al. 1994, Proc. Natl. Acad. Sci. USA 91: 9022-9026.
  • a benzodiazepine library see, e.g., Bunin et al, 1994. Proc. Natl. Acad. Sci. USA 91: 4708-4712
  • Peptoid libraries (Simon et al. 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371) can also be used.
  • Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley et al, 9%9, Adv. Exp. Med. Biol. 251: 215-218; Scott et al, 1990, Science 249: 386- 390: Fowlkes et al. , 1992, BioTechniques 13: 422-427; Oldenburg et al. , 1992. Proc. Natl. Acad. Sci. USA 89: 5393-5397; Yu et al, 1994. Cell 76: 933-945; Staudt et al. 1988.
  • screening can be carried out by contacting the library members with a LYST:LYST-IP complex, or with a LYST-2. LIPl.
  • molecules that bind to a LYST:LYST-IP complex, or to an individual LYST-2 or novel LIP protein can be screened for by using the modified yeast two hybrid system described infra, and exemplified in EXAMPLES, infra.
  • agents that modulate i.e., inhibit, antagonize or agonize
  • LYST:LYST-IP complex activity can be screened for using a binding inhibition assay, wherein agents are screened for their ability to inhibit formation of a LYST:L YST-IP complex under aqueous, or physiological, binding conditions in which LYST:LYST-IP complex formation occurs in the absence of the agent to be tested.
  • Agents that interfere with the formation of LYST:L YST-IP complexes are identified as antagonists of complex formation.
  • Agents that eliminate the formation of LYST:L YST-IP complexes are identified as inhibitors of complex formation.
  • Agents that enhance the formation of LYST:L YST-IP complexes are identified as agonists of complex formation .
  • Methods for screening may involve labeling the complex proteins with radioligands (e.g., ,25 I or 3 H), magnetic ligands (e.g., paramagnetic beads covalently attached to photobiotin acetate), fluorescent ligands (e.g., fluorescein or rhodamine) or enzyme ligands (e.g., luciferase or beta-galactosidase).
  • radioligands e.g., ,25 I or 3 H
  • magnetic ligands e.g., paramagnetic beads covalently attached to photobiotin acetate
  • fluorescent ligands e.g., fluorescein or rhodamine
  • enzyme ligands e.g., luciferase or beta-galactosidase
  • one binding partner is a small fragment or peptidomimetic that is not retained by a commercially available filter. Upon binding, the labeled species is then unable to pass through the filter, providing for a simple assay of complex formation.
  • Suitable labeling includes, but is not limited to, radiolabeling by inco ⁇ oration of radiolabeled amino acids, e.g., 3 H-leucine or 33 S-methionine. radiolabeling by post-translational iodination with l25 I or I3I I using the chloramine T method. Bolton-Hunter reagents, etc.. labeling with 32 P using a kinase and inorganic radiolabeled phosphorous, biotin labeling with photobiotin-acetate and sunlamp exposure, etc.
  • the free species is labeled.
  • each can be labeled with a distinguishable marker such that isolation of both moieties can be followed to provide for more accurate quantitation. and to distinguish the formation of homomeric from heteromeric complexes.
  • Typical binding conditions are. for example, but not by way of limitation, in an aqueous salt solution of 10-250 mM NaCl. 5-50 mM Tris-HCl, pH 5-8, and 0.5% Triton X-100 or other detergent that improves the specificity of interaction.
  • Metal chelators and/or divalent cations may be added to improve binding and/or reduce proteolysis. Reaction temperatures may range around 4, 10, 15, 22, 25, 37, or 42 degrees Celsius, and time of incubation is typically at least 15 seconds, but longer times are preferred to allow binding equilibrium to occur.
  • Particular LYST:LYST-IP complexes can be assayed using routine protein binding assays to determine optimal binding conditions for reproducible binding.
  • the physical parameters of complex formation can be analyzed by quantitation of complex formation using assay methods specific for the label used, e.g., liquid scintillation spectroscopy for radioactivity detection, enzyme activity measurements for enzyme labeling, etc.
  • the reaction results are then analyzed utilizing Scatchard analysis, Hill analysis, and other methods commonly known in the art (see, e.g., PROTEINS, STRUCTURES, AND MOLECULAR PRINCIPLES, 2nd Edition (1993) Creighton, Ed., W.H. Freeman and Company, New York).
  • one of the binding species is immobilized on a filter, in a microtiter plate well, in a test tube, to a chromatography matrix, etc..
  • Proteins can be covalently immobilized using any method well known in the art, for example, but not limited to the method of Kadonaga and Tjian (1986, Proc. Natl. Acad. Sci. USA 83: 5889-5893, 1986), i.e.. linkage to a cyanogen-bromide derivatized substrate such as CNBr-Sepahrose 4B. Where needed, the use of spacers can reduce steric hindrance by the substrate.
  • Non-covalent attachment of proteins to a substrate include, but are not limited to. attachment of a protein to a charged surface, binding with specific antibodies, binding to a third unrelated interacting protein.
  • immobilized LYST or LYST-2 is used to assay for binding with a radioactively-labeled LYST-IP in the presence and absence of a compound to be tested for its ability to modulate LYST:LYST-IP complex formation.
  • the binding partners are allowed to bind under aqueous, or physiological, conditions (e.g., the conditions under which the original interaction was detected).
  • the LYST-IP is immobilized and contacted with the labeled LYST or LYST-2 protein or derivative thereof under binding conditions.
  • Assays of agents for competition for binding of one member of a LYST:L YST-IP complex (or derivatives thereof) with the other member of the LYST:L YST-IP complex (labeled by any means, e.g., those means described supra), are provided to screen for competitors of LYST:L YST-IP complex formation.
  • blocking agents to inhibit non-specific binding of reagents to other protein components, or abso ⁇ tive losses of reagents to plastics, immobilization matrices, etc. are included in the assay mixture.
  • Blocking agents include, but are not restricted to, bovine serum albumin, beta-casein, nonfat dried milk. Denhardt's reagent, Ficoll, polyvinylpyrolidine, nonionic detergents (NP40, Triton X-100, Tween 20, Tween 80, etc.), ionic detergents (e.g., SDS, LDS. etc.). polyethylene glycol. etc. Appropriate blocking agent concentrations are utilized to allow LYST:LYST-IP complex formation.
  • One aspect of the present invention provides methods for assaying and screening fragments, derivatives and analogs of LYST or LYST-2 interacting proteins (for binding to a LYST or LYST-2 peptide).
  • Derivatives, analogs and fragments of LYST-IPs that interact with LYST or LYST-2 can be identified by means of a yeast two hybrid assay system (Fields and Song, 1989, Nature 340: 245-246 and U.S. Patent No. 5,283,173). Because the interactions are screened for in yeast, the intermolecular protein interactions detected in this system occur under physiological conditions that mimic the conditions in mammalian cells (Chien et al, 1991, Proc. Natl. Acad. Sci. USA 88: 9578-9581).
  • the improved yeast two hybrid system is based upon the detection of expression of a reporter gene, the transcription of which is dependent upon the reconstitution of a transcriptional regulator by the interaction of two proteins, each fused to one half of the transcriptional regulator.
  • the "bait” (LYST or LYST-2 or derivative or analog) and “prey” (proteins to be tested for ability to interact with the bait) proteins are expressed as fusion proteins to a DNA binding domain, and to a transcriptional regulatory domain, respectively, or vice versa.
  • the prey has a complexity in the range of about 25 to about 100.000. about 100 to about 100.000. about 50.000 to about 100.000. or about 100,000 to about 500.000.
  • the prey population can be one or more nucleic acids encoding mutants of a LYST-IP (e.g., as generated by site-directed mutagenesis or another method of making mutations in a nucleotide sequence).
  • the prey populations are proteins encoded by DNA. e.g.. cDNA or genomic DNA or synthetically generated DNA.
  • the populations can be expressed from chimeric genes comprising cDNA sequences from an un-characterized sample of a population of cDNA from mammalian RNA.
  • recombinant biological libraries expressing random peptides can be used as the source of prey nucleic acids.
  • the invention provides methods of screening for inhibitors or enhancers of the protein interactants identified herein. Briefly, the protein-protein interaction assay can be carried out as described herein, except that it is done in the presence of one or more candidate molecules. An increase or decrease in reporter gene activity relative to that present when the one or more candidate molecules are absent indicates that the candidate molecule has an effect on the interacting pair.
  • inhibition of the interaction is selected for (i.e., inhibition of the interaction is necessary for the cells to survive), for example, where the interaction activates the URA3 gene, causing yeast to die in medium containing the chemical 5- fluoroorotic acid (Rothstein. 1983, Meth. Enzymol 101: 167-180).
  • the identification of inhibitors of such interactions can also be accomplished, for example, but not by way of limitation, using competitive inhibitor assays, as described supra.
  • proteins of the bait and prey populations are provided as fusion (chimeric) proteins (preferably by recombinant expression of a chimeric coding sequence) comprising each protein contiguous to a pre-selected sequence.
  • the pre-selected sequence is a DNA binding domain.
  • the DNA binding domain can be any DNA binding domain, as long as it specifically recognizes a DNA sequence within a promoter.
  • the DNA binding domain is of a transcriptional activator or inhibitor.
  • the pre-selected sequence is an activator or inhibitor domain of a transcription factor.
  • the regulatory domain alone (not as a fusion to a protein sequence) and the DNA-binding domain alone (not as a fusion to a protein sequence) preferably do not detectably interact (so as to avoid false positives in the assay).
  • the assay system further includes a reporter gene operably linked to a promoter that contains a binding site for the DNA binding domain of the transcriptional activator (or inhibitor). Accordingly, in the present method of the present invention, binding of a LYST or LYST-2 fusion protein to a prey fusion protein leads to reconstitution of a transcriptional activator (or inhibitor) which activates (or inhibits) expression of the reporter gene.
  • the activation (or inhibition) of transcription of the reporter gene occurs intracellularly, e.g., in prokaryotic or eukaryotic cells, preferably in cell culture.
  • the promoter that is operably linked to the reporter gene nucleotide sequence can be a native or non-native promoter of the nucleotide sequence, and the DNA binding site(s) that are recognized by the DNA binding domain portion of the fusion protein can be native to the promoter (if the promoter normally contains such binding site(s)) or non-native to the promoter.
  • one or more tandem copies (e.g., 4 or 5 copies) of an appropriate DNA binding site can be introduced upstream of the TATA box in the desired promoter (e.g., in the area of about position -100 to about -400).
  • 4 or 5 tandem copies of the 17 base pair UAS (GAL4 DNA binding site) are introduced upstream of the TATA box in the desired promoter, which is upstream of the desired coding sequence for a selectable or detectable marker.
  • the GAL1-10 promoter is operably fused to the desired nucleotide sequence; the GAL1-10 promoter already contains 5 binding sites for GAL4.
  • the transcriptional activation binding site of the desired gene(s) can be deleted and replaced with GAL4 binding sites (Bartel et al, 1993, BioTechniques 14: 920-924, Chasman et al, 1989, Mol. Cell. Biol. 9: 4746-4749).
  • the reporter gene preferably contains the sequence encoding a detectable or selectable marker, the expression of which is regulated by the transcriptional activator, such that the marker is either turned on or off in the cell in response to the presence of a specific interaction.
  • the assay is carried out in the absence of background levels of the transcriptional activator (e.g., in a cell that is mutant or otherwise lacking in the transcriptional activator).
  • more than one reporter gene is used to detect transcriptional activation, e.g., one reporter gene encoding a detectable marker and one or more reporter genes encoding different selectable markers.
  • the detectable marker can be any molecule that can give rise to a detectable signal, e.g., a fluorescent protein or a protein that can be readily visualized or that is recognizable by a specific antibody.
  • the selectable marker can be any protein molecule that confers the ability to grow under conditions that do not support the growth of cells not expressing the selectable marker, e.g., the selectable marker is an enzyme that provides an essential nutrient and the cell in which the interaction assay occurs is deficient in the enzyme and the selection medium lacks such nutrient.
  • the reporter gene can either be under the control of the native promoter that naturally contains a binding site for the DNA binding protein, or under the control of a heterologous or synthetic promoter.
  • the activation domain and DNA binding domain used in the assay can be from a wide variety of transcriptional activator proteins, as long as these transcriptional activators have separable binding and transcriptional activation domains.
  • the GAL4 protein of S. cerevisiae (Ma et al. 1987. Cell 48: 847-853)
  • the GCN4 protein of S. cerevisiae Hope and Struhl, 1986, Cell 46: 885-894
  • the ARD1 protein of S. cerevisiae Thukral et al, 1989. Mol.
  • DNA binding domain and activation domains that are employed in the fusion proteins need not be from the same transcriptional activator.
  • a GAL4 or LexA DNA binding domain is employed.
  • a GAL4 or he ⁇ es simplex virus VP16 (Triezenberg et al, 1988, Genes Dev. 2: 730-742) activation domain is employed.
  • amino acids 1-147 of GAL4 (Ma et al, 1987, Cell 48: 847-853; Ptashne et al, 1990, N ⁇ twre 346: 329-331) is the D ⁇ A binding domain, and amino acids 41 1-455 of VP16 (Triezenberg et al, 1988, Genes Dev. 2: 730-742; Cress et al, 1991, Science 251: 87-90) comprise the activation domain.
  • the yeast transcription factor GAL4 is reconstituted by protein-protein interaction and the host strain is mutant for GAL4.
  • the D ⁇ A-binding domain is Acel ⁇ and/or the activation domain is Acel, the D ⁇ A binding and activation domains of the Acel protein, respectively.
  • Acel is a yeast protein that activates transcription from the CUP I operon in the presence of divalent copper.
  • CUP I encodes metallothionein. which chelates copper, and the expression of CUP 1 protein allows growth in the presence of copper, which is otherwise toxic to the host cells.
  • the reporter gene can also be a CUP I -lacZ fusion that expresses the enzyme beta-galactosidase (detectable by routine chromogenic assay) upon binding of a reconstituted Acel ⁇ transcriptional activator (see Chaudhuri et al, 1995. EE7?S Letters 357: 221-226).
  • the D ⁇ A binding domain of the human estrogen receptor is used, with a reporter gene driven by one or three estrogen receptor response elements (Le Douarin et al. 1995. Nucl Acids. Res. 23: 876- 878).
  • the D ⁇ A binding domain and the transcriptional activator/inhibitor domain each preferably has a nuclear localization signal (see Ylikomi et al. 1992. EMBO J. 11: 3681-3694. Dingwall and Laskey, 1991 , TIBS 16: 479-481) functional in the cell in which the fusion proteins are to be expressed.
  • the fusion constructs can further contain sequences encoding affinity tags such as glutathione-S-transferase or maltose-binding protein or an epitope of an available antibody, for affinity purification (e.g., binding to glutathione. maltose, or a particular antibody specific for the epitope. respectively) (Allen et al. 1995, 77RS 20: 51 1- 516).
  • the fusion constructs further comprise bacterial promoter sequences for recombinant production of the fusion protein in bacterial cells.
  • the host cell in which the interaction assay occurs can be any cell, prokaryotic or eukaryotic. in which transcription of the reporter gene can occur and be detected, including, but not limited to, mammalian (e.g., monkey, mouse, rat, human, bovine), chicken, bacterial, or insect cells, and is preferably a yeast cell.
  • mammalian e.g., monkey, mouse, rat, human, bovine
  • chicken bacterial, or insect cells
  • Expression constructs encoding and capable of expressing the binding domain fusion proteins, the transcriptional activation domain fusion proteins, and the reporter gene product(s) are provided within the host cell, by mating of cells containing the expression constructs, or by cell fusion, transformation, electroporation, microinjection, etc.
  • the DNA binding domain is the GAL4 DNA binding domain
  • the activation domain is the he ⁇ es simplex virus VP16 transcriptional activation domain
  • the reporter gene contains the desired coding sequence operably linked to a minimal promoter element from the adenovirus E1B gene driven by several GAL4 DNA binding sites (see Fearon et al, 1992, Proc. Natl Acad. Sci. USA 89: 7958-7962).
  • the host cell used should not express an endogenous transcription factor that binds to the same DNA site as that recognized by the DNA binding domain fusion population.
  • the host cell is mutant or otherwise lacking in an endogenous, functional form of the reporter gene(s) used in the assay.
  • yeast strains or derivative strains made therefrom which can be used are N 105, N 106. N 1051. N 1061, and YULH, as described in EXAMPLES, infra.
  • Other exemplary strains that can be used in the assay of the invention also include, but are not limited to. the following:
  • Y190 MATa. ura3-52. his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4 ⁇ . gal80 ⁇ . cyh r 2.
  • CG-1945 MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901. Ieu2-3, 112. gal4-542, gal80-538, cyh r 2.
  • CG-1945 contains H7S5 and IacZ reporter genes driven by GAL4 binding sites.
  • Y187 MAT-a, ura3-52, his3-200, ade2-101, trpl-901, leu2-3,112, gal4a, gal80a,
  • Y187 contains a IacZ reporter gene driven by GAL4 binding sites.
  • SFY526 contains
  • HIS3 and IacZ reporter genes driven by GAL4 binding sites were HIS3 and IacZ reporter genes driven by GAL4 binding sites.
  • ⁇ F7c MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::GAL1 UAS rMERS(x3) -CYCl-lacZ, available from Clontech, Palo Alto, CA.
  • HF7c contains HIS3 and IacZ reporter genes driven by GAL4 binding sites.
  • YRG-2 MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3,112, gal4-542, gal80-538, LYS2::GALl UA:r GALl rATA -HIS3, URA3::GALl UAsr neglect iersMf CYCl-lacZ, available from
  • YRG-2 contains H7S3 and IacZ reporter genes driven by GAL4 binding sites. Many other strains commonly known and available in the art can be used.
  • cells mutant in the reporter gene may be selected by known methods, or the cells can be made mutant in the target reporter gene by known gene-disruption methods prior to introducing the reporter gene (Rothstein, 1983,
  • plasmids encoding the different fusion protein populations can be introduced simultaneously into a single host cell (e.g., a haploid yeast cell) containing one or more reporter genes, by co-transformation, to conduct the assay for protein-protein interactions.
  • a single host cell e.g., a haploid yeast cell
  • the two fusion protein populations are introduced into a single cell either by mating (e.g., for yeast cells) or cell fusions (e.g., of mammalian cells).
  • mating e.g., for yeast cells
  • cell fusions e.g., of mammalian cells.
  • conjugation of haploid yeast cells of opposite mating type that have been transformed with a binding domain fusion expression construct (preferably a plasmid) and an activation (or inhibitor) domain fusion expression construct (preferably a plasmid), respectively will deliver both constructs into the same diploid cell.
  • the mating type of a yeast strain may be manipulated by transformation with the HO gene (Herskowitz and Jensen. 1991. Meth. Enzymoi 194: 132-
  • a yeast interaction mating assay is employed using two different types of host cells, strain-type a and alpha of the yeast Saccharomyces cerevisiae.
  • the host cell preferably contains at least two reporter genes, each with one or more binding sites for the DNA-binding domain (e.g., of a transcriptional activator).
  • the activator domain and DNA binding domain are each parts of chimeric proteins formed from the two respective populations of proteins.
  • One strain of host cells for example the a strain, contains fusions of the library of nucleotide sequences with the DNA-binding domain of a transcriptional activator, such as GAL4.
  • the hybrid proteins expressed in this set of host cells are capable of recognizing the DNA- binding site in the promoter or enhancer region in the reporter gene construct.
  • the second set of yeast host cells for example, the alpha strain, contains nucleotide sequences encoding fusions of a library of DNA sequences fused to the activation domain of a transcriptional activator.
  • the fusion protein constructs are introduced into the host cell as a set of plasmids.
  • These plasmids are preferably capable of autonomous replication in a host yeast cell and preferably can also be propagated in E. coli.
  • the plasmid contains a promoter directing the transcription of the DNA binding or activation domain fusion genes, and a transcriptional termination signal.
  • the plasmid also preferably contains a selectable marker gene, permitting selection of cells containing the plasmid.
  • the plasmid can be single-copy or multi-copy. Single-copy yeast plasmids that have the yeast centromere may also be used to express the activation and DNA binding domain fusions ( ⁇ lledge et ai, 1988. Gene 70: 303- 312).
  • the fusion constructs are introduced directly into the yeast chromosome via homologous recombination.
  • the homologous recombination for these pu ⁇ oses is mediated through yeast sequences that are not essential for vegetative growth of yeast, e.g., the MER2, MER1, ZIPI, REC102, o ⁇ ME14 gene.
  • Bacteriophage vectors can also be used to express the DNA binding domain and/or activation domain fusion proteins. Libraries can generally be prepared faster and more easily from bacteriophage vectors than from plasmid vectors.
  • the present invention provides a method of detecting one or more protein-protein interactions comprising (a) recombinantly expressing LYST or LYST-2 or a derivative or analog thereof in a first population of yeast cells being of a first mating type and comprising a first fusion protein containing the LYST or LYST-2 sequence and a DNA binding domain, wherein said first population of yeast cells contains a first nucleotide sequence operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that an interaction of said first fusion protein with a second fusion protein, said second fusion protein comprising a transcriptional activation domain, results in increased transcription of said first nucleotide sequence: (b) negatively selecting to eliminate those yeast cells in said first population in which said increased transcription of said
  • the bait LYST or LYST-2 sequence and the prey library of chimeric genes are combined by mating the two yeast strains on solid media for a period of approximately 6-8 hours. In a less preferred embodiment, the mating is performed in liquid media.
  • the resulting diploids contain both kinds of chimeric genes, i.e., the DNA-binding domain fusion and the activation domain fusion.
  • Preferred reporter genes include the URA3, HIS3 and/or the IacZ genes (see. e.g., Rose and Botstein. 1983, Meth. Enzymol 101: 167-180) operably linked to GAL4 DNA-binding domain recognition elements.
  • reporter genes comprise the functional coding sequences for, but not limited to, Green Fluorescent Protein (GFP) (Cubitt et al, 1995. Trends Biochem. Sci. 20: 448-455). luciferase, LEU2, LYS2, ADE2. TRPl, CANT CYH2, GUS. CUP! or chloramphenicol acetyl transferase (CAT). Expression of LEU2, LYS2, ADE2 and TRPl are detected by growth in a specific defined media: GUS and CAT can be monitored by well known enzyme assays: and CAN1 and CYH2 are detected by selection in the presence of canavanine and cycloheximide. With respect to GFP. the natural fluorescence of the protein is detected. In a specific embodiment, transcription of the reporter gene is detected by a linked replication assay. For example, as described by Vasavada et al. 1991. Proc. Natl. Acad. Sci.
  • reporter genes that encode proteins can be detected by immunoassay, i.e., by detecting the immunospecific binding of an antibody to such protein, which antibody can be labeled, or alternatively, which antibody can be incubated with a labeled binding partner to the antibody, so as to yield a detectable signal.
  • immunoassay i.e., by detecting the immunospecific binding of an antibody to such protein, which antibody can be labeled, or alternatively, which antibody can be incubated with a labeled binding partner to the antibody, so as to yield a detectable signal.
  • Alam and Cook (1990. Anal. Biochem. 188: 245-254) disclose non-limiting examples of detectable marker genes that can be operably linked to a transcriptional regulatory region responsive to a reconstituted transcriptional activator, and thus used as reporter genes.
  • the activation of reporter genes like URA3 or HIS3 enables the cells to grow in the absence of uracil or histidine, respectively, and hence serves as a selectable marker.
  • the cells exhibiting protein-protein interactions are selected by the ability to grow in media lacking a nutritional component, such as uracil or histidine (referred to as -URA (minus URA) and -HIS (minus HIS) medium, respectively).
  • the -HIS medium preferably contains 3- amino- 1,2,4-triazole (3-AT), which is a competitive inhibitor of the HIS3 gene product, and thus, requires higher levels of transcription in the selection (see. Durfee et al, 1993, Genes Dev. 7: 555-569).
  • 3-azauracil. which is an inhibitor of the URA3 gene product, can be included in -URA medium (Le Douarin et ai, 1995, Nucl. Acids Res.
  • URA3 gene activity can also be detected and/or measured by determining the activity of its gene product, orotidine-51-monophosphate decarboxylase (Pierrat et ai. 1992, Gene 119: 237-245, Wolcott et al, 1966, Biochem. Biophys. Acta 122: 532-534).
  • the activities of the reporter genes like GFP or IacZ are monitored by measuring a detectable signal (e.g., fluorescent or chromogenic, respectively) that results from the activation of these reporter genes. For example.
  • IacZ transcription can be monitored by incubation in the presence of a chromogenic substrate, such as X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside), of its encoded enzyme, ⁇ -galactosidase.
  • a chromogenic substrate such as X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside)
  • ⁇ -galactosidase a chromogenic substrate
  • the pool of all interacting proteins isolated by this manner from mating the LYST or LYST-2 sequence product and the librar ' identifies the "LYST interactive population".
  • false positives arising from transcriptional activation by the DNA binding domain fusion proteins in the absence of a transcriptional activator domain fusion protein are prevented or reduced by negative selection for such activation within a host cell containing the DNA binding fusion population, prior to exposure to the activation domain fusion population.
  • a host cell containing the DNA binding fusion population prior to exposure to the activation domain fusion population.
  • URA3 as a reporter gene
  • negative selection is carried out by incubating the cell in the presence of 5-fluoroorotic acid (5-FOA, which kills URA+ cells (Rothstein, 1983. Meth. Enzymol. 101:
  • Negative selection involving the use of a selectable marker as a reporter gene and the presence in the cell medium of an agent toxic or growth inhibitory to the host cells in the absence of reporter gene transcription is prefe ⁇ ed. since it allows a higher rate of processing than other methods.
  • negative selection can also be carried out on the activation domain fusion population prior to interaction with the DNA binding domain fusion population, by similar methods, either alone or in addition to negative selection of the DNA binding fusion population.
  • Negative selection can also be carried out on the recovered LYST:LYST-IP complex by known methods (see, e.g., Bartel et al, 1993, BioTechniques 14: 920-924) although pre-negative selection (prior to the interaction assay), as described above, is preferred.
  • each plasmid encoding a protein (peptide or polypeptide) fused to the activation domain can be transformed back into the original screening strain, either alone or with a plasmid encoding only the DNA-binding domain, the DNA-binding domain fused to the detected interacting protein, or the DNA-binding domain fused to a protein that does not affect transcription or participate in the protein-protein interaction.
  • a positive interaction detected with any plasmid other than that encoding the DNA-binding domain fusion to the detected interacting protein is deemed a false positive and is eliminated from the screen.
  • the LYST or LYST-2 plasmid population is transformed in a yeast strain of a first mating type (a or alpha), and the second plasmid population (containing the library of DNA sequences) is transformed in a yeast strain of a different mating type. Both strains are preferably mutant for URA3 and HIS3. and contain H7S5, and optionally IacZ, as reporter genes.
  • the first set of yeast cells are positively selected for the LYST plasmids and are negatively selected for false positives by incubation in medium lacking the selectable marker (e.g., tryptophan) and containing 5-FOA.
  • Yeast cells of the second mating type are transformed with the second plasmid population, and are positively selected for the presence of the plasmids containing the library of fusion proteins. Selected cells are pooled. Both groups of pooled cells are mixed together and mating is allowed to occur on a solid phase. The resulting diploid cells are then transfe ⁇ ed to selective media that selects for the presence of each plasmid and for activation of reporter genes.
  • the DNA sequences encoding the pairs of interactive proteins are isolated by a method wherein either the DNA-binding domain hybrids or the activation domain hybrids are amplified, in separate respective reactions.
  • the amplification is carried out by polymerase chain reaction (PCR) (see, U.S. Patent Nos. 4.683,202; 4,683,195; and 4.889,818; Innis et al. 1990, PCR PROTOCOLS. Academic Press. Inc.. San Diego, CA) using pairs of oligonucleotide primers specific for either the DNA-binding domain hybrids or the activation domain hybrids.
  • PCR polymerase chain reaction
  • This PCR reaction can also be performed on pooled cells expressing interacting protein complexes, preferably pooled a ⁇ ays of interactants.
  • Other amplification methods known in the art can be used, including but not limited to ligase chain reaction (see European Patent No. 320,308), use of Q ⁇ replicase, or methods listed in Kricka et al, 1995, MOLECULAR PROBING, BLOTTING, AND SEQUENCING, Academic Press, New York, Chapter 1.
  • the plasmids encoding the DNA-binding domain hybrid and the activation domain hybrid proteins can also be isolated and cloned by any of the methods well known in the art.
  • a shuttle e.g., yeast to E. coli
  • the genes can be recovered by transforming the yeast DNA into E. coli and recovering the plasmids from E. coli (see, e.g., Hoffman et al, 1987, Gene 57: 267-272).
  • the yeast vector can be isolated, and the insert encoding the fusion protein subcloned into a bacterial expression vector, for growth of the plasmid in E. coli.
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited cows. pigs, horses, chickens, cats, dogs, etc.. and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject.
  • Formulations and methods of administration that can be employed when the Therapeutic comprises a nucleic acid are described above; additional appropriate formulations and routes of administration can be selected from among those described hereinbelow.
  • a Therapeutic of the invention e.g., encapsulation in liposomes, microparticles. microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu,
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural. and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g , in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249: 1527-1533, Treat et ai. 1989, In: LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.). Liss, New York, pp. 353-365, Lopez-Berestein. ibid., pp. 317-327, see generally ibid.).
  • the Therapeutic can be delivered in a controlled release system.
  • a pump may be used (see Langer. supra. Sefton. 1987, CRC Crit Ref Biomed. Eng. 14: 201. Buchwald et ai. 1980. Swrgerv 88: 507. Saudek et ai. 1989, N. Engl J. Med. 321: 574).
  • polymeric materials can be used (see Langer and
  • a controlled release system can be placed in proximity of the therapeutic target, i.e.. the brain, thus requiring only a fraction of the systemic dose (see. e.g., Goodson, 1984, IN MEDICAL
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • nucleic acid Therapeutic can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination.
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil. soybean oil, mineral oil. sesame oil and the like. Water is a prefe ⁇ ed ca ⁇ ier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid ca ⁇ iers. particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt. rice, flour, chalk, silica gel.
  • composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules. powders, sustained-release formulations and the like.
  • the composition can be formulated as a
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in REMINGTON'S PHARMACEUTICAL SCIENCES, by E.W.
  • compositions will contain a therapeutically effective amount of the Therapeutic. l o preferably in purified form, together with a suitable amount of ca ⁇ ier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions for intravenous administration are solutions in sterile isotonic
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • a solubilizing agent such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition 20 administered by infusion it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
  • the Therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, fe ⁇ ic hydroxides, isopropylamine. triethylamine, 2-ethylamino ethanol. histidine, procaine, etc.
  • treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro assays optionally may be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to about 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of about 0.5% to about 10% by weight; oral formulations preferably contain about 10% to about 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • animal models for diseases and disorders involving LYST:LYST-IP complexes, and individual LYST-2, LIPl, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, or LIP10 proteins are provided. These include, but are not limited to, atopic disorders including asthma, autoimmune disorders, degenerative disorders including neurodegenerative disorders, cell proliferative disorders including tumorigenesis and tumor spread, disorders caused by viral infection, disorders involving pigmentation disorders, and disorders caused by platelet dysfunction.
  • Such animals can be initially produced by promoting homologous recombination or insertional mutagenesis between LYST or LYST-2 and/or LYST-IP genes, or between an individual LYST-2 or novel LIP gene in the chromosome, and exogenous LYST or LYST-2 and/or LYST-IP genes, or individual LYST-2 or novel LIP genes that have been rendered biologically inactive or deleted (preferably by insertion of a heterologous sequence, e.g., an antibiotic resistance gene).
  • homologous recombination is carried out by transforming embryo-derived stem (ES) cells with a vector containing the insertionally inactivated Z.
  • LYST-2 and/or LYST-IP gene or an individual LYST-2 or novel LIP gene, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst. and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal ("knockout animal") in which a LYST or LYST-2 and/or a LYST-IP gene, or an individual LYST-2 or novel LIP gene, has been inactivated or deleted (see Capecchi. 1989. Science 244: 1288-1292).
  • the chimeric animal can be bred to produce additional knockout animals. Such animals can be mice, hamsters, sheep, pigs, cattle, etc., and are preferably non-human mammals. In a specific embodiment, a knockout mouse is produced.
  • Such knockout animals are expected to develop or be predisposed to developing diseases or disorders involving, but not restricted to, cell proliferative disorders including cancer and benign hypertrophy, various disorders involving cellular apoptosis and cellular differentiation, autoimmune diseases, etc., and thus can have use as animal models of such diseases, viral diseases and disorders, e.g. , to screen for or test molecules (e.g. , potential Therapeutics) for the ability to inhibit cell proliferative, autoimmune, viral and other diseases.
  • test molecules e.g. , potential Therapeutics
  • transgenic animals that have inco ⁇ orated and express (or overexpress or misexpress) a functional LYST or LYST-2 and/or LYST-IP gene, e.g., by introducing the LYST or LYST-2 and or LYST-IP genes under the control of heterologous promoters (i. e. , promoters that are not the native LYST or LYST-2 or LYST-IP promoter) that either overexpress the protein or proteins, or express them in tissues not normally expressing the complexes or proteins, can have use as animal models of diseases and disorders characterized by elevated levels of a LYST:LYST-IP complex, or by elevated levels of an individual LYST-2 or novel LIP protein.
  • Such animals can be used to screen for or test molecules for the ability to treat or prevent the diseases and disorders cited above.
  • the invention provides a recombinant non-human animal in which both the endogenous LYST or LYST-2 gene and an endogenous LYST-IP gene have been deleted or inactivated by homologous recombination or insertional mutagenesis of said animal or an ancestor thereof.
  • the invention provides a recombinant non-human animal containing both a LYST gene or LYST-2 gene and a LYST-IP gene, in which the LYST or LYST-2 gene is under the control of a promoter that is not the native LYST or LYST-2 gene promoter and the LYST-IP gene is under the control of a promoter that is not the native LYST-IP gene promoter.
  • the invention provides a recombinant non-human animal containing a transgene comprising a nucleic acid sequence encoding a chimeric protein comprising a fragment of LYST or LYST-2 of at least 6 amino acids fused via a covalent bond to a fragment of a LYST-IP of at least 6 amino acids.
  • the invention also provides a recombinant non-human animal in which an endogenous LYST-2, LIPl, LIP2. LIP3. L1P4. L1P5, LIP6, LIP7, LIP8. LIP9. or LIPIO gene has been deleted or inactivated by homologous recombination or insertional mutagenesis of said animal or an ancestor thereof.
  • yeast two hybrid system was used to identify protein interactions.
  • Yeast is an eukaryote, and therefore, any intramolecular protein interactions detected in this type of system demonstrate protein interactions that occur under physiological conditions (Chien et al, 1991, Proc. Natl Acad. Sci. USA 88: 9578-9581).
  • Expression vectors were constructed to encode two hybrid proteins.
  • one hybrid consisted of the DNA binding domain of the yeast transcriptional activator Gal4 fused to a portion of LYST or LYST-2.
  • the other hybrid consisted of the Gal4 activator domain fused to "prey" protein sequences encoded by a mammalian cDNA library.
  • LYST portion of LYST was fused to the Gal4 activator domain, and the prey protein sequences of the mammalian cDNA library were fused to the DNA binding domain, but the assay was otherwise identically performed.
  • Each of the vectors was then inserted into complementary (a and alpha) mating types of yeast using methods known in the art (Chien et al, 1991, Proc. Natl. Acad. Sci. USA 88: 9578-9581). Mating was carried out to express both vector constructs within the same yeast cells, thus allowing interaction to occur. Interaction between the bait and prey domains led to transcriptional activation of reporter genes containing cis-binding elements for Gal4.
  • the reporter genes encoding the indicator protein beta-galactosidase, and metabolic markers for uracil and histidine auxotrophy, were included in specific fashion in one or the other of the yeast strains used in the mating. In this way, yeast were selected for successful mating, expression of both fusion constructs, and expression of LYST, LYST-2, or LYST-IPs. Yeast clones that contained interacting proteins were picked and grown in individual wells of microtiter plates. The plasmids containing the LYST-IP sequences were then isolated and characterized.
  • the prey cDNAs were obtained from a fetal brain cDNA library of 1 x 10 7 independent isolates (Clontech #HL4029AH. Palo Alto, CA). The library was synthesized from Xhol-dT15 primed fetal brain mRNA (from five male/female 19-22 week fetuses) that was directionally cloned into either pAD-GAL4.
  • a yeast Gal4 activation domain cloning vector including the LEU2 gene for selection in yeast deficient in leucine biosynthesis, or pBD-GAL4.
  • a yeast Gal4 DNA-binding domain cloning vector including the TRPl gene for selection in yeast deficient in tryptophane biosynthesis.
  • HL4008AB HL4008AB
  • N106r yeast cells were transformed with 200 ⁇ g of the pasmid DNA and 10' yeast transformants were used in screens.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGBT9BS (Yang et al, 1995, Nucleic Acids Res. 23: 1152-1156).
  • a second bait was encoded by the LYST nucleotide sequence of nucleotides 3190-4032 (GenBank Accession No. U67615) and was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC CTG TTC AGA AGT CAC AAA GAG G-3' (SEQ ID NO:27) and the reverse primer 5'-G GAA TTC TCA TTG AGT GAG GTT TTC GAG TAA G-3' (SEQ ID NO:28) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGBT9BS (Yang et al. 1995).
  • a third bait was encoded by the LYST nucleotide sequence of nucleotides 6586-7449 (GenBank Accession No. U67615) and was amplified from the full length LYST cDNA by PCR using the forward 5'-GTG GAT CCC CCA TAT GAA GGA GAG AAC TCC TCT AAT ATT ATT CCA-3' (SEQ ID NO:29) and the reverse primer 5'-G GAA TTC TCA TCC CAT GTT TCT CAC ATC TTC CAG-3' (SEQ ID NO:30) by standard techniques.
  • a fourth bait was encoded by the L YST nucleotide sequence of nucleotides 9037-9585 (GenBank Accession No. U67615) and was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC GAT CCA ACA GAA GGG CCA AAT C-3' (SEQ ID NO: 0
  • the bait fragment 190-1056 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC
  • the amplified fragment was cloned between the BamHI and Eco RI restriction sites of pGAD- GH (Clonentech):
  • the bait fragment 774-1424 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC ACA CTA ACT GAG TTC CTA GCA GGC TTT GGG GAC TGC-3' (SEQ ID NO:33) and the reverse primer 5'-GAG AGA GAG AGA GAG AGA CTC GAG TCA ATC CAT ACA ACA GCA TAT TCC AAT G-3' (SEQ ID NO:34) by standard techniques.
  • the amplified fragment was cloned between the BamHI and Xhol restriction sites of pGAD-GH (Clonentech).
  • the bait fragment 4009-4821 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC CTC GAA AAC CTC ACT CAA GGG-3' (SEQ ID NO:35) and the reverse primer 5'-G GAA TTC TCA GGA GCC CAG TGA AAT TAT ATG-3' (SEQ ID NO:36) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-GH (Clonentech).
  • the bait fragment 6586-7449 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC CCA TAT GAA GGA GAG AAC TCC TCT AAT ATT ATT CCA-3' (SEQ ID NO:29) and the reverse primer 5'-G GAA TTC TCA TCC CAT GTT TCT CAC ATC TTC CAG-3' (SEQ ID NO:30) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-GH (Clonentech. Palo Alto. CA).
  • the bait fragment 9037-9585 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC GAT CCA ACA GAA GGG CCA AAT C-3' (SEQ ID NO:31 ) and the reverse primer 5'-G GAA TTC TCA TGT CAG AGC GGT GAT GTT ACC-3' (SEQ ID NO:32) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-GH (Clonentech).
  • the bait fragment 9502-10590 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC AAG GTT CGT GAT GAT GTA TAC CAC-3' (SEQ ID NO:37) and the reverse primer 5'-G GAA TTC TCA CAA GCC TTT TAT CCA TGA CAA AGG-3' (SEQ ID NO:38) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-GH.
  • the bait fragment 10576-1 161 1 was amplified from the full length LYST cDNA by PCR using the forward primer 5'-GTG GAT CCC TGG ATA AAA
  • GAA GTT CAT TCG CAT TCA CCC-3' (SEQ ID NO:40) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-GH.
  • LYST-2 bait protein (FIG. 1. SEQ ID NO:2).
  • the baits were encoded by the LYST-2 nucleotide sequence (FIG. 1. SEQ ID NO: l) of nucleotides 3-794.
  • the bait fragment 3-794 was amplified from the full length LYST-2 cDNA by PCR using the forward primer 5 ' -GCG GAA CTA GTG ACT TCT GAT GTA AAG GAA C-3' (SEQ ID NO:41 ) and the reverse primer 5'-GCG GAA TTC TCA ATA GCG ATT ATC TGC GTG TAC-3' (SEQ ID NO:42) by standard techniques.
  • the bait fragment 774-1424 was amplified from the full length LYST-2 cDNA by PCR using the forward primer 5' -GCG GAA CTA GTG GTA ACA GCA GAT AAT CGC TAT-3' (SEQ ID NO:43) and the reverse primer 5'-GCG GAA TTC TCA CCA GGG TTA AAT GTA GAG TTG- 3' (SEQ ID NO:44) by standard techniques.
  • the amplified fragment was cloned between the BamHI and EcoRI restriction sites of pGAD-WM.
  • the bait sequence was confirmed by nucleic acid sequencing to confirm that PCR amplification reproduced an accurate copy of the LYST or LYST-2 sequence.
  • cells were grown until mid-to-late log phase on media that selected for the presence of the appropriate plasmids.
  • the two mating strains, alpha and a. were then diluted in YAPD media (Sherman et al. eds.. 1991, GETTING STARTED WITH YEAST. Vol. 194. Academic Press. New York), filtered onto nitrocellulose membranes, and incubated at 30°C for 6-8 hours.
  • the cells were then transfe ⁇ ed to media selective for the desired diploids. i.e.. yeast harboring reporter genes for beta-galactosidase. uracil auxotrophy. and histidine auxotrophy. and expression of the vectors encoding the bait and prey.
  • the mating products were plated on SC (synthetic complete) media (Kaiser. Michaelis and Mitchell. Eds.
  • This medium is herein refe ⁇ ed to as SCS medium, for SC Selective medium.
  • GenBank # U67615 (LYST), or GenBank or FIG. # in column 2 (LYST-IPs).
  • t #AA designates the number of amino acids in the region of LYST-IP. for which the % identity/similarity is shown Table Ic. Protein interactions identified by reverse yeast tvvo-hybrid screens using LYST-2 baits
  • YULH yeast cells were created that express the individual plasmids encoding the binding domain fusions of all LYST-interacting preys.
  • the transformed haploid yeast cells were plated on SCS plates, grown overnight, and examined for growth. Cells that did not grow contained LYST interacting proteins that were not self-activating false positives. No growth was found for all proteins tested, confirming that they were not "self-activating" proteins, that is, these proteins require interaction with a second protein domain for a functional activation complex.
  • a matrix mating test was performed by spreading bait- containing haploid yeast over the surface of individual YPAD plates, followed by superimposing prey-containing haploids in a 12 x 8 matrix, and incubating the plates overnight at 30°C. Each plate contained positive (known protein interaction pairs) and negative controls. Resulting diploid colonies were replica-plated both on SCS plates (to select for activating and binding domain plasmids. diploids, and protein interactions ) and SC-Leu-T ⁇ plates (that were used for a filter-lift beta-galactosidase assay). Baits and preys from LacZ+ diploids growing on SCS were sequenced.
  • the human EST assemblies were done by using public available EST assembly databases such as the National Center for Biotechnology Information (N. C.B.I.) Blast 2.0 software program (Gish 1994-1997; Altschul et al. 1990, J. Mol. Biol. 215: 403-410). Sequences that aligned with 95% or greater identity at the nucleic acid level over at least 30 nucleotides of their termini were utilized if the alignment resulted in 5' extension or 3 ' extension of the EST sequence. Once this first assembly was complete, the extended sequence was again subject to a Blast comparison to detect new homologies to the added extensions. The sequence was extended in both directions until new related sequences that allowed extension of the assembled sequence were no longer detected.
  • N. C.B.I. National Center for Biotechnology Information
  • the assembled EST sequence was subjected to further searches using the BlastX 2.0 software program for the identification of protein coding regions by database similarity search (Gish 1994-1997; Gish and States 1993, Nat. Genet. 3: 266-272).
  • This BlastX program uses translates the DNA sequence in all six reading frames and compares the translated protein sequence with those in protein databases. The statistical significance is estimated under the assumption that the equivalent of one entire reading frame in the query sequence codes for protein and that significant alignments will involve only coding reading frames. Sequences producing high-scoring segment pairs are shown.
  • sequences were analyzed for open reading frames using proprietary software that translates the DNA sequence in all six reading frames of the (assembled) DNA sequence using the standard genetic code.
  • the interacting ESTs were obtained from directionally-cloned libraries, and thus the direction of translation of the assembled EST is known as 5 * to 3 ' and the open reading frame is also known.
  • ORFs the possible open reading frames found in the co ⁇ ect frame was analyzed.
  • Open reading frames greater than 50 amino acids following an initiator codon or an ORF with no initiator methionine encoded at the 5 ' end were determined to be possible protein products, and were compared to sequences in protein data bases using the BLASTP 2.0 software program (Gish 1994-1997; Altschul et ai. 1990. J. Molec. Biol. 215: 403-410). Protein sequences with identities and similarities to known protein sequences are shown.
  • BLIMPS software program found matches to entries in the BLOCKS.
  • the BLOCKS analysis aligns similar sequence domains found in proteins and reveals the co ⁇ esponding protein families.
  • BLASTP found matches to entries in the PRODOM database.
  • the PRODOM analysis displays the alignment that constitutes protein domains with high identities and similarities.
  • LIP-1 a novel protein, which was designated LIP-1.
  • Amino acids 1 1-194 of LIP-1 show 61% identity and 78% similarity to the C-terminal region (amino acids 254-436) of mouse Tcp-10 protein (GenBank accession No. X58170).
  • LIP- 1 also shows 66% identity to human tcpl Oa protein (GenBank accession No. U03399) (nucleotides 89 to 194 of LIP-1 and nucleotides 286 to 390 of U03399). Therefore.
  • LIP-1 represents a novel protein with homology to mouse and human Tcp-10 proteins. Interestingly, another LYST-interactant. LIP-7 (see later in this chapter), shows homology to the amino- terminal region of Tcp-10. The nucleotide and amino acid sequences of LIP-1 are shown in Figure 2.
  • This assembly contains a number of published ESTs including nucleotides 1 to 698 from EST AI 141700 (co ⁇ esponding to nucleotides 112 to 781 in human assembly hs6793_0), nucleotides 1 to 634 from EST W67725 (co ⁇ esponding to nucleotides 22 to 675 in human assembly hs6793_0), and nucleotides 1 to 604 from EST AA203533 (co ⁇ esponding to nucleotides 98 to 692 in human assembly hs6793_0).
  • the overlap of the prey sequences and human assembly hs6793_0 created a 850 contig that could not be further extended by database searches and was used for further analysis. This contig showed no significant similarity to any gene of known function by BlastN.
  • LIP-2 An open reading frame could be translated from nucleotides 1 to 552 of the contig.
  • the resulting 184 amino acids co ⁇ espond to a C-terminal or core region of a novel protein which was called LIP-2.
  • Amino acids 24-133 of LIP-2 show 40% identity and 56% similarity to amino acids 2-1 17 of bacterial ribosomal protein L17 (GenBank accession No. M26414). Therefore.
  • LIP-2 represents a novel human protein with similarity to ribosomal protein LI 7.
  • the nucleotide and amino acid sequences of LIP-2 are shown in Figure 3.
  • LIP3 (EST cg50136.clO) Two identical sequences (cg50136.cl0 and cg50136.gl0). interacting with LYST (9037- 9585 and 9502-10590), showed significant identity to published ESTs.
  • An extended expressed sequence of 141 1 nucleotides was assembled from nucleotides 1 to 447 of human fetal lung EST W40354 (Hillier et al. 1995. The WashU-Merck EST Project, direct submission to GenBank)nucleotides 1 to 382 from human endothelial cell EST AA446837 (Hillier et al.. 1997. WashU-Merck EST Project 1997. Unpublished) (co ⁇ esponding to nucleotides 328 to 878 in the extended sequence), nucleotides 1 to 435 from cg50136.cl0 (co ⁇ esponding to nucleotides 345 to
  • nucleotides 1 to 422 from EST N49053 from a human multiple sclerosis library (Hillier et al. 1995. The WashU-Merck EST Project, direct submission to
  • GenBank (co ⁇ esponding to nucleotides 779 to 1201 in the extended sequence), and nucleotides
  • nucleotides 1410 nucleotides was used for subsequent analysis. Nucleotides 213 to 599 of the extended expressed sequence showed 74% identity to nucleotides 3589 to 3975 of rat Olf-1/EBF associated Zn finger protein Roaz (GenBank accession No. U92564).
  • LIP-3 An open reading frame could be translated from nucleotides 1 to 594. These 198 amino acids represent a core or C-terminal region of a novel protein, which was designated LIP-3.
  • Amino acids 26 to 197 of LIP-3 show 72% identity and 83% similarity to amino acids 1023 to
  • LIP-3 represents a novel human protein with homologies to rat Olf-1/EBF associated Zn finger protein Roaz.
  • the nucleotide and amino acid sequences of LIP-3 are shown in Figure 4.
  • EST W22541 contained cg50136.a7 and could be extended using ESTs AA009453 (505 nucleotides), AA482060 (457 nucleotides), AI193517 (314 nucleotides) and AA252768 (485 nucleotides) to a 921 nucleotide sequence. Nucleotides 627 to 397 of this assembly were 68% identical to nucleotides 843 to 1070 of human hnRNP-El mRNA (GenBank accession No. X78137).
  • Open reading frames could be translated from this sequence in all six frames.
  • the longest ORF could be translated in frame +2 from nucleotide 71 to 898. 276 amino acids.
  • As the protein translation depicted does not begin with an "ATG" initiator methionine codon.
  • ATG initiator methionine codon.
  • This ORF was also identified to have the most similarity to known proteins using BlastX.
  • LIP-4 represents a novel, protein with similarity to human ribonuclear proteins.
  • the nucleotide and amino acid sequences of LIP-4 are shown in Figure 5.
  • One identified prey sequence (cg50175.c7, 450 nucleotides), interacting with LYST, was highly homologous to a number of known ESTs [98% identical to human cDNA clone AA173372 which is 480 nucleotides and was initially obtained from a Stratagene neuroepithelium (#937231) library (Hillier et al, 1997.
  • LIP-5 has a high probability of containing a phosphorylase pyridoxal-phosphate attachment site (strength - 1682. score - 1067). LIP-5 represents a novel human protein. The nucleotide and amino acid sequences of LIP-5 are shown in Figure 6.
  • Nucleotides 166 to 486 of cg50136.f9 were 99% identical to nucleotides 1 to 320 of EST AA460131 (588 nucleotides; Hillier et al, 1997, WashU-NCI human EST Project, direct submission to GenBank). This sequence was obtained from a human total fetus (9 weeks) cDNA library.
  • nucleotides 1-486 of cg50136.f9 and nucleotides 321-588 of AA460131 was used for subsequent analysis.
  • the extended sequence showed similarity to several viral nonstructural proteins: nucleotides 2 to 488 showed 83% identity to nucleotides 170 to 653 of the nonstructural protein Ns2-3 of a border disease virus strain (pestivirus type 3) (GenBank accession No. U43603) and nucleotides 43 to 406 showed 90% identity to nucleotides 17 to 378 of proteins p54, p80 and pl25 of the bovine viral dia ⁇ hea virus (GenBank accession No. Z54331).
  • LIP-6 An open reading frame could be translated from nucleotides 2 to 583, 194 amino acids.
  • An open reading frame with a Kozak consensus start methionine could be translated from nucleotides 1 1 to 583, 191 amino acids.
  • These 191 amino acids represent a novel protein, which was called LIP-6.
  • Amino acids 3 to 141 of LIP-6 show 92% identity and 93% similarity to amino acids 64 to 198 of the viral (pestivirus type 3) nonstructural protein Ns2-3 (GenBank accession No. U43603).
  • Amino acids 28 to 132 are 97% homologous to amino acids 22 to 126 of the nonstructural protein pi 25 of the bovine viral dia ⁇ hea virus (GenBank accession No. Z54331).
  • LIP-6 represents a novel human protein with similarity to viral nonstructural proteins.
  • the nucleotide and amino acid sequences of LIP-6 are shown in Figure 7.
  • LIP-7 (EST cg50136.a5)
  • LIP-7 An open reading frame could be translated from nucleotides 2 to 586. These 195 amino acids co ⁇ espond to a C-terminal or core region of a novel protein, which was called LIP-7. Amino acids 59 to 139 of LIP-7 showed 30% identity and 59% similarity to amino acids 162 to 404 of the human T-complex protein 10A (TCPIOA) (GenBank accession No. U03399). Amino acids 10 to 149 of LIP-7 showed 29% identity and 54% similarity to amino acids 4039 to 4461 of the human trichohyalin (TRHY) gene (GenBank accession No. L09190). Amino acids 6 to 186 of LIP-7 showed 28% identity and 51% similarity to amino acids 2387 to 2890 of the human neuronal kinesin heavy chain (GenBank accession No. U06698).
  • cytoskeletal proteins e.g. myosin I isoform, kinesin precursor, cytokeratin, caldesmon, NUF1, tropomyosin, neurofilament protein, kinesin-like protein KIF1. myosin like protein MLP1, troponin T
  • proteins involved in vesicular transport intracellular protein transport protein, nuclear fusion protein Bikl, synaptonemal complex protein.
  • BLOCKS analysis showed homologies to postsynaptic proteins, gas vesicles protein, clusterin proteins, clathrin light chain proteins, tropomyosin protein and intermediate filament protein.
  • LIP-7 represents a novel protein with homologies to the amino-terminal region of Tcp-10 and with homologies to cytoskeletal and vesicular transport proteins. It is interesting that the LYST-interactant LIP-1 shows similarity to the C-terminal region of Tcp-10.
  • the nucleotide and amino acid sequences of LIP-7 are shown in Figure 8.
  • LIP-8 (EST cg50136.d2) Two identified sequences (cg50136c6 and cg50136d2). interacting with LYST (6586- 7449). showed no significant homologies to published sequences. The interacting sequence of 386 nucleotides. named cg50136d2 ⁇ . was used for further analysis. Nucleotides 27 to 244 of cg50136d2 ⁇ were 69% homologous to nucleotides 1 1 15 to 1335 of the human RNA for the cellular oncogene c-fes (GenBank accession No. X52192).
  • LIP-8 represents a novel protein.
  • the nucleotide and amino acid sequences of LIP-8 are shown in Figure 9.
  • nucleotides 2 to 324 of cg50175.cl 1 were 96% identical to nucleotides 73 to 394 of the expressed sequence H51347 (428 nucleotides; Hillier et al, 1995, WashU-NCI human EST Project, direct submission to GenBank). obtained from a human adult brain cDNA library and nucleotides 324 to 43 of cg50175.cl 1 were 98% identical to nucleotides 188 to 468 of the expressed sequence AA628560 (468 nucleotides; Hillier et al, 1997.
  • Open reading frames could be translated from this sequence in all six frames.
  • the longest ORF could be translated in frame 3 from nucleotide 78 to 716. 213 amino acids.
  • This contig included EST R17261 (373 bp) initially obtained from an infant brain library (Hillier et al. 1995. WashU-NCI human EST Project, direct submission to GenBank); nucleotides 18 to 365 of the contig were 91% identical to nucleotides 1 to 348 of EST R 17261.
  • This contig cgl52106_fl 1+ could not be extended further.
  • Contig cgl52106_fl l + showed 60% nucleotide identity and 60% nucleotide similarity to cat glyceraldehyde-3- phosphate dehydrogenase mRNA. partial eds (GenBank accession No. AF054608).
  • Amino acids 1 to 33 of Contig cgl52106_fl 1+ were 40% identical and 65% similar to amino acids 33 to 64 of chicken tyrosine kinase VEGFR-3.
  • the nucleotide and amino acid sequences of LIP-10 are shown in Figure 11.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Diabetes (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des complexes de la protéine LYST ou LYST-2, les protéines étant identifiées pour leur interaction avec LYST ou LYST-2 par un système d'analyse hybride à la levure. Les protéines interagissant avec LYST ou LYST-2 sont les protéines 14-3-3, HS1, Hrs, BMK1, KB07, Efs, XAP-4, OS9, caséine-kinase II bêta SU, calmoduline, troponine I, importine bêta, Fte-1, HERR1, imogène 38, atrophine-1, norbine, HBF-G2, DGS-I, GBDR1, la protéine contenant OPA, la protéine M4, LIP1, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, et LIP10. L'invention concerne donc des complexes de LYST ou LYST-2 et des protéines 14-3-3, la prétine HS1, Hrs, BMK1, KB07, Efs, XAP-4, OS9, caséine-kinase II bêta SU, calmoduline, troponine I, importine bêta, Fte-1, HERR1, imogène 38, atrophine-1, norbine, HBF-G2, DGS-I, GBDR1, la protéine contenant OPA, la protéine M4, LIP1, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, et LIP10, ainsi que des dérivés, des fragments, et des analogues de ceux-ci. Cette invention concerne également des acides nucléiques codant pour les protéines LIP1, LIP2, LIP3, LIP4, LIP5, LIP6, LIP7, LIP8, LIP9, et LIP10, ainsi que des dérivés, des fragments, et des analogues de ceux-ci. L'invention concerne en outre des méthodes de criblage de ces complexes ou de ces protéines, ces méthodes permettant de déterminer l'efficacité de ces complexes ou de ces protéines pour prévenir et/ou traiter certaines maladies et certains troubles, en particulier les maladies atopiques, les maladies auto-immunes, les maladies neurodégénératives, le cancer, les troubles de la pigmentation, les dysfonctionnements plaquettaires, et les maladies virales.
PCT/US1999/006831 1998-04-03 1999-03-29 Complexes de proteine lyst et proteines a interaction lyst WO1999051741A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU32140/99A AU3214099A (en) 1998-04-03 1999-03-29 Lyst protein complexes and lyst interacting proteins
EP99914251A EP1066378A2 (fr) 1998-04-03 1999-03-29 Complexes de proteine lyst et proteines a interaction lyst
JP2000542454A JP2002510490A (ja) 1998-04-03 1999-03-29 Ｌｙｓｔタンパク質複合体およびｌｙｓｔ相互作用タンパク質
CA002325562A CA2325562A1 (fr) 1998-04-03 1999-03-29 Complexes de proteine lyst et proteines a interaction lyst

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5495698A 1998-04-03 1998-04-03
US09/054,956 1998-04-03

Publications (2)

Publication Number Publication Date
WO1999051741A2 true WO1999051741A2 (fr) 1999-10-14
WO1999051741A3 WO1999051741A3 (fr) 1999-12-23

Family

ID=21994612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/006831 WO1999051741A2 (fr) 1998-04-03 1999-03-29 Complexes de proteine lyst et proteines a interaction lyst

Country Status (5)

Country Link
EP (1) EP1066378A2 (fr)
JP (1) JP2002510490A (fr)
AU (1) AU3214099A (fr)
CA (1) CA2325562A1 (fr)
WO (1) WO1999051741A2 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
WO2015168674A1 (fr) * 2014-05-02 2015-11-05 Research Institute At Nationwide Children's Hospital Compositions et procédés d'immunomodulation d'anti-lyst
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
WO2017106338A1 (fr) * 2015-12-14 2017-06-22 Rowan University Biomarqueurs d'autoanticorps de la maladie d'alzheimer au stade précoce, antigènes cibles et utilisations diagnostiques associées
CN111269919A (zh) * 2020-03-19 2020-06-12 四川农业大学 玉米基因ZmLIP2及其应用
US10780107B2 (en) 2016-06-23 2020-09-22 Nitto Denko Corporation Agent for inducing cell death, agent for suppressing cell proliferation, and pharmaceutical composition used for treatment of disease resulting from abnormal cell proliferation
US11541149B2 (en) 2015-12-11 2023-01-03 Research Institute At Nationwide Children's Hospital Systems and methods for optimized patient specific tissue engineering vascular grafts
US12186455B2 (en) 2008-01-08 2025-01-07 Yale University Compositions and methods for promoting patency of vascular grafts

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005094571A1 (fr) * 2004-03-31 2005-10-13 Japan Science And Technology Agency Animal génétiquement modifié et méthode de mesure de l'exocytose à l'aide de l'animal
GB0903913D0 (en) 2009-03-06 2009-04-22 Medical Res Council Compositions and methods

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029407A2 (fr) * 1995-03-23 1996-09-26 Incyte Pharmaceuticals, Inc. Homologues de cellubrevines
WO1997028262A1 (fr) * 1996-02-01 1997-08-07 University Of Florida Compositions de genes lyst1 et lyst2 et leurs procedes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029407A2 (fr) * 1995-03-23 1996-09-26 Incyte Pharmaceuticals, Inc. Homologues de cellubrevines
WO1997028262A1 (fr) * 1996-02-01 1997-08-07 University Of Florida Compositions de genes lyst1 et lyst2 et leurs procedes d'utilisation

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BARBOSA M D F S ET AL: "IDENTIFICATION OF THE HOMOLOGOUS BEIGE AND CHEDIAK-HIGASHI SYNDROME GENES" NATURE, vol. 382, no. 6588, 18 July 1996 (1996-07-18), pages 262-265, XP002032769 ISSN: 0028-0836 cited in the application *
BARBOSA M D F S ET AL: "THE RAB PROTEIN FAMILY: GENETIC MAPPING OF SIX RAB GENES IN THE MOUSE" GENOMICS, vol. 30, no. 3, 10 December 1995 (1995-12-10), pages 439-444, XP000674357 ISSN: 0888-7543 *
CHIEN C -T ET AL: "THE TWO-HYBRID SYSTEM: A METHOD TO IDENTIFY AND CLONE GENES FOR PROTEINS THAT INTERACT WITH A PROTEIN OF INTEREST" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, November 1991 (1991-11), pages 9578-9582, XP002913781 ISSN: 0027-8424 cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,14 March 1994 (1994-03-14), XP002120655 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,15 January 1995 (1995-01-15), XP002120657 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,2 March 1995 (1995-03-02), XP002120650 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,25 August 1995 (1995-08-25), XP002120652 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,29 January 1994 (1994-01-29), XP002120653 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,31 January 1996 (1996-01-31), XP002120656 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,31 May 1991 (1991-05-31), XP002120651 HINXTON, GB cited in the application *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES,4 June 1997 (1997-06-04), XP002120654 HINXTON, GB cited in the application *
PEROU C M ET AL: "COMPLEMENTATION ANALYSIS OF CHEDIAK-HIGASHI SYNDROME: THE SAME GENE MAY BE RESPONSABLE FOR THE DEFECT IN ALL PATIENTS AND SPECIES" SOMATIC CELL AND MOLECULAR GENETICS, vol. 19, no. 5, 1 September 1993 (1993-09-01), pages 459-468, XP000674425 *
PEROU C M ET AL: "IDENTIFICATION OF THE MURINE BEIGE GENE BY YAC COMPLEMENTATION AND POSITIONAL CLONING" NATURE GENETICS, vol. 13, no. 3, 1 July 1996 (1996-07-01), pages 303-308, XP000674628 ISSN: 1061-4036 cited in the application *
PEVSNER J ET AL: "Mammalian homologues of yeast vacuolar protein sorting (vps) genes implicated in Golgi-to-lysosome trafficking" GENE, vol. 183, no. 1, 1 January 1996 (1996-01-01), page 7-14 XP004062720 ISSN: 0378-1119 *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
EP2053409A1 (fr) 2003-11-20 2009-04-29 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US12186455B2 (en) 2008-01-08 2025-01-07 Yale University Compositions and methods for promoting patency of vascular grafts
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9365643B2 (en) 2012-01-27 2016-06-14 AbbVie Deutschland GmbH & Co. KG Antibodies that bind to repulsive guidance molecule A (RGMA)
US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
RU2728703C2 (ru) * 2014-05-02 2020-07-31 Рисерч Инститьют Эт Нэшнуайд Чилдрен`С Хоспитал Композиции и способы для анти-lyst иммуномодуляции
CN106456771B9 (zh) * 2014-05-02 2021-07-20 国家儿童医院研究所 用于抗lyst免疫调节的组合物和方法
AU2015252805B2 (en) * 2014-05-02 2018-05-10 Research Institute At Nationwide Children's Hospital Compositions and methods for anti-LYST immunomodulation
CN106456771A (zh) * 2014-05-02 2017-02-22 国家儿童医院研究所 用于抗lyst免疫调节的组合物和方法
AU2018205184B2 (en) * 2014-05-02 2020-04-30 Research Institute At Nationwide Children's Hospital Compositions and methods for anti-lyst immunomodulation
WO2015168674A1 (fr) * 2014-05-02 2015-11-05 Research Institute At Nationwide Children's Hospital Compositions et procédés d'immunomodulation d'anti-lyst
KR20170007334A (ko) * 2014-05-02 2017-01-18 리서치 인스티튜트 앳 네이션와이드 칠드런스 하스피탈 항-ｌｙｓｔ 면역조절을 위한 조성물 및 방법
KR102492647B1 (ko) * 2014-05-02 2023-01-30 더 리서치 인스티튜트 앳 네이션와이드 칠드런스 하스피탈 항-ｌｙｓｔ 면역조절을 위한 조성물 및 방법
CN106456771B (zh) * 2014-05-02 2021-06-15 国家儿童医院研究所 用于抗lyst免疫调节的组合物和方法
US11541149B2 (en) 2015-12-11 2023-01-03 Research Institute At Nationwide Children's Hospital Systems and methods for optimized patient specific tissue engineering vascular grafts
US12115282B2 (en) 2015-12-11 2024-10-15 Research Institute At Nationwide Children's Hospital Systems and methods for optimized patient specific tissue engineering vascular grafts
WO2017106338A1 (fr) * 2015-12-14 2017-06-22 Rowan University Biomarqueurs d'autoanticorps de la maladie d'alzheimer au stade précoce, antigènes cibles et utilisations diagnostiques associées
US11619632B2 (en) 2015-12-14 2023-04-04 Rowan University Early-stage Alzheimer's disease autoantibody biomarkers, target antigens and diagnostic uses thereof
US10780107B2 (en) 2016-06-23 2020-09-22 Nitto Denko Corporation Agent for inducing cell death, agent for suppressing cell proliferation, and pharmaceutical composition used for treatment of disease resulting from abnormal cell proliferation
CN111269919A (zh) * 2020-03-19 2020-06-12 四川农业大学 玉米基因ZmLIP2及其应用

Also Published As

Publication number Publication date
WO1999051741A3 (fr) 1999-12-23
AU3214099A (en) 1999-10-25
JP2002510490A (ja) 2002-04-09
EP1066378A2 (fr) 2001-01-10
CA2325562A1 (fr) 1999-10-14

Similar Documents

Publication Publication Date Title
US6627405B1 (en) 53BP2 complexes
EP1066378A2 (fr) Complexes de proteine lyst et proteines a interaction lyst
US6521412B1 (en) HsReq*1 and hsReq*2proteins and use thereof to detect CDK2
AU778692B2 (en) NLK1 -interacting proteins
WO2000050590A1 (fr) Proteine capable d'interaction avec un polypeptide mdm et procedes d'utilisation
AU770000B2 (en) Hermansky pudlak syndrome protein-interacting proteins and methods of use thereof
US20030023034A1 (en) p27 (Kip1) -FKBP-12 protein complexes
JP2003517821A (ja) ２７８６、ヒトアミノペプチダーゼ
CA2319782A1 (fr) Complexes proteiques de retinoblastome et proteines interagissant avec le retinoblastome
US20050239064A1 (en) Methods and compositions based on protein interactions with mastermind
JP2001508313A (ja) Ｃ▲ｉｉ▼ｔａと相互作用する蛋白質及びその利用方法
AU3291799A (en) Retinoblastoma protein complexes and retinoblastoma interacting proteins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09582687

Country of ref document: US

ENP Entry into the national phase in:

Ref document number: 2325562

Country of ref document: CA

Ref country code: CA

Ref document number: 2325562

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2000 542454

Kind code of ref document: A

Format of ref document f/p: F

NENP Non-entry into the national phase in:

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: 32140/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1999914251

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999914251

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1999914251

Country of ref document: EP