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WO2001081545A2 - Regulation of gene expression by hspbp1 - Google Patents

Regulation of gene expression by hspbp1 Download PDF

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Publication number
WO2001081545A2
WO2001081545A2 PCT/US2001/013422 US0113422W WO0181545A2 WO 2001081545 A2 WO2001081545 A2 WO 2001081545A2 US 0113422 W US0113422 W US 0113422W WO 0181545 A2 WO0181545 A2 WO 0181545A2
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WIPO (PCT)
Prior art keywords
hspbpl
expression
protein
hsp70
extra
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PCT/US2001/013422
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French (fr)
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WO2001081545A3 (en
Inventor
Vincent Guerriero
Deborah A. Raynes
Luke J. Whitesell
Meghan E. Kreeger
Original Assignee
Vincent Guerriero
Raynes Deborah A
Whitesell Luke J
Kreeger Meghan E
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Priority claimed from US09/444,336 external-priority patent/US6410713B1/en
Application filed by Vincent Guerriero, Raynes Deborah A, Whitesell Luke J, Kreeger Meghan E filed Critical Vincent Guerriero
Priority to AU2001255697A priority Critical patent/AU2001255697A1/en
Publication of WO2001081545A2 publication Critical patent/WO2001081545A2/en
Publication of WO2001081545A3 publication Critical patent/WO2001081545A3/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention generally relates to the field of molecular medicine and more particularly to a method of regulating the expression of extra-nuclear genetic material in eukaryotic cells through HspBPl.
  • Hsps heat stress proteins
  • Hsps are named according to their molecular weight .
  • the most studied has a molecular weight of 70 kDa and is therefore called Hsp70.
  • Hsp70 and a related protein called Hsc70 help the cell survive stress events by binding to partially denatured proteins and assisting to refold these proteins into more stable native structures .
  • members of the Hsp70 family have come to be known as molecular " chaperones .”
  • Hsp70 plays a role in DNA replication, transport of proteins across membranes, binding of proteins to the endoplasmic reticulum, and uncoating clathrin coated vesicles. S. Lindquist and E.A. Craig, Annual Revue of Genetics. 22:631-77 (1988). Furthermore, Hsp70 is known to associate with non-esterified fatty acids, palmitic acid, stearic acid, and myristic acid and to be involved in signal transduction pathways in the cytoplasm. Hohfeld, Jorg, et al . , Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/Hsp40 Reaction Cycle . Cell vol. 83, 589-598 (November 17, 1995) .
  • Hsp70 has been its role as a "chaperone, "a protein that stabilizes other proteins against aggregation and that mediates the folding of newly translated polypeptides in the cytosol and organelles.
  • Proper functioning of Hsp70 as a protein chaperone is dependent on its bound nucleotide state. Specifically, the ATP form of Hsp70 binds substrate very poorly and therefore must be converted to the ADP form before the misfolded protein can bind. Then, the high affinity of Hsp70 for ATP is utilized to "power" the protein folding and other functions of Hsp70 by the generation of energy through the hydrolysis of bound ATP.
  • Hsp40 which stimulates ATPase activity of Hsp70 resulting in converting ATP-Hsp70 to ADP-Hsp70, which has a greater affinity for the misfolded substrate.
  • Hsp40 which stimulates ATPase activity of Hsp70 resulting in converting ATP-Hsp70 to ADP-Hsp70, which has a greater affinity for the misfolded substrate.
  • Hip binds to Hsp70 and stabilizes the ADP-Hsp70 form, resulting in greater substrate affinity.
  • H ⁇ hfeld, J., Minami, Y., and Hartl, F-U. A novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle . Cell 83, 589-598(1995).
  • Another regulator, the protein Hop facilitates interaction between Hsp70 and Hsp90.
  • Two other regulatory factors, Bag-1 and Hap46 stimulate the exchange of ATP for ADP, which results in lower substrate affinity. See Takayama, S., D. N. Bimstone, S. Matsuzawa, B. C. Freeman, C.
  • HspBPl Hsp70 binding protein
  • HspBPl dramatically alters the activity of Hsp70 by binding to its ATPase domain and inhibiting the ability of Hsp70 to refold a denatured protein.
  • CHIP another protein called CHIP with similar inhibitory activity.
  • HspBPl is newly discovered, very little is known about its activity in the cell other then its binding with Hsp70. Thus, to better understand the full role that HspBPl may play in normal physiology and disease, there remains a need to further characterize the activity of HspBPl .
  • One aspect of the invention relates to the unexpected discovery that the expression of co-chaperone HspBPl polypeptides can down regulate the expression of extra- nuclear genetic material in eukaryotic cells.
  • this aspect of the invention is not meant to be limited to a particular mechanism of action, the expression of HspBPl polypeptides are thought to inhibit expression of extra- nuclear genetic material by excluding it from the transcriptional machinery in the nucleus .
  • Another aspect of the invention involves the expression of HspBPl anti- sense polynucleotides in eukaryotic cells to up-regulate extra-nuclear genetic material expression.
  • HspBPl polypeptides and polynucleotides may have regulatory consequences for many different physiological pathways or disease conditions, such as development, apoptosis, cellular stress, viral infection, heart disease, and cancer, as well as for the efficacy of gene therapy treatments.
  • a second object of the invention is to provide a method for inhibiting the expression of transiently transfected plasmids .
  • a third object of the invention is to provide a method for inhibiting the expression of viral genomic material within eukaryotic cells.
  • a fourth object of the invention is to provide a method of up-regulating the expression of proteins introduced into eukaryotic cells that are undergoing gene therapy through the expression of anti-sense HspBPl polynucleotide sequences .
  • a fifth object of the invention is to provide the amino acid sequence for substantially purified HspBPl.
  • a sixth object of the invention is to provide pharmaceutical compositions based on the human HspBPl or peptide fragments thereof.
  • the invention generally features a method of using human heat-shock protein-binding protein 1 (HspBPl) , having the amino acid sequence shown in SEQ ID N0:1, to regulate the expression of extra-nuclear genetic material. Furthermore, the invention features a method of using HspBPl anti-sense polynucleotides, having the nucleotide sequence shown in SEQ ID NO: 2, to regulate the expression of extra-nuclear genetic material. Finally, the invention features pharmaceutical compositions comprising substantially purified HspBPl .
  • HspBPl human heat-shock protein-binding protein 1
  • Fig. 1. contains graphs A-D showing that HspBPl inhibits the expression of transiently transfected EGFP gene but not the stably-integrated EGFP gene.
  • HspBPl inhibits the expression of transiently transfected EGFP gene regulated by the CMV promoter.
  • NIH 3T3 fibroblasts were transfected using cationic lipid reagent (Lipofectamine Plus, BRL) per manufacturer' s recommendations using: No DNA, a mixture of pcDNA (1.5 ⁇ g) and pEGFP (0.5 ⁇ g) or a mixture of pHspBPl (1.5 ⁇ g) and pEGFP (0.5 ⁇ g).
  • Cells were lysed in non-ionic detergent 5 buffer 18 hours post-transfection. Protein concentrations were determined using BCA reagent (Pierce) , and EGFP fluorescence was quantitated using a microplate fluorometer (BioRad) . Results from each transfection were calculated as fluorescent units per mg of cellular 10 protein. The mean and standard deviation of values from triplicate transfections for each condition are depicted.
  • HspBPl does not inhibit the expression of stably 15 integrated EGFP gene regulated by CMV promoter.
  • SKNSH cells were stably transfected with pEGFP plasmid regulated by the CMV promoter. Cells were then transiently transfected with either 1.5 ⁇ g pcDNA and 0.5 ⁇ g pDSred-Nl (plasmid containing cDNA for a red fluorescent protein) or 20 15 ⁇ g pcDNA and 5 ⁇ g pHspBPl . Cells were lysed in non-ionic detergent buffer 18 hours post-transfection. Protein concentrations were determined using BCA reagent (Pierce) and EGFP fluorescence was quantitated using a microplate fluorometer (BioRad) . Results from each transfection were 25 calculated as fluorescent units per mg of cellular protein. The mean and standard deviation of values from triplicate transfections for each condition are depicted.
  • HspBPl inhibits the expression of transiently expressed heat responsive reporter plasmid.
  • NIH 3T3 cells were cotransfected with 1.5 ⁇ g of either pcDNA3.1 or pHspBPl and 0.5 ⁇ g of the heat responsive reporter construct Y9.
  • Y9 is a plasmid containing the EGFP coding sequence and a
  • Transfected cells were heat shocked in a 42°C water bath for 150 min. 24 hours post transfection. Cells were lysed with non-ionic detergent buffer 18 hours following heat shock and EGFP levels were analyzed on a fluorometer.
  • HspBPl does not inhibit the expression of stably integrated heat responsive promoter.
  • NIH 3T3 cells were stably transfected with Y9 reporter construct containing the minimal heat responsive reporter. Cells were then transiently co-transfected with either 1.5 ⁇ g pcDNA or pHspBPl with 0.5 ⁇ g pcDNA . Cells were heat shocked and analyzed as above.
  • Fig. 2 is a panel of Western blot data showing the inhibition of EGFP expression.
  • NIH 3T3 fibroblasts were transfected with the mixtures of plasmid DNA as indicated.
  • pEGFP-HspBPl refers to a plasmid encoding EGFP fused in frame to the 5' end of the full-length HspBPl coding sequence.
  • Cells were lysed 24 hrs post-transfection, and lOO ⁇ g of total cellular protein per lane were fractionated by SDS-PAGE. Following electrophoretic transfer to nitrocellulose, the membrane was probed with an anti-EGFP antibody and reactivity visualized using horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (Pierce) .
  • Fig. 3 is a depiction of a Northern blot analysis of cells transfected with HspBPl.
  • NIH 3T3 cells were transfected with pEGFP and pHspBPl in either the sense (lane 1) or anti-sense orientation (lane 2) . Cells were allowed to grow for 24 hrs . , then harvested and total RNA was isolated. Triplicate northern blots were probed with 32 P- labeled cDNAs for either Hsp70 (A) , HspBPl (B) or EGFP (C) .
  • Fig. 4 illustrates data showing HspBPl binding to DNA.
  • the DNA fragments employed were end-labeled using T4 DNA polymerase. Labeled DNA and the indicated added proteins were combined and incubated on a non-denaturing polyacrylamide gel . The gel was dried and then exposed to film. Lane 1 had no protein added. Lanes 2-5 were samples incubated with 1, 2, 4 and 8 ⁇ g of HspBPl. Lanes 6-9 were incubated with 1, 2, 4, and 8 ⁇ g of bovine serum albumin.
  • Fig. 5 depicts data showing the effect of Hsp70 on HspBPl gelshift.
  • Gel shift assays were performed as in Fig.4. Samples in A and B were incubated with 0, 1, 2, 3, 4 ⁇ g of HspBPl in lanes 1-5. In addition, samples in B were also incubated with 2 ⁇ g Hsp70.
  • Fig. 6 schematically depicts the protein structure of HspBPl in comparison with the protein structure of Importin.
  • extra-nuclear genetic material is meant to define sources of genes or genetic information found outside of a nucleus, including, but not limited to, plasmids, viral genomes, retro-viral genomes, and expression vectors .
  • the invention is based, in part, on providing a eukaryotic cell with a HspBPl polypeptide sequence (SEQ ID NO: 1) to inhibit the expression of extra-nuclear genetic material .
  • a human HspBPl SEQ ID NO:l
  • HspBPl inhibits the expression of mRNA and protein from genetic material (such as a plasmid) located outside of the nucleus.
  • HspBPl and Hsp70 cooperate to inhibit transcription. Yet, while Hsp70 has been localized in the nucleus, there have been no reports of Hsp70 binding to DNA. However, one of the Hsp70 binding proteins, Hap46 binds DNA and stimulates transcription, thus acting as a general transcription activator (Zeiner, M., Niyaz, Y. and Gehring, U. The hsp70 -associating protein Hap46 binds to DNA and stimulates transcription . Proc. Natl . Acad. Sci. 96:10194- 10199 (1999)) .
  • Bag- 1 a related protein called Bag- 1 recently has been shown to bind to and stimulate transcription of the human polyomavirus JC virus promoter (Devireddy LR, Kumar KU, Pater MM, et al . BAG-1, a novel Bel -2 -interacting protein, activates expression of human JC virus . J. Gen. Virol. 81:351-357, 2000). Thus, it was of interest to better characterize the activity of HspBPl and its relationship with Hsp70.
  • the invention encompasses polypeptides comprising amino acid sequences of HspBPl (SEQ ID N0:1; GenBank Acession Number AF093420) .
  • the invention also encompasses polynucleotides which encode HspBPl (SEQ ID NO: 2) and anti-sense versions thereof.
  • any nucleic acid sequence which encodes an amino acid sequence of a HspBPl (SEQ ID NO:l) can be used to produce recombinant molecules which express HspBPl .
  • the invention further encompasses HspBPl variants.
  • a preferred variant is one having at least 90% amino acid sequence similarity to the HspBPl amino acid sequences identified by SEQ ID NO:l. Most preferably, however, is a HspBPl variant having at least 95% amino acid sequence similarity to SEQ ID NO:l.
  • nucleotide sequences which encode HspBPl and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring transcription sequences under appropriately selected conditions of stringency, it can be advantageous to produce nucleotide sequences encoding HspBPl or its derivatives possessing a substantially different codon usage.
  • codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater stability or half-life, than transcripts produced from the naturally occurring sequence.
  • a DNA sequence, or portions thereof, encoding HspBPl and its derivatives may be produced entirely by synthetic chemistry. Subsequently, the synthetic nucleotide sequence may be inserted into any of the many available DNA vectors and cell systems using reagents that are commonly available. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HspBPl or any portion thereof .
  • Natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein.
  • One may, for example, screen a peptide library for inhibitors of HspBPl activity by encoding a chimeric HspBPl that can be detected by a commercially available antibody.
  • a fusion protein may be engineered to contain a cleavage site located between the HspBPl encoding sequence and the heterologous protein sequence, so that HspBPl may be cleaved and purified away from the heterologous moiety.
  • Methods well known in the art can be used to construct expression vectors containing sequences encoding HspBPl and appropriate transcriptional and translational control elements.
  • constitutive or inducible promoter elements such as the tetracycline-inducible promoter sold by Clone Tech, may be utilized.
  • Methods also may include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination in a variety of expression vector/host systems, such as bacteria transformed with recombinant bacteriophage or plasmids or insect cell systems infected with viral expression vectors such as the baculovirus . These methods are described in standard laboratory references, such as Sambrook, J. et al . Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Plainview, N.Y. (1989) .
  • Altered nucleic acids encoding HspBPl that may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HspBPl .
  • the protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in functionally equivalent HspBPl .
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of HspBPl is retained. For example, negatively charged amino acids aspartic acid and glutamic acid might be substituted for one another.
  • alleles encoding HspBPl are also included within the scope of the invention.
  • an "allele” or “allelic sequence” is an alternative form of the nucleic acid sequence encoding HspBPl . Alleles result from a mutation, i.e. a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence .
  • HspBPl may be used for research or therapeutically .
  • examples include, but are not limited to, administering HspBPl through the introduction of an expression vector into a subject for in vivo therapy, administering a vector expressing anti-sense of a polynucleotide encoding HspBPl, introducing HspBPl polypeptide into cells by means of a membrane transport/import signal attached to the polypeptide, or administering HspBPl polypeptide as part of a pharmaceutical composition.
  • appropriate agents for use in combination with HspBPl for therapy may include any conventional pharmaceutical carrier such as saline or buffered saline (intravenous dosing) and dextrose or water (oral dosing) . Further details on techniques for formulation and administration may be found in the latest edition of Remington ' s Pharmaceutical Sciences (Maack Publishing Co., Easton, PA) .
  • NIH3T3 mouse fibroblast cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) . Cells were cultured in 75cm 2 flasks in RPMI 1640 supplemented with 10% Fetal Calf Serum (Irvine Scientific, Santa Ana, CA) , lOmM HEPES and 2mM L-glutamine. Cells were maintained in a 37° atmosphere of 6% C0 2 . Cells were passaged when 70% confluent and used for experiments within 15 passages. NIH3T3 cells were stably transfected with a pEGFP vector encoding a G418 resistance gene.
  • NIH 3T3 cells were stably transfected with a vector containing EGFP under the regulation of a minimal hsp70 promotor, Y9.
  • the vector was provided by Thomas Tsang (University of Arizona, Arlington, Arizona) and the stably transfected cells were selected.
  • Cells transfected with non-stress inducible reporter constructs were assayed 18 hours post-transfection.
  • Cells transiently or stably transfected with a stress-inducible plasmid were heat shocked 18 hours post transfection by floating 60mm sealed tissue culture dishes containing 42 °C media in a 42 °C water bath for 120 minutes. Cells were returned to normal culture conditions and assayed 18 hours post heat-stress.
  • cells were rinsed with phosphate buffered saline and lysed in TNES (50mM Tris pH 7.5, 2mM EDTA, lOOmM NaCl, 1% NP40) containing protease inhibitors (20ug/mL aprotenin, 20ug/mL leupeptin, lmM PMSF) . Lysates were cleared of insoluble fractions by centrifugation at TNES (50mM Tris pH 7.5, 2mM EDTA, lOOmM NaCl, 1% NP40) containing protease inhibitors (20ug/mL aprotenin, 20ug/mL leupeptin, lmM PMSF) . Lysates were cleared of insoluble fractions by centrifugation at
  • ⁇ -galactosidase activity was assayed using 0- nitrophenyl- ⁇ -D-galactopyranoside (ONPG) as a substrate.
  • Equal volumes of cell lysate and 2X buffer 120mM Na 2 HP0 4 ,80mM NaH 2 P0 4 , 2mM MgC12, lOOmM ⁇ -mercaptoethanol
  • ONPG 0- nitrophenyl- ⁇ -D-galactopyranoside
  • the membrane was blocked in 3% nonfat powdered milk and probed with a 1:1000 dilution of mouse primary antibody against EGFP (Quantum Biotechnologies, Montreal, Canada) at room temperature for 1 hour followed by a 1:10,000 dilution of horse radish peroxidase- conjugated goat anti-mouse secondary antibody at room temperature for 1 hour. Immunoreactivity was detected using chemiluminescent substrate and exposure of membrane to Kodak Xar-5 film.
  • RNA was transfected with pEGFP and pHspBPl in either the sense or anti-sense orientation. Cells were allowed to grow for 24 hrs., then harvested in TRIZOL (Life Technologies).
  • Electrophoretic Mobility-Shift Assays were performed to deduce DNA binding as follows: The vector pcDNA 3.1 myc/his A " was digested with the restriction enzyme Apol . This resulted in 4 fragments of 2404, 1444, 848 and 827 bp in length. The 827 fragment contained the CMV immediate /early promoter. Both the 848 and 827 bp fragments were isolated by electro-elution. The fragments were end- labeled with 32 P using T4 DNA polymerase (Promega Corp., Madsion , WI . ) according to the supplier's protocol. Labeled DNA and the indicated added proteins were combined in a final volume of 20ul and a final buffer of 20 mM
  • Hepes pH 7.5
  • 100 mM KC1 100 mM MgCl 2
  • 25 mM DTT 25 mM DTT
  • 4% glycerol 4% glycerol
  • NIH3T3 cells were grown on coverslips and transfected as previously described with pcDNA and rhodamine labeled pEGFP (Gene Therapy Systems, San Diego, CA) or HspBPl and rhodamine labeled pEGPF. 24 hours post-transfection, cells were washed and mounted onto slides using an aqueous mounting medium. Slides were analyzed using a Nikon Eclipse TE300 microscope and images were acquired using a laser scanning confocal unit (model MRC1024, Bio-Rad) , a 15-milliwatt krypton-argon laser, and a 60X oil 1.4 NA oil immersion objective.
  • GFP fluorescence was excited using the 488-nm laser line and collected using a standard fluorescein isothiocyanate filter set (530+/-30nm) .
  • fluorescence associated with rhodamine-labeled plasmid was excited using the 568-nm laser line and collected using a standard Texas Red filter set (605 +/- 32nm) .
  • Images were acquired as a Z series of 20 sections at O.l ⁇ m per section and collected using a Kalman average. Pixel saturation was less than 10% and gain and iris settings were equivalent for all images.
  • HspBPl is a newly described protein and therefore very little is known about its activity in the cell.
  • Initial experiments were performed to determine the effect of overexpression of HspBPl.
  • pEGFP reporter plasmid
  • pHspBPl expression plasmid containing HspBPl
  • Fig.lA reporter protein activity
  • HspBPl expression results in inhibition of EGFP expression.
  • HspBPl is inhibiting the promoter activity of the EGFP plasmid.
  • EGFP and HspBPl are expressed off of the same promoter (CMV) , therefore it is expected that expression of both proteins would be inhibited.
  • CMV promoter
  • HspBPl protein levels were constructed with HspBPl fused to EGFP (EGFP-HspBPl) and transfected into cells.
  • EGFP-HspBPl EGFP-HspBPl
  • the anti-EGFP antibody detects both EGFP and EGFP
  • EGF-HspBPl When co-transfected with pEGFP, EGF-HspBPl inhibited expression of EGFP (compare lanes 1 & 4) .
  • the fusion protein EGFP-HspBPl was detectable.
  • HspBPl can interact with extra-nuclear DNA and either directly regulate transcription or in some other manner alter the ability of the DNA to be expressed. Therefore, we tested that ability of HspBPl to bind DNA using a gelshift assay (Fig.4) . HspBPl does cause a shift of the labeled DNA(Fig. 4) and less HspBPl is needed to cause this shift in the presence of Hsp70 (Fig. 5) . Hsp70 alone did not bind to the DNA. The presence of the CMV promoter was not required for DNA binding, therefore, it seemed unlikely that HspBPl was regulating transcription. Moreover, in vi tro transcription assays did not result in inhibition by HspBPl (data not shown) .
  • HspBPl can bind DNA but not specifically to a promoter region.
  • HspBPl inhibits reporter plasmid expression in transiently transfected cells but not stably transfected cells
  • Rhodamine labeled pEGFP was used to determine if HspBPl caused nuclear exclusion of the reporter plasmid.
  • the data clearly demonstrated that HspBPl causes plasmid DNA to be excluded from the nucleus.
  • HspBPl causes exclusion from the nucleus
  • Fig.4 points to a direct interaction with the introduced genetic material.
  • Hsp70 has been reported to play a role in nuclear transport of some proteins. See Yang, J. and DeFranco, D.B. Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 tumor antigen . Mol. Cell. Biol. 8: 5088-5098 (1999); Fujihara, S.M. and Nadler, S.G. Modulation of nuclear protein import . Biochem. Pharm. 56: 157-161 (1998); Shulga, N.
  • Hsp70 has been reported to play a role in the nuclear import of adenovirus DNA (Saphire, A.C.S., Guan, T., Schirmer, E.C., Nemerow, G.R., and Gerace, L. Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70. J. Biol. Chem. 275: 4298-4304 (2000) .
  • HspBPl could block this transport by binding to and inhibiting Hsp70 activity.
  • Hap46 Hsp70 binding protein
  • the hsp70 -associating protein Hap46 binds to DNA and stimulates transcription .
  • Hap46 contains a positively charged sequence required for DNA binding as well as a putative nuclear localization sequence.
  • HspBPl does not contain these features and therefore further research is needed to define the regions of the molecule that bind DNA.
  • HspBPl lacks clear amino acid homologies to other proteins. However, structural similarity could provide some insight into function.
  • the program 3D-PSSM (Kelley, L.A., MacCallum, R.M., Sternberg, J.E. Enhanced genome annotation using strucuture profiles in the program 3D- PSSM. J. Mol. Biol. 299, 499-520 (2000)) was used to predict the 3-dimensional structure of HspBPl.
  • Both karyopherin o. and importin o. are nuclear transporter proteins that bind to the NLS (nuclear localization signal) of proteins and transport these proteins to the nucleus .
  • NLS nuclear localization signal
  • HspBPl excludes plasmid from the nucleus, therefore, a predicted structure that is related to other nuclear transport proteins is consistent with our results.
  • proteins involved with export of proteins and tRNA from the nucleus (reviewed by G ⁇ rlich, D. and Kutay, U. Transport between the cell nucleus and the cytoplasm. Ann. Rev. Cell Dev. Biol. 15: 607-660 (1999)), so it is possible that HspBPl might be exporting plasmid rather than inhibiting uptake.
  • HspBPl can serve as an endogenous protective mechanism to prevent foreign genetic material from entering the nucleus. This mechanism would, for example, prevent viral genomes from entering the nucleus . Of course, such activity would also inhibit the efficacy of desirable gene uptake, such as through gene replacement therapy.
  • identification of a protein that promotes exclusion of genetic material from the nucleus is a novel finding that may provide insight into the variation of transfection efficiencies among various cell types.
  • HIV virus One particularly attractive target for HspBPl is the HIV virus.
  • PIC preintegration complex
  • the ability of HIV to transport its preintegration complex (PIC) into the nucleus of an infected cell during the interphase is one unique feature of this virus that separates it from the other retroviruses, which rely on the breakdown of the nuclear envelope during mitosis for delivery of their genome into the nucleus.
  • nuclear import is critical for HIV replication in non-dividing cells, such as macrophages, as well as in slowly dividing populations, such as primary T lymphocytes .
  • Recent evidence has shown that the import of the PIC is dependent on the HIV-encoded protein Vpr. Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M.
  • Vpr is a PIC protein that can associate with the nuclear import molecule karyopherin ⁇ in the cell .
  • MA matrix protein
  • MA contains a nuclear localization sequence (NLS) that can bind to karyopherin ⁇ , and, thus, facilitate the transport of the PIC into the nucleus.
  • NLS nuclear localization sequence
  • Hsp70 has been shown to replace Vpr of HIV during nuclear import of the PIC.
  • Agostini et al Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex.
  • Exp. Cell Res. 259: (2)398- 403 (2000) Hsp70 and Vpr bind to a the amino-terminal portion of karopherin ct .
  • Karyopherin ⁇ then has a region that can bind Hsp70 or Vpr and this binding stimulates the interaction between PIC and karopherin ⁇ . Since HspBPl binds to and inhibits the activity of Hsp70 (Raynes, D. and Guerriero, V.
  • HspBPl can be used to inhibit the import of the PIC into the nucleus.
  • Hsc70 this protein is very similar to Hsp70 and also binds HspBPl
  • HspBPl is required for the import of adenovirus DNA into the nucleus.
  • Structure prediction tools indicate with a greater than 95% confidence level that HspBPl has a structure similar to karyopherin and importin which are in
  • HspBPl the armadillo repeat family (Fig. 6) . Both of these proteins are nuclear transporter proteins that bind to the NLS of proteins and transport these proteins to the nucleus. Thus, over-expression of HspBPl should exclude viral genomes from the nucleus since the predicted
  • HspBPl can regulate nuclear transport. Accordingly, HspBPl is a novel target for the development of an anti-viral drug. The fact that HspBPl inhibits extra-nuclear genetic material uptake strongly suggests that this protein can inhibit the uptake of viral nucleic acid into the nucleus .
  • a major technical block to gene replacement therapy is the method to introduce the normal gene into the recipient' s genome.
  • the gene must enter the cell and then be delivered to the nucleus where the cell' s genetic material, DNA, is stored.
  • DNA DNA
  • Non-viral vectors may offer a solution to this problem. Plasmids are small pieces of DNA that can be introduced into cells but do not contains proteins like viruses that can cause an immune response. If plasmids could be used for gene replacement therapy, then the problems associated with viruses would be eliminated.
  • the plasmid DNA would contain the corrected gene and deliver the gene to the nucleus of the cells where is would become incorporated into the cell' s genome. A major block in this process is delivering the plasmid DNA to nucleus. In some cells, this process is very efficient, yet in others it is practically impossible. Very little information is available on the mechanism of plasmid DNA uptake into the nucleus and how this process is regulated.
  • HspBPl has the ability to prevent plasmid DNA from going into the nucleus . This is the first time a protein has been identified with this activity, and, therefore, has opened the door to blocking this endogenous activity to facilitate plasmid uptake into the nucleus . It is possible that the endogenous expression of this protein in various cells and tissues prevents or hinders plasmid uptake. Accordingly, lowering the endogenous levels of HspBPl in cells would allow an increase in nuclear plasmid uptake. Additionally, lowering the endogenous levels of HspBPl may also facilitate viral genome uptake into cells and lower the amount of virus that is used in gene replacement therapy. A lower viral dose would decrease the chance for an immune response. Thus, expressing the anti-sense polyn ⁇ cT.eg ⁇ ides of HspBPl as, for example, described in example 1, would up-regulate the expression of extra-nuclear genetic material .

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Abstract

The invention relates to the discovery of a new gene expression regulatory activity for the human heat-shock protein-binding protein HspBP1, SEQ ID NO:1 (Fig 1). More specifically, the invention relates to a method of providing HspBP1 polynucleotides (SEQ ID NO:2) and polypeptides (SEQ ID NO:1) to regulate the expression of extra-nuclear genetic material in eukaryotic cells.

Description

REGULATION OF GENE EXPRESSION BY HSPBP1
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based on U.S. Provisional Application 60/200,083, filed by the same inventors on April 27, 2000. This application also is a continuation-in-part of U.S. Application 09/444,336, entitled "DNA Encoding Proteins that Inhibit Hsp70 Function," filed on 11/19/99, which is based on U.S. Provisional Application 60/109,351, filed on 11/20/98 and entitled "Inhibition of HSP 70 ATPase Activity and Protein Renaturation by a Novel HSP-Binding Protein." The contents of these applications are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
Field of Invention
This invention generally relates to the field of molecular medicine and more particularly to a method of regulating the expression of extra-nuclear genetic material in eukaryotic cells through HspBPl.
Description of the Related Art
Practically all organisms respond to heat by inducing the synthesis of a group of proteins called the hea -shock proteins. Although the details of this response vary among organisms, the involvement of the Hsp70 and Hsp90 gene families is known to be highly conserved. More recently, it has come to be known that heat-shock proteins can be induced by a variety of stress-related stimuli besides heat. Hence, such proteins commonly are more broadly referred to as heat stress or, sometimes, stress proteins . All eukaryotic cells have heat stress proteins (Hsps) that provide protection from various environmental stresses, such as anoxia, heat, ethanol and certain heavy metal ions. This exposure stimulates increased expression and activity by these proteins, with the increased levels helping the cells to survive.
The Hsps are named according to their molecular weight . The most studied has a molecular weight of 70 kDa and is therefore called Hsp70. Hsp70 and a related protein called Hsc70 help the cell survive stress events by binding to partially denatured proteins and assisting to refold these proteins into more stable native structures . Thus, members of the Hsp70 family have come to be known as molecular " chaperones ."
In terms of function, studies have shown that Hsp70 plays a role in DNA replication, transport of proteins across membranes, binding of proteins to the endoplasmic reticulum, and uncoating clathrin coated vesicles. S. Lindquist and E.A. Craig, Annual Revue of Genetics. 22:631-77 (1988). Furthermore, Hsp70 is known to associate with non-esterified fatty acids, palmitic acid, stearic acid, and myristic acid and to be involved in signal transduction pathways in the cytoplasm. Hohfeld, Jorg, et al . , Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/Hsp40 Reaction Cycle . Cell vol. 83, 589-598 (November 17, 1995) .
Perhaps the best studied function of Hsp70 has been its role as a "chaperone, " a protein that stabilizes other proteins against aggregation and that mediates the folding of newly translated polypeptides in the cytosol and organelles. Proper functioning of Hsp70 as a protein chaperone is dependent on its bound nucleotide state. Specifically, the ATP form of Hsp70 binds substrate very poorly and therefore must be converted to the ADP form before the misfolded protein can bind. Then, the high affinity of Hsp70 for ATP is utilized to "power" the protein folding and other functions of Hsp70 by the generation of energy through the hydrolysis of bound ATP.
In the last decade, a number of proteins have been described that bind to and regulate the activity of Hsp70. One of these co-chaperones is Hsp40, which stimulates ATPase activity of Hsp70 resulting in converting ATP-Hsp70 to ADP-Hsp70, which has a greater affinity for the misfolded substrate. See Cyr D.M., Langer, T., and Douglas, M.G. DNAJ- like proteins -molecular chaperones and specific regulators of Hsp70. Trends in Biochem. 19, 176-181 (1994); and Silver, P.A., and Way, J.C. Eukaryotic DnaJ homologs and the specifici ty of Hsp70 activity. Cell 74, 5-6 (1993) . Another is called Hip. Hip binds to Hsp70 and stabilizes the ADP-Hsp70 form, resulting in greater substrate affinity. Hδhfeld, J., Minami, Y., and Hartl, F-U. A novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle . Cell 83, 589-598(1995).
Another regulator, the protein Hop, facilitates interaction between Hsp70 and Hsp90. Smith, DF, Sullivan, WP, Marion, TN, Zaitsu, K, Madden, B McCormick, DJ and Toft, D.O. Identification of a 60-kilodal ton stress -related protein, p60, which interacts wi th hsp90 and hsplO . Mol . Cell. Biol . 13:869-876 (1993). Two other regulatory factors, Bag-1 and Hap46, stimulate the exchange of ATP for ADP, which results in lower substrate affinity. See Takayama, S., D. N. Bimstone, S. Matsuzawa, B. C. Freeman, C. Aime-Sempe, Z. Xie, R. I. Morimoto, and J. C. Reed. BAG-1 modulates the chaperone activi ty of Hsp70/Hsc70. EMBO J. 16:4887-4896 (1997); and Zeiner, M., M. Gebauer, and U. Gehring. Mammalian protein RAP46 : an interaction partner and modulator of 70 kDa heat shock proteins . EMBO J. 16:5483-5490(1997). Despite the fact that these regulators of Hsp70 have been discovered and characterized, the functional regulation of Hsp70 is not well understood. Hence, the recent isolation and characterization of a novel human cDNA that codes for a Hsp70 binding protein called HspBPl has provided for further progress in this area. Raynes, D.A. and Guerriero, V. Isolation and Characterization of Iso forms HspBPl , an Inhibi tor of Hsp70. Biochimica et Biophysica Acta 1490, 203-207 (2000) .
HspBPl dramatically alters the activity of Hsp70 by binding to its ATPase domain and inhibiting the ability of Hsp70 to refold a denatured protein. Recently, another protein called CHIP with similar inhibitory activity has been described. Ballinger CA, Connell P, Wu YX, Hu ZY, Thompson LJ, Yin LY, Patterson C. Identification of CHIP, a novel tetratricopeptide repeat -containing protein that interacts with heat shock proteins and negatively regulates chaperone functions . Mol . Cell. Bio.19 -.4535- 4545 (1999) .
Because HspBPl is newly discovered, very little is known about its activity in the cell other then its binding with Hsp70. Thus, to better understand the full role that HspBPl may play in normal physiology and disease, there remains a need to further characterize the activity of HspBPl .
BRIEF SUMMARY OF THE INVENTION
One aspect of the invention relates to the unexpected discovery that the expression of co-chaperone HspBPl polypeptides can down regulate the expression of extra- nuclear genetic material in eukaryotic cells. Although this aspect of the invention is not meant to be limited to a particular mechanism of action, the expression of HspBPl polypeptides are thought to inhibit expression of extra- nuclear genetic material by excluding it from the transcriptional machinery in the nucleus . Another aspect of the invention involves the expression of HspBPl anti- sense polynucleotides in eukaryotic cells to up-regulate extra-nuclear genetic material expression. Thus, HspBPl polypeptides and polynucleotides may have regulatory consequences for many different physiological pathways or disease conditions, such as development, apoptosis, cellular stress, viral infection, heart disease, and cancer, as well as for the efficacy of gene therapy treatments.
It is a general object of the invention to provide a method for regulating the expression of extra-nuclear genetic material through the use of HspBPl polypeptides and polynucleotides .
A second object of the invention is to provide a method for inhibiting the expression of transiently transfected plasmids .
A third object of the invention is to provide a method for inhibiting the expression of viral genomic material within eukaryotic cells.
A fourth object of the invention is to provide a method of up-regulating the expression of proteins introduced into eukaryotic cells that are undergoing gene therapy through the expression of anti-sense HspBPl polynucleotide sequences .
A fifth object of the invention is to provide the amino acid sequence for substantially purified HspBPl.
A sixth object of the invention is to provide pharmaceutical compositions based on the human HspBPl or peptide fragments thereof.
In accordance with these objectives, the invention generally features a method of using human heat-shock protein-binding protein 1 (HspBPl) , having the amino acid sequence shown in SEQ ID N0:1, to regulate the expression of extra-nuclear genetic material. Furthermore, the invention features a method of using HspBPl anti-sense polynucleotides, having the nucleotide sequence shown in SEQ ID NO: 2, to regulate the expression of extra-nuclear genetic material. Finally, the invention features pharmaceutical compositions comprising substantially purified HspBPl .
Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiments and particularly pointed out in the claims. However, such drawings and description disclose only some of the various ways in which the invention may be practiced.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. contains graphs A-D showing that HspBPl inhibits the expression of transiently transfected EGFP gene but not the stably-integrated EGFP gene.
A. HspBPl inhibits the expression of transiently transfected EGFP gene regulated by the CMV promoter. NIH 3T3 fibroblasts were transfected using cationic lipid reagent (Lipofectamine Plus, BRL) per manufacturer' s recommendations using: No DNA, a mixture of pcDNA (1.5μg) and pEGFP (0.5μg) or a mixture of pHspBPl (1.5μg) and pEGFP (0.5μg). Cells were lysed in non-ionic detergent 5 buffer 18 hours post-transfection. Protein concentrations were determined using BCA reagent (Pierce) , and EGFP fluorescence was quantitated using a microplate fluorometer (BioRad) . Results from each transfection were calculated as fluorescent units per mg of cellular 10 protein. The mean and standard deviation of values from triplicate transfections for each condition are depicted.
B. HspBPl does not inhibit the expression of stably 15 integrated EGFP gene regulated by CMV promoter. SKNSH cells were stably transfected with pEGFP plasmid regulated by the CMV promoter. Cells were then transiently transfected with either 1.5μg pcDNA and 0.5 μg pDSred-Nl (plasmid containing cDNA for a red fluorescent protein) or 20 15μg pcDNA and 5μg pHspBPl . Cells were lysed in non-ionic detergent buffer 18 hours post-transfection. Protein concentrations were determined using BCA reagent (Pierce) and EGFP fluorescence was quantitated using a microplate fluorometer (BioRad) . Results from each transfection were 25 calculated as fluorescent units per mg of cellular protein. The mean and standard deviation of values from triplicate transfections for each condition are depicted.
30 C. HspBPl inhibits the expression of transiently expressed heat responsive reporter plasmid. NIH 3T3 cells were cotransfected with 1.5 μg of either pcDNA3.1 or pHspBPl and 0.5μg of the heat responsive reporter construct Y9. Y9 is a plasmid containing the EGFP coding sequence and a
35 minimal hsp70 promoter. Transfected cells were heat shocked in a 42°C water bath for 150 min. 24 hours post transfection. Cells were lysed with non-ionic detergent buffer 18 hours following heat shock and EGFP levels were analyzed on a fluorometer.
D. HspBPl does not inhibit the expression of stably integrated heat responsive promoter. NIH 3T3 cells were stably transfected with Y9 reporter construct containing the minimal heat responsive reporter. Cells were then transiently co-transfected with either 1.5μg pcDNA or pHspBPl with 0.5μg pcDNA . Cells were heat shocked and analyzed as above.
Fig. 2 is a panel of Western blot data showing the inhibition of EGFP expression. NIH 3T3 fibroblasts were transfected with the mixtures of plasmid DNA as indicated. pEGFP-HspBPl refers to a plasmid encoding EGFP fused in frame to the 5' end of the full-length HspBPl coding sequence. Cells were lysed 24 hrs post-transfection, and lOOμg of total cellular protein per lane were fractionated by SDS-PAGE. Following electrophoretic transfer to nitrocellulose, the membrane was probed with an anti-EGFP antibody and reactivity visualized using horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (Pierce) .
Fig. 3 is a depiction of a Northern blot analysis of cells transfected with HspBPl. NIH 3T3 cells were transfected with pEGFP and pHspBPl in either the sense (lane 1) or anti-sense orientation (lane 2) . Cells were allowed to grow for 24 hrs . , then harvested and total RNA was isolated. Triplicate northern blots were probed with 32 P- labeled cDNAs for either Hsp70 (A) , HspBPl (B) or EGFP (C) .
Fig. 4 illustrates data showing HspBPl binding to DNA. The DNA fragments employed were end-labeled using T4 DNA polymerase. Labeled DNA and the indicated added proteins were combined and incubated on a non-denaturing polyacrylamide gel . The gel was dried and then exposed to film. Lane 1 had no protein added. Lanes 2-5 were samples incubated with 1, 2, 4 and 8μg of HspBPl. Lanes 6-9 were incubated with 1, 2, 4, and 8 μg of bovine serum albumin.
Fig. 5 depicts data showing the effect of Hsp70 on HspBPl gelshift. Gel shift assays were performed as in Fig.4. Samples in A and B were incubated with 0, 1, 2, 3, 4 μg of HspBPl in lanes 1-5. In addition, samples in B were also incubated with 2μg Hsp70.
Fig. 6 schematically depicts the protein structure of HspBPl in comparison with the protein structure of Importin.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in art of the invention. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which might be used in connection with the invention.
The term " extra-nuclear genetic material" is meant to define sources of genes or genetic information found outside of a nucleus, including, but not limited to, plasmids, viral genomes, retro-viral genomes, and expression vectors .
The invention is based, in part, on providing a eukaryotic cell with a HspBPl polypeptide sequence (SEQ ID NO: 1) to inhibit the expression of extra-nuclear genetic material . During the course of" investigating the regulatory consequences of a human HspBPl (SEQ ID NO:l), it was unexpectedly discovered that HspBPl inhibits the expression of mRNA and protein from genetic material (such as a plasmid) located outside of the nucleus.
One possible explanation for this novel activity is that HspBPl and Hsp70 cooperate to inhibit transcription. Yet, while Hsp70 has been localized in the nucleus, there have been no reports of Hsp70 binding to DNA. However, one of the Hsp70 binding proteins, Hap46 binds DNA and stimulates transcription, thus acting as a general transcription activator (Zeiner, M., Niyaz, Y. and Gehring, U. The hsp70 -associating protein Hap46 binds to DNA and stimulates transcription . Proc. Natl . Acad. Sci. 96:10194- 10199 (1999)) . In addition, a related protein called Bag- 1 recently has been shown to bind to and stimulate transcription of the human polyomavirus JC virus promoter (Devireddy LR, Kumar KU, Pater MM, et al . BAG-1, a novel Bel -2 -interacting protein, activates expression of human JC virus . J. Gen. Virol. 81:351-357, 2000). Thus, it was of interest to better characterize the activity of HspBPl and its relationship with Hsp70.
Although many different methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and material are now described.
The invention encompasses polypeptides comprising amino acid sequences of HspBPl (SEQ ID N0:1; GenBank Acession Number AF093420) . The invention also encompasses polynucleotides which encode HspBPl (SEQ ID NO: 2) and anti-sense versions thereof. Thus, any nucleic acid sequence which encodes an amino acid sequence of a HspBPl (SEQ ID NO:l) can be used to produce recombinant molecules which express HspBPl . The invention further encompasses HspBPl variants. A preferred variant is one having at least 90% amino acid sequence similarity to the HspBPl amino acid sequences identified by SEQ ID NO:l. Most preferably, however, is a HspBPl variant having at least 95% amino acid sequence similarity to SEQ ID NO:l.
It will be appreciated by those skilled in the art that, as a result of the degeneracy of the genetic code, a multitude of HspBPl-encoding nucleotide sequences, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. The invention contemplates every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices . These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence encoding naturally occurring HspBPl, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode HspBPl and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring transcription sequences under appropriately selected conditions of stringency, it can be advantageous to produce nucleotide sequences encoding HspBPl or its derivatives possessing a substantially different codon usage. For example, codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HspBPl and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater stability or half-life, than transcripts produced from the naturally occurring sequence.
As known by one skilled in the art, a DNA sequence, or portions thereof, encoding HspBPl and its derivatives may be produced entirely by synthetic chemistry. Subsequently, the synthetic nucleotide sequence may be inserted into any of the many available DNA vectors and cell systems using reagents that are commonly available. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HspBPl or any portion thereof .
Natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. One may, for example, screen a peptide library for inhibitors of HspBPl activity by encoding a chimeric HspBPl that can be detected by a commercially available antibody. In addition, a fusion protein may be engineered to contain a cleavage site located between the HspBPl encoding sequence and the heterologous protein sequence, so that HspBPl may be cleaved and purified away from the heterologous moiety.
Methods well known in the art can be used to construct expression vectors containing sequences encoding HspBPl and appropriate transcriptional and translational control elements. For example, constitutive or inducible promoter elements, such as the tetracycline-inducible promoter sold by Clone Tech, may be utilized. Methods also may include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination in a variety of expression vector/host systems, such as bacteria transformed with recombinant bacteriophage or plasmids or insect cell systems infected with viral expression vectors such as the baculovirus . These methods are described in standard laboratory references, such as Sambrook, J. et al . Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Plainview, N.Y. (1989) .
Altered nucleic acids encoding HspBPl that may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HspBPl . The protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in functionally equivalent HspBPl . Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of HspBPl is retained. For example, negatively charged amino acids aspartic acid and glutamic acid might be substituted for one another.
Also included within the scope of the invention are alleles encoding HspBPl. As used herein, an "allele" or "allelic sequence" is an alternative form of the nucleic acid sequence encoding HspBPl . Alleles result from a mutation, i.e. a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence .
Many ways exist in the art by which HspBPl may be used for research or therapeutically . Examples include, but are not limited to, administering HspBPl through the introduction of an expression vector into a subject for in vivo therapy, administering a vector expressing anti-sense of a polynucleotide encoding HspBPl, introducing HspBPl polypeptide into cells by means of a membrane transport/import signal attached to the polypeptide, or administering HspBPl polypeptide as part of a pharmaceutical composition. Depending on the route of administration, appropriate agents for use in combination with HspBPl for therapy may include any conventional pharmaceutical carrier such as saline or buffered saline (intravenous dosing) and dextrose or water (oral dosing) . Further details on techniques for formulation and administration may be found in the latest edition of Remington ' s Pharmaceutical Sciences (Maack Publishing Co., Easton, PA) .
In order to further illustrate the invention, the following actual and prophetic examples are provided. However, these examples are not intended in any way to limit the invention.
EXAMPLE 1: HspBPl-Mediated Inhibition of Extra-Nuclear Genetic Material Expression
Experimental Method:
Cell Cultures and Stably Transfected Cell Lines NIH3T3 mouse fibroblast cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) . Cells were cultured in 75cm2 flasks in RPMI 1640 supplemented with 10% Fetal Calf Serum (Irvine Scientific, Santa Ana, CA) , lOmM HEPES and 2mM L-glutamine. Cells were maintained in a 37° atmosphere of 6% C02. Cells were passaged when 70% confluent and used for experiments within 15 passages. NIH3T3 cells were stably transfected with a pEGFP vector encoding a G418 resistance gene. A polyclonal population of cells were selected after 14 days of culturing cells in 10 cm dishes with 500ug/ml G418. NIH 3T3 cells were stably transfected with a vector containing EGFP under the regulation of a minimal hsp70 promotor, Y9. The vector was provided by Thomas Tsang (University of Arizona, Tucson, Arizona) and the stably transfected cells were selected.
Cells were plated at 50% confluency (55x10s cells) in 60mm dishes 24 hours prior to transfection using a polycationic lipid Lipofectamine Plus (BRL Life Technologies, Rockville, MD) . Transfections were conducted in serum- free medium with 2ug total DNA, with cotransfections containing a 3:1 ratio of experimental or control plasmid (pHspBPl, pHspBPl-GFP, pHspBPl-RFP, pcDNA 3.1+) to reporter or filler plasmid (pEGFP, pDSred-Nl, pcmv-β, pcDNA 3.1+) . All other conditions were carried out per manufacturer's recommendations. Cells transfected with non-stress inducible reporter constructs were assayed 18 hours post-transfection. Cells transiently or stably transfected with a stress-inducible plasmid were heat shocked 18 hours post transfection by floating 60mm sealed tissue culture dishes containing 42 °C media in a 42 °C water bath for 120 minutes. Cells were returned to normal culture conditions and assayed 18 hours post heat-stress.
For determinations of fluorescence and β-galactosidase activity, cells were rinsed with phosphate buffered saline and lysed in TNES (50mM Tris pH 7.5, 2mM EDTA, lOOmM NaCl, 1% NP40) containing protease inhibitors (20ug/mL aprotenin, 20ug/mL leupeptin, lmM PMSF) . Lysates were cleared of insoluble fractions by centrifugation at
16,0005xg for 20 minutes at 4°C. Equal volumes of lysates were analyzed for fluorescence in 96-well clear bottom plates using a Fluoromark microplate flourometer (Bio Rad, Hercules, CA) . EGFP activity was read at excitation and emission wavelengths of 485 nm and 538 nm respectively. RFP activity was read at excitation and emission wavelengths of 544 nm and 590 nm respectively. Samples were normalized to ug of protein concentration as quantified using a BCA protein assay kit (Pierce, Rockford, IL) and a Specta plate reader (Tecan, Durrham, NC) . β-galactosidase activity was assayed using 0- nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate. Equal volumes of cell lysate and 2X buffer (120mM Na2HP04,80mM NaH2P04, 2mM MgC12, lOOmM β-mercaptoethanol) containing 1.33mg/mL ONPG were incubated in a 37 °C water bath for 20 minutes. Samples' absorbances were measured at dual wavelengths of 450 nm and 690 nm in a Spectra plate reader.
To visualize proteins through western blotting, cells were lysed in TNES and protein concentration of supernatant fractions was determined as previously described. lOOug of cellular protein from each sample was diluted in 15:1 Laemmli sample buffer, heated at 95 °C for 5 minutes and cooled at 4°C for 5 minutes. All samples were then fractionated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane by semi-dry electroblotting. The membrane was blocked in 3% nonfat powdered milk and probed with a 1:1000 dilution of mouse primary antibody against EGFP (Quantum Biotechnologies, Montreal, Canada) at room temperature for 1 hour followed by a 1:10,000 dilution of horse radish peroxidase- conjugated goat anti-mouse secondary antibody at room temperature for 1 hour. Immunoreactivity was detected using chemiluminescent substrate and exposure of membrane to Kodak Xar-5 film.
To visualize RNA through Northern blot analysis, NIH 3T3 cells were transfected with pEGFP and pHspBPl in either the sense or anti-sense orientation. Cells were allowed to grow for 24 hrs., then harvested in TRIZOL (Life
Technologies) and total RNA was isolated according to the supplied protocol. Triplicate northern blots were probed with 32 P labeled cDNAs for either Hsp70, HspBPl or EGFP. Blots were then exposed to film and the images were recorded using a digital camera.
Electrophoretic Mobility-Shift Assays were performed to deduce DNA binding as follows: The vector pcDNA 3.1 myc/his A" was digested with the restriction enzyme Apol . This resulted in 4 fragments of 2404, 1444, 848 and 827 bp in length. The 827 fragment contained the CMV immediate /early promoter. Both the 848 and 827 bp fragments were isolated by electro-elution. The fragments were end- labeled with 32P using T4 DNA polymerase (Promega Corp., Madsion , WI . ) according to the supplier's protocol. Labeled DNA and the indicated added proteins were combined in a final volume of 20ul and a final buffer of 20 mM
Hepes (pH 7.5), 100 mM KC1, 10 mM MgCl2 , 25 mM DTT, and 4% glycerol . Samples were incubated for 30 min. at room temperature and then analyzed on a non-denaturing polyacrylamide gel . The gel was dried and then exposed to film.
For analysis by confocal microscopy, NIH3T3 cells were grown on coverslips and transfected as previously described with pcDNA and rhodamine labeled pEGFP (Gene Therapy Systems, San Diego, CA) or HspBPl and rhodamine labeled pEGPF. 24 hours post-transfection, cells were washed and mounted onto slides using an aqueous mounting medium. Slides were analyzed using a Nikon Eclipse TE300 microscope and images were acquired using a laser scanning confocal unit (model MRC1024, Bio-Rad) , a 15-milliwatt krypton-argon laser, and a 60X oil 1.4 NA oil immersion objective. GFP fluorescence was excited using the 488-nm laser line and collected using a standard fluorescein isothiocyanate filter set (530+/-30nm) . Simultanequsly, fluorescence associated with rhodamine-labeled plasmid was excited using the 568-nm laser line and collected using a standard Texas Red filter set (605 +/- 32nm) . Images were acquired as a Z series of 20 sections at O.lμm per section and collected using a Kalman average. Pixel saturation was less than 10% and gain and iris settings were equivalent for all images.
Results :
HspBPl is a newly described protein and therefore very little is known about its activity in the cell. Initial experiments were performed to determine the effect of overexpression of HspBPl. Surprisingly, when the reporter plasmid (pEGFP) was co-transfected with an expression plasmid containing HspBPl (pHspBPl) there was extremely low reporter protein activity (Fig.lA) . Controls in which the expression vector did not contain a cDNA insert (pcDNA) resulted in expression of EGFP. If, however, the same reporter plasmid was stably integrated into the cell' s genome, transfection with pHspBPl had no effect (Fig. IB) . In both the transient and stably integrated reporter plasmid experiments, expression is regulated by the CMV promoter.
To determine if this result was promoter specific, similar experiments were performed using a different promoter, the heat shock promoter. In transient transfections, expression of the reporter gene (pEGFP) was heat sensitive (Fig. IC, black bars) and when co-expressed with HspBPl the final level of EGFP activity was greatly reduced (Fig. IC, grey bars) . Once again, when EGFP cDNA under the heat-shock promoter was stably integrated into the cell' s genome, HspBPl did not inhibit expression (Fig. ID) , in fact there was a stimulation of expression. Taken together, these results indicate that HspBPl regulates gene expression differently in transiently transfected cells vs. stably transfected cells and this regulation is not promoter specific.
The next series of experiments were designed to determine if the inhibition of transiently transfected plasmid expression was due to inhibition of protein folding, protein expression and/or mRNA expression. Hsp70 is known to participate in the folding of newly synthesized proteins and HspBPl can inhibit this activity. Therefore, the loss of EGFP activity might be due to HspBPl inhibiting Hsp70 activity in the cell and EGFP could be produced but not folded properly. Western blot analysis was used to determine relative levels of EGFP and HspBPl in cells. As shown in Fig. 2, EGFP is expressed when pEGFP is transfected without HspBPl containing plasmid (lane 1) . However, the expression of the EGFP is greatly reduced when co-transfected with pHspBPl (lane 2 ) . These experiments suggest that HspBPl expression results in inhibition of EGFP expression. One possibility is that HspBPl is inhibiting the promoter activity of the EGFP plasmid. However, EGFP and HspBPl are expressed off of the same promoter (CMV) , therefore it is expected that expression of both proteins would be inhibited. A possible explanation for HspBPl expression but not EGFP is that the HspBPl protein is more stable.
To follow HspBPl protein levels plasmids were constructed with HspBPl fused to EGFP (EGFP-HspBPl) and transfected into cells. The anti-EGFP antibody detects both EGFP and
EGFP-HspBPl. When co-transfected with pEGFP, EGF-HspBPl inhibited expression of EGFP (compare lanes 1 & 4) .
However, the fusion protein EGFP-HspBPl was detectable.
These results are consistent with HspBPl having a longer half-life than EGFP and fusion of HspBPl to EGFP results in a more stable fusion protein.
Northern blot analysis was used next to determine if the inhibition of EGFP mRNA levels mimic inhibition of protein expression as would be expected for transcriptional regulation. Cells transfected with both pHspBPl and pEGFP containing vectors showed very little mRNA for either of these proteins (Fig. 3B& C, lane 1) . The mRNA levels are clearly expressed when cells were transfected with pHspBPl in the anti-sense orientation (Fig.3 B& C, lane2) . Blots were also probed for Hsp70, and these levels were not greatly altered by HspBPl. These results are consistent with HspBPl inhibiting expression of the reporter mRNA as well as itself . Even though no detectable mRNA for HspBPl can be observed, when fused to EGFP the fusion protein can be detected (Fig. 2) .
The decrease in mRNA and protein levels suggests that HspBPl can interact with extra-nuclear DNA and either directly regulate transcription or in some other manner alter the ability of the DNA to be expressed. Therefore, we tested that ability of HspBPl to bind DNA using a gelshift assay (Fig.4) . HspBPl does cause a shift of the labeled DNA(Fig. 4) and less HspBPl is needed to cause this shift in the presence of Hsp70 (Fig. 5) . Hsp70 alone did not bind to the DNA. The presence of the CMV promoter was not required for DNA binding, therefore, it seemed unlikely that HspBPl was regulating transcription. Moreover, in vi tro transcription assays did not result in inhibition by HspBPl (data not shown) .
The above results suggests that HspBPl can bind DNA but not specifically to a promoter region. Taken together with the fact that HspBPl inhibits reporter plasmid expression in transiently transfected cells but not stably transfected cells, it is possible that HspBPl inhibits reporter plasmid transport into the nucleus or increases export of the plasmid from the nucleus resulting in nuclear exclusion. Rhodamine labeled pEGFP was used to determine if HspBPl caused nuclear exclusion of the reporter plasmid. The data clearly demonstrated that HspBPl causes plasmid DNA to be excluded from the nucleus. These results are consistent with the inhibition of extra- nuclear genetic material expression, but not stably integrated genetic material within the nucleus, and with the inhibition of reporter protein and mRNA expression.
We speculate that when two plasmids are transfected, initially both enter the nucleus and are expressed. However, when enough HspBPl is produced, this results in exclusion of both plasmids from the nucleus and the arresting of protein expression. A longer half-life for HspBPl would explain why it can be detected when fused to EGFP, but EGFP alone is not detectable (Fig. 2, lanes 2& 4) . Hsp70 may also be involved since inclusion of this protein increases HspBPl binding to DNA (Fig. 5) . It is possible that HspBPl either directly prevents extra- nuclear genetic material uptake into the nucleus or it may facilitate transport of non-stably integrated material out of the nucleus. Localization studies have shown that HspBPl is in both the nucleus and cytoplasm (data not shown) .
The mechanism by which HspBPl causes exclusion from the nucleus is unknown, but the fact that HspBPl can directly bind DNA (Fig.4) points to a direct interaction with the introduced genetic material. Hsp70 has been reported to play a role in nuclear transport of some proteins. See Yang, J. and DeFranco, D.B. Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 tumor antigen . Mol. Cell. Biol. 8: 5088-5098 (1999); Fujihara, S.M. and Nadler, S.G. Modulation of nuclear protein import . Biochem. Pharm. 56: 157-161 (1998); Shulga, N. , Mosammaparast, N., Wozniak, R., and Goldfarb, D.S. Yeast Nucleoporins involved in passive nuclear envelope permabili ty. J. Cell Biol. 149: 1027-1028 (2000). Addionally, Hsp70 has been reported to play a role in the nuclear import of adenovirus DNA (Saphire, A.C.S., Guan, T., Schirmer, E.C., Nemerow, G.R., and Gerace, L. Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70. J. Biol. Chem. 275: 4298-4304 (2000) . Thus, HspBPl could block this transport by binding to and inhibiting Hsp70 activity.
Recently, Zeiner et al . reported that another Hsp70 binding protein called Hap46 can bind to DNA and stimulates transcription. Zeiner, M., Niyaz, Y. and Gehring, U. The hsp70 -associating protein Hap46 binds to DNA and stimulates transcription . Proc. Natl. Acad. Sci. 96:10194-10199 (1999). Hap46 contains a positively charged sequence required for DNA binding as well as a putative nuclear localization sequence. However, HspBPl does not contain these features and therefore further research is needed to define the regions of the molecule that bind DNA.
HspBPl lacks clear amino acid homologies to other proteins. However, structural similarity could provide some insight into function. The program 3D-PSSM (Kelley, L.A., MacCallum, R.M., Sternberg, J.E. Enhanced genome annotation using strucuture profiles in the program 3D- PSSM. J. Mol. Biol. 299, 499-520 (2000)) was used to predict the 3-dimensional structure of HspBPl. The results (Fig. 6) indicated with a greater than 95% confidence level that HspBPl has a structure similar to karyopherin o. and importin α which are in the armadillo repeat family.
Both karyopherin o. and importin o. are nuclear transporter proteins that bind to the NLS (nuclear localization signal) of proteins and transport these proteins to the nucleus . We report here that HspBPl excludes plasmid from the nucleus, therefore, a predicted structure that is related to other nuclear transport proteins is consistent with our results. There are also proteins involved with export of proteins and tRNA from the nucleus (reviewed by Gόrlich, D. and Kutay, U. Transport between the cell nucleus and the cytoplasm. Ann. Rev. Cell Dev. Biol. 15: 607-660 (1999)), so it is possible that HspBPl might be exporting plasmid rather than inhibiting uptake.
The questions that are raised by this structural similarity disclosed herein revolve around the need to identify a binding site on HspBPl that interacts with DNA and the need to determine if this interaction has physiological importance in the cell. One possibility is that HspBPl can serve as an endogenous protective mechanism to prevent foreign genetic material from entering the nucleus. This mechanism would, for example, prevent viral genomes from entering the nucleus . Of course, such activity would also inhibit the efficacy of desirable gene uptake, such as through gene replacement therapy. Finally, the identification of a protein that promotes exclusion of genetic material from the nucleus is a novel finding that may provide insight into the variation of transfection efficiencies among various cell types.
Example 2 : HspBPl and Inhibition of Viral Infection
One area of research for the development of new therapies for viral infection that has received little attention is the transport of the viral genome into the nucleus (reviewed in Whittaker, G.R., Kann, M., and Helenius, A. 2000. Viral Entry into the Nucleus. Ann. Rev. Cell Dev. Biol. 16: 627-651). In fact, many viruses replicate in the nucleus of their host cell, and therefore must transport their genome into this organelle. Accordingly, the "nuclear exclusion" (prevention of nuclear uptake of extra-nuclear genetic material) activity of HspBPl can be used to inhibit viral infection by preventing the viral genome from entering the nucleus of the infected celjL .
One particularly attractive target for HspBPl is the HIV virus. The ability of HIV to transport its preintegration complex (PIC) into the nucleus of an infected cell during the interphase is one unique feature of this virus that separates it from the other retroviruses, which rely on the breakdown of the nuclear envelope during mitosis for delivery of their genome into the nucleus. Moreover, there is evidence that nuclear import is critical for HIV replication in non-dividing cells, such as macrophages, as well as in slowly dividing populations, such as primary T lymphocytes . Recent evidence has shown that the import of the PIC is dependent on the HIV-encoded protein Vpr. Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M. A., Ratner, L., Lane, C. M., Moore, M. S., Blobel, G., and Bukrinsky, M. Viral protein R regulates nuclear import of the HIV-1 pre -integration complex. EMBO J. 17,909B917 (1998) . Vpr is a PIC protein that can associate with the nuclear import molecule karyopherin α in the cell .
Another viral protein that is part of the PIC is matrix protein (MA) . MA contains a nuclear localization sequence (NLS) that can bind to karyopherin α, and, thus, facilitate the transport of the PIC into the nucleus. The binding of Vpr to karopherin o. has been shown to increase the affinity of karopherin o. for the NLS, which then promotes nuclear import.
Recently, the cellular molecular chaperone Hsp70 has been shown to replace Vpr of HIV during nuclear import of the PIC. Agostini et al . Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex. Exp. Cell Res. 259: (2)398- 403 (2000) . Hsp70 and Vpr bind to a the amino-terminal portion of karopherin ct . Karyopherin α then has a region that can bind Hsp70 or Vpr and this binding stimulates the interaction between PIC and karopherin α . Since HspBPl binds to and inhibits the activity of Hsp70 (Raynes, D. and Guerriero, V. Inhibi tion of Hsp70 ATPase Activi ty and protein renaturation by a Novel Hsp70-binding Protein . J. Biol. Chem. 273: 883 (1998); Raynes, D.A. and Guerriero, V. Isolation and Characterization of Iso forms HspBPl, an Inhibi tor of Hsp70. Biochimica et Biophysica Acta 5 1490:203-207(2000)), HspBPl can be used to inhibit the import of the PIC into the nucleus. In addition, Hsc70 (this protein is very similar to Hsp70 and also binds HspBPl) is required for the import of adenovirus DNA into the nucleus. Saphire, A.C., Guan, T., Schirmer, E.C., 10 Nemerow, G.R., and Gerace, L. Nuclear Import of Adenovirus DNA In Vitro Involves the Nuclear Protein Import Pathway and Hsc70. J. Biol. Chem. 275:4298-4304(1999).
It has also been shown that cells containing high levels 15 of Hsp70 support increased viral gene expression and cytopathic effects. Vasconcelos DY, Cai XH, Oglesbee MJ. Constitutive overe pression of the major inducible 70 kDa heat shock protein mediates large plaque formation by measles virus . Journal of General Virology. 79: 2239- 202247(1998). Finally, the activation of the heat-shock response by an adenovirus is essential for viral replication. Glotzer JB, Saltik M, Chiocca S, Michou Al, Moseley P, Cotten M. Activation of heat-shock response by an adenovirus is essential for virus replication . 25 Nature 407:207-211(2000) .
Structure prediction tools (see example 1) indicate with a greater than 95% confidence level that HspBPl has a structure similar to karyopherin and importin which are in
30 the armadillo repeat family (Fig. 6) . Both of these proteins are nuclear transporter proteins that bind to the NLS of proteins and transport these proteins to the nucleus. Thus, over-expression of HspBPl should exclude viral genomes from the nucleus since the predicted
35 structure that is related to other nuclear transport proteins strongly suggests that HspBPl can regulate nuclear transport. Accordingly, HspBPl is a novel target for the development of an anti-viral drug. The fact that HspBPl inhibits extra-nuclear genetic material uptake strongly suggests that this protein can inhibit the uptake of viral nucleic acid into the nucleus .
Example 3: HspBPl Inhibition as an Adjuvant for Gene Replacement Therapy
Many people are born with genetic diseases that are caused by defective genes . The normal course of action is to treat the symptoms when possible. This is a temporary solution for the patient and therefore requires constant medical attention. The decoding of the human genome is a major milestone that will increase the potential for a new type of therapy called gene replacement. The overall goal will be to insert a normal gene into the patient' s genome to replace the defective gene that is causing the symptoms of the disease. The anticipated result would be permanent cure, and, therefore, no further treatment would be required.
A major technical block to gene replacement therapy is the method to introduce the normal gene into the recipient' s genome. The gene must enter the cell and then be delivered to the nucleus where the cell' s genetic material, DNA, is stored. Currently, much work is being done using viruses to accomplish this task. These viruses have been modified in an attempt to make them harmless to the patient. However, recent trials have indicated that the patient' s immune system responds very dramatically to these introduced viruses, having potentially very negative effects. Marshall, E. Gene Therapy on Trial . Science 288:951-972(2000) .
It may be possible to deliver the new gene using an approach that does not require viruses . Non-viral vectors may offer a solution to this problem. Plasmids are small pieces of DNA that can be introduced into cells but do not contains proteins like viruses that can cause an immune response. If plasmids could be used for gene replacement therapy, then the problems associated with viruses would be eliminated. The plasmid DNA would contain the corrected gene and deliver the gene to the nucleus of the cells where is would become incorporated into the cell' s genome. A major block in this process is delivering the plasmid DNA to nucleus. In some cells, this process is very efficient, yet in others it is practically impossible. Very little information is available on the mechanism of plasmid DNA uptake into the nucleus and how this process is regulated. See Dean DA, Dean BS, Muller S, Smith LC. Sequence requirements for plasmid nuclear import. Exper. Cell Res. 253:713-722 (1999); Wilson GL, Dean BS, Wang G, Dean DA. Nuclear import of plasmid DNA in digi tonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences . J. Biol. Chem. 274: 22025-22032(1999) .
As disclosed herein, it has been discovered that HspBPl has the ability to prevent plasmid DNA from going into the nucleus . This is the first time a protein has been identified with this activity, and, therefore, has opened the door to blocking this endogenous activity to facilitate plasmid uptake into the nucleus . It is possible that the endogenous expression of this protein in various cells and tissues prevents or hinders plasmid uptake. Accordingly, lowering the endogenous levels of HspBPl in cells would allow an increase in nuclear plasmid uptake. Additionally, lowering the endogenous levels of HspBPl may also facilitate viral genome uptake into cells and lower the amount of virus that is used in gene replacement therapy. A lower viral dose would decrease the chance for an immune response. Thus, expressing the anti-sense polynμcT.eg^ides of HspBPl as, for example, described in example 1, would up-regulate the expression of extra-nuclear genetic material .
While these examples are contemplated to illustrate the preferred modes of practicing the invention, it will be understood by those in the art that numerous alternative methodologies may be successfully practiced in lieu of the preferred method described herein.

Claims

We claim:
1. A method of regulating expression of extra-nuclear genetic material in eukaryotic cells, comprising:
(a) providing said eukaryotic cells with an
5 expression vector harboring a polynucleotide sequence encoding HspBPl, and
(b) expressing the polynucleotide sequence of step (a) .
102. The method of claim l, wherein said extra-nuclear genetic material comprises a transfected plasmid.
3. The method of claim 1, wherein said extra-nuclear genetic material comprises a genome of a virus.
15
4. The method of claim 3, wherein said virus is selected from a group consisting of human immunodeficiency virus, adenovirus, and measles virus.
20 5. The method of claim 1, wherein HspBPl is constitutively expressed in step (b) .
6. The method of claim 1, wherein HspBPl is conditionally expressed in step (b) .
25
7. The method of claim 1, wherein said polynucleotide sequence of step (a) comprises SEQ ID NO: 2.
8. The method of claim 1, wherein said polynucleotide 30 sequence of step (a) comprises an anti-sense sequence of
SEQ ID NO: 2.
9. The method of claim 8, wherein said anti-sense sequence comprises an adjuvant to nuclear uptake of
35 nucleic acids during gene replacement therapy.
10. The method of claim l, wherein the expression of the polynucleotide sequence of step (b) results in a HspBPl polypeptide.
11. The method of claim 10, wherein the HspBPl 5 polypeptide comprises SEQ ID NO:l.
12. A method of inhibiting expression of extra-nuclear genetic material in eukaryotic cells, comprising the step of providing said eukaryotic cells with a HspBPl
10 polypeptide sufficient to inhibit said expression.
13. The method of claim 12, wherein said extra-nuclear genetic material comprises a plasmid.
1514. The method of claim 12, wherein said extra-nuclear genetic material comprises a genome of a virus.
15. The method of claim 14 , wherein said virus is selected from the group consisting of human
20 immunodeficiency virus, adenovirus, or measles virus.
16. The method of claim 12, wherein a HspBPl polypeptide is provided to said eukaryotic cells through an expression vector harboring polynucleotides encoding HSPBPl.
25
17. The method of claim 16, wherein said polynucleotides comprise SEQ ID N0:2.
18. The method of claim 12, wherein a HspBPl polypeptide 30 comprises a pharmaceutical composition including HspBPl in conjunction with a suitable pharmaceutical carrier.
19. A substantially purified human HspBPl polypeptide or immunologically active fragments thereof .
35
20. The polypeptide of claim 19, wherein said HspBPl polypeptide comprises SEQ ID NO:l.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014100883A1 (en) 2012-12-28 2014-07-03 União Brasileira De Educação E Assistência, Mantenedora Da Pucrs Hsp70 binding compound, use, antitumour pharmaceutical composition, gene construct for expressing an hsp70 binder, production process, process for evaluating tumour cells, method of sensitization of tumour cells to chemotherapeutic drugs

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2000031114A1 (en) * 1998-11-20 2000-06-02 Vincent Guerriero DNA ENCODING PROTEINS THAT INHIBIT Hsp70 FUNCTION

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Publication number Priority date Publication date Assignee Title
WO2000031114A1 (en) * 1998-11-20 2000-06-02 Vincent Guerriero DNA ENCODING PROTEINS THAT INHIBIT Hsp70 FUNCTION

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014100883A1 (en) 2012-12-28 2014-07-03 União Brasileira De Educação E Assistência, Mantenedora Da Pucrs Hsp70 binding compound, use, antitumour pharmaceutical composition, gene construct for expressing an hsp70 binder, production process, process for evaluating tumour cells, method of sensitization of tumour cells to chemotherapeutic drugs
EP3459967A1 (en) 2012-12-28 2019-03-27 União Brasileira De Educaçao E Assistência- Mantenedora Da Pucrs Use of an hspbp1 fragment as antitumor agent and for sensitization of tumour cells to chemotherapeutic drugs
US10508139B2 (en) 2012-12-28 2019-12-17 União Brasileira De Educação E Assistência, Mantenedora Da Pucrs Compound, use, anti-tumor pharmaceutical composition, gene construct for polypeptide expression, process for the production, process for the evaluation of tumor cells, method of sensitization of tumor cells to chemotherapeutic

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