WO2001081545A2 - Regulation de l'expression genique par hspbp1 - Google Patents

Regulation de l'expression genique par hspbp1 Download PDF

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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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hspbpl
expression
protein
hsp70
extra
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PCT/US2001/013422
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WO2001081545A3 (fr
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Vincent Guerriero
Deborah A. Raynes
Luke J. Whitesell
Meghan E. Kreeger
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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/fr
Publication of WO2001081545A3 publication Critical patent/WO2001081545A3/fr

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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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  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la découverte d'une nouvelle activité régulatrice de l'expression génique pour la protéine de liaison de la protéine du stress HspBP1, SEQ ID NO :1 (fig.1). L'invention porte, plus spécifiquement sur un procédé de production de polynucléotides (SEQ ID NO:2) et de polypeptides (SEQ ID NO:1), pour la régulation de l'expression du matériel génétique extra-nucléaire dans des cellules eucaryotes.
PCT/US2001/013422 1999-11-19 2001-04-27 Regulation de l'expression genique par hspbp1 WO2001081545A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001255697A AU2001255697A1 (en) 2000-04-27 2001-04-27 Regulation of gene expression by hspbp1

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/444,336 US6410713B1 (en) 1998-11-20 1999-11-19 DNA encoding proteins that inhibit Hsp70 function
US20008300P 2000-04-27 2000-04-27
US60/200,083 2000-04-27

Publications (2)

Publication Number Publication Date
WO2001081545A2 true WO2001081545A2 (fr) 2001-11-01
WO2001081545A3 WO2001081545A3 (fr) 2002-03-21

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WO (1) WO2001081545A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014100883A1 (fr) 2012-12-28 2014-07-03 União Brasileira De Educação E Assistência, Mantenedora Da Pucrs Composé ligand de hsp70, utilisation, composition pharmaceutique antitumorale, construction génétique pour l'expression d'un ligand de hsp70, procédé de production, procédé d'évaluation de cellules tumorales, méthode de sensibilisation de cellules tumorales, méthode de sensibilisation de cellules tumorales à des agents chimiothérapeutiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031114A1 (fr) * 1998-11-20 2000-06-02 Vincent Guerriero PROTEINES CODANT POUR L'ADN QUI INHIBENT LA FONCTION Hsp70

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031114A1 (fr) * 1998-11-20 2000-06-02 Vincent Guerriero PROTEINES CODANT POUR L'ADN QUI INHIBENT LA FONCTION Hsp70

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAYNES ET AL.: 'Inhibition of Hsp70 ATPase activity and protien renaturation by a novel HSP70-binding protein' J. BIOLOGICAL CHEMISTRY vol. 273, no. 49, 04 December 1998, pages 32883 - 32888, XP002947011 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014100883A1 (fr) 2012-12-28 2014-07-03 União Brasileira De Educação E Assistência, Mantenedora Da Pucrs Composé ligand de hsp70, utilisation, composition pharmaceutique antitumorale, construction génétique pour l'expression d'un ligand de hsp70, procédé de production, procédé d'évaluation de cellules tumorales, méthode de sensibilisation de cellules tumorales, méthode de sensibilisation de cellules tumorales à des agents chimiothérapeutiques
EP3459967A1 (fr) 2012-12-28 2019-03-27 União Brasileira De Educaçao E Assistência- Mantenedora Da Pucrs Fragment de hspbp1 comme agent antitumoral et pour la sensibilisation de cellules tumorales à des agents chimiothérapeutiques
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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