Cell death agonists

Abstract

A Bcl-2 associated protein (Bax) and uses thereof.

What is claimed is:  
     
         1 . A purified and isolated Bcl-2 associated protein (Bax).  
     
     
         2 . The protein of  claim 1 , wherein the protein is a homodimer.  
     
     
         3 . The protein of  claim 1 , wherein the protein has the amino acid sequence shown in FIGS. 3, 5,  6  or  7 .  
     
     
         4 . The protein of  claim 1 , wherein the protein has a 21 kD and a sequence of 192 amino acids.  
     
     
         5 . A purified and isolated protein fragment having the amino acid sequence of Domain I or Domain II of FIG. 7.  
     
     
         6 . The protein of  claim 5 , wherein the fragment is present in Bax.  
     
     
         7 . The protein of  claim 5 , wherein the fragment is present in Bcl-2.  
     
     
         8 . The protein of  claim 5 , wherein at least one amino acid position in Domain I or II is deleted or replaced by another amino acid.  
     
     
         9 . The protein of  claim 8 , wherein the glycine present in the amino acid sequence NWGR is replaced with alanine or glutamic acid.  
     
     
         10 . The protein of  claim 5 , wherein at least one amino acid position in Domain I or II is deleted or replaced by a hydrocarbon.  
     
     
         11 . The protein of  claim 10 , wherein the hydrocarbon is selected from the group consisting of a straight or branched chain C6-12 alkyl group, a five or six membered, substituted or unsubstituted cyclic hydrocarbon or heterocyclic ring system and mixtures thereof.  
     
     
         12 . An associated protein complex, which comprises: Bax protein coupled with Bcl-2.  
     
     
         13 . The associated protein of  claim 12 , wherein the protein complex is in the form of a heterodimer.  
     
     
         14 . The associated protein complex of  claim 12 , wherein the protein complex is composed of a protein according to  claim 5 .  
     
     
         15 . A DNA isolate consisting essentially of a DNA sequence encoding Bax.  
     
     
         16 . A DNA isolate consisting essentially of a DNA sequence encoding a fragment of Bax or Bcl-2, wherein the fragment is a protein according to  claim 5 .  
     
     
         17 . A composition consisting of DNA molecules which encode the Bax protein.  
     
     
         18 . A composition consisting of DNA molecules which encode a fragment of the protein according to  claim 5 .  
     
     
         19 . A pharmaceutical composition containing the protein Bax, which comprises: pharmaceutically effective amounts of Bax and a pharmaceutically suitable carrier.  
     
     
         20 . A purified and isolated human Bax protein having seven Ser/Thr residues.  
     
     
         21 . A purified and isolated murine Bax protein having seven Ser/Thr residues.  
     
     
         22 . A composition consisting of αRNA molecules which encode a protein having 192 amino acids and being a 21 kD Bax protein.  
     
     
         23 . A composition consisting of βRNA molecules which encode a protein having 218 amino acids and being a 24 kD Bax protein.  
     
     
         24 . The composition of  claim 23 , wherein the protein lacks a hydrophobic terminus and is cytosolic in form.  
     
     
         25 . A composition consisting of 1.0 kb and 1.5 kb γRNA molecules which encode a 4.5 kD Bax protein having 41 amino acids.  
     
     
         26 . A cell line which contains a 1.0 kb α RNA molecule which expresses a 21 kD Bax protein.  
     
     
         27 . The cell line of  claim 26 , wherein the protein is associated with Bcl-2.  
     
     
         28 . The cell line of  claim 26 , wherein the cell line is FL5.12.  
     
     
         29 . A cell line which contains 1.0 kb α and 1.5 kb β RNA molecules which expresses a 21 kD and 24 kD protein.  
     
     
         30 . The cell line of  claim 29  wherein the proteins are associated with Bcl-2.  
     
     
         31 . The cell line of  claim 29  wherein the cell line is RL 7.  
     
     
         32 . A method for controlling the cell death repressor activity of Bcl-2, which comprises: adding an effective amount of a Bax protein to a cell containing Bcl-2 activity to enable the formation of an associated heterodimer comprising Bax-Bcl-2.  
     
     
         33 . The method of  claim 32 , wherein the Bax or Bcl-2 protein is a fragment having the amino acid sequence of Domain I or II of FIG. 7.  
     
     
         34 . The method of  claim 33 , wherein the fragment is present in Bax.  
     
     
         35 . The method of  claim 33 , wherein the fragment is present in Bcl-2.  
     
     
         36 . The method of  claim 33 , wherein at least one amino acid position in Domain I or II is deleted or replaced by another amino acid.  
     
     
         37 . The method of  claim 36 , wherein the glycine present in the amino acid sequence NWGR is replaced with alanine or glutamic acid.  
     
     
         38 . The method of  claim 33 , wherein at least one amino acid position in Domain I or II is deleted or replaced by a hydrocarbon.  
     
     
         39 . The method of  claim 38 , wherein the hydrocarbon is selected from the group consisting of a straight or branched chain C6-12 alkyl group, a five or six membered, substituted or unsubstituted cyclic hydrocarbon or heterocyclic ring system, and mixture thereof.  
     
     
         40 . A method for assaying for the predisposition for an apoptotic cell death, which comprises: collecting a specimen to be tested; contacting the specimen with a material reactive with a Bcl-2 or Bax protein; and determining the presence or absence of Bcl-2 or Bax protein and their ratio in the specimen.  
     
     
         41 . The method of  claim 40 , wherein the assay is a labeled assay using an EIA, ELISA or RIA tag.  
     
     
         42 . The method of  claim 40 , wherein the specimen collected is a tissue specimen.  
     
     
         43 . The method of  claim 42 , wherein the tissue specimen is selected from the group consisting of lung, stomach, heart, spleen, smooth muscle, intestine, pancreas, liver, brain, lymphnode, thymus, skin, kidney, hair follicle, breast or prostate tissue.  
     
     
         44 . The use of the protein Bax as claimed in  claim 1  for performing immunochemical methods for the detection and determination of the protein or its associated protein Bcl-2, in order to monitor cell growth or to detect or monitor the course of diseases.  
     
     
         45 . The use of the protein Bax as claimed in  claim 5  for performing immunochemical methods for the detection and determination of the protein or its associated protein Bcl-2, in order to monitor cell growth or to detect or monitor the course of diseases.  
     
     
         46 . A method for the treatment of a neurodegenerative disease, an immunodeficiency, or an ischemia, which comprises: administering an effective amount of an agent to a patient which regulates the ratio of Bcl-2 to Bax to promote the survival of cells.  
     
     
         47 . The method of  claim 46 , wherein the agent is selected from the group consisting of Bax, domain I, domain II, fragments thereof, analogs thereof and mixtures thereof.  
     
     
         48 . A method for the treatment of hyperplasias, hypertrophies, cancers and autoimmunity disorders, which comprises: administering an effective amount of an agent to a patient to regulate the ratio of Bcl-2 to Bax so as to favor the Bax in the ratio and promote cell death.  
     
     
         49 . The method of  claim 48 , wherein the agent is selected from the group consisting of Bax, domain I, domain II, fragments thereof, analogues thereof, and mixtures thereof.  
     
     
         50 . An isolated Bax polypeptide comprising a polypeptide sequence of at least 6 consecutive amino acids which is substantially identical to a polypeptide sequence shown in FIG. 3, 5,  6  or  7 .  
     
     
         51 . The isolated Bax polypeptide of  claim 50 , wherein the Bax polypeptide comprises domain I and domain II.  
     
     
         52 . The isolated Bax polypeptide of  claim 50 , which is approximately 218 to 192 amino acids long and is substantially identical to a naturally-occurring full-length Bax polypeptide.  
     
     
         53 . The isolated Bax polypeptide of  claim 50  which is a naturally-occurring polypeptide sequence in mammals.  
     
     
         54 . The isolated Bax polypeptide of  claim 53  which is a human Bax polypeptide.  
     
     
         55 . The isolated Bax polypeptide of  claim 54 , which is approximately 192 amino acids long and is substantially identical to the full-length Bax polypeptide sequence shown in FIG. 3.  
     
     
         56 . The isolated Bax polypeptide of  claim 50 , wherein said polypeptide comprises the 192 amino acid long human Bax sequence shown in FIG. 3.  
     
     
         57 . An isolated polynucleotide encoding a Lyar polypeptide of  claim 50 .  
     
     
         58 . The isolated polynucleotide of  claim 57 , comprising a nucleotide sequence encoding the 192 residue polypeptide sequence shown in FIG. 3.  
     
     
         59 . An isolated Bax polynucleotide comprising a polynucleotide sequence that is substantially identical to a naturally-occurring human or murine Bax polynucleotide sequence.  
     
     
         60 . The polynucleotide according to  claim 59 , wherein the naturally-occurring polynucleotide sequence is a sequence of at least 25 consecutive polynucleotides shown in FIG. 3 or FIG. 5 or  6 .  
     
     
         61 . The polynucleotide according to  claim 60  which encodes a Bax polypeptide.  
     
     
         62 . The isolated polynucleotide of  claim 61 , further comprising an operably linked transcriptional regulatory sequence.  
     
     
         63 . An isolated nonhuman mammalian cell comprising a polynucleotide encoding a human Bax polypeptide.  
     
     
         64 . A method for identifying candidate Bax modulatory agents comprising: 
 combining in aqueous binding conditions an agent, a Bax polypeptide capable of binding to a Bcl-2 polypeptide under the aqueous binding conditions, a Bcl-2 polypeptide capable of binding to a Bax polypeptide under the aqueous binding conditions;    determining whether the agent inhibits formation of Bax/Bcl-2 heterodimers; and    identifying agents which inhibit formation of Bax/Bcl-2 heterodimers as candidate Bax modulatory agents.    
     
     
         65 . The method of  claim 64 , wherein the Bax polypeptide is a full-length human Bax α isoform polypeptide or a full-length human Bax β isoform.  
     
     
         66 . The method of  claim 64 , wherein the Bcl-2 polypeptide is a full-length human Bcl-2 polypeptide.  
     
     
         67 . A method for identifying candidate Bax modulatory agents, comprising: 
 combining in aqueous binding conditions an agent, a Bax polypeptide capable of binding to a Bax polypeptide forming a Bax/Bax homodimer under the aqueous binding conditions; and    determining whether the agent inhibits formation of Bax/Bax homodimers; and    identifying agents which inhibit formation of Bax/Bax homodimers as candidate Bax modulatory agents.    
     
     
         68 . The method of  claim 67 , wherein the Bax polypeptide is a full-length human Bax α isoform polypeptide or a full-length human Bax β isoform.  
     
     
         69 . The method according to  claim 67 , wherein the candidate Bax modulatory agent is not cytotoxic to mammalian cells which do not express detectable amounts of Bax mRNA or protein.  
     
     
         70 . An antisense polynucleotide comprising a polynucleotide that is complementary to a sequence of at least 30 nucleotides that is substantially identical to a sequence shown in FIG. 3, 5,  6  or FIG. 7.  
     
     
         71 . A human or murine Bax protein encoded by a recombinant polypeptide in a host cell.  
     
     
         72 . An isolated host cell comprising an extrachromosomal polynucleotide comprising a human or murine Bax encoding sequence.  
     
     
         73 . A composition comprising a substantially pure protein complex comprising a Bax polypeptide and a Bcl-2 polypeptide.  
     
     
         74 . The composition of  claim 73 , wherein the protein complex comprises a mammalian Bax and mammalian Bcl-2 and the complex is formed in a yeast cell.  
     
     
         75 . The composition of  claim 73 , wherein the Bax polypeptide is shorter than a naturally-occurring Bax polypeptide.  
     
     
         76 . The composition of  claim 73 , wherein the Bcl-2 polypeptide is shorter than a naturally-occurring Bcl-2 polypeptide.  
     
     
         77 . A composition comprising a protein complex comprising a Bax polypeptide and a Bcl-2 polypeptide, wherein said Bax polypeptide and Bcl-2 polypeptide are encoded by a recombinant polynucleotide expressed in a host cell.  
     
     
         78 . A method of screening for candidate Bax-modulating agents, comprising: 
 performing a heterodimerization assay which includes a Bax polypeptide with a Bcl-2 polypeptide and an agent; and    determining whether the agent inhibits or augments the heterodimerization.    
     
     
         79 . The method of  claim 78 , wherein the heterodimerization assay comprises an in vitro binding reaction.  
     
     
         80 . The method of  claim 78 , wherein the heterodimerization assay comprises a two-hybrid assay in yeast or an  E. coli /BCCP interactive system.  
     
     
         81 . The method of  claim 80 , wherein the two-hybrid assay comprises a human Bax polypeptide sequence fused to a GAL4 protein and a human Bcl-2 polypeptide sequence fused to a GAL4 protein.  
     
     
         82 . The method of  claim 78 , wherein a Bax or Bcl-2 polypeptide is immobilized.  
     
     
         83 . The method of  claim 78 , wherein the agent is a peptide having the sequence of NWGR of Domain I or a compound derived by molecular modeling of this sequence.  
     
     
         84 . A method for altering apoptosis of a cell, comprising administering to a cell an agent that inhibits Bax/Bcl-2 heterodimer formation.  
     
     
         85 . The method of  claim 84 , wherein said cell is derived from the hematopoietic lineage, neural lineage, epithelial lineage, or mesodermal lineage.  
     
     
         86 . The method of  claim 84 , wherein the cell is a neoplastic cell.  
     
     
         87 . The method of  claim 85 , wherein the cell is a leukocyte, a nerve, a hormore response epithelial cell, or a muscle cell.  
     
     
         88 . A transgenic nonhuman animal comprising a transgene encoding a Bax polypeptide.  
     
     
         89 . A host cell comprising a polynucleotide encoding a Bax polypeptide and capable of expressing a Bax polypeptide.  
     
     
         90 . A substantially purified Bax polypeptide isolated from a host cell of  claim 89 .  
     
     
         91 . A Bax polypeptide labeled with a radioisotope or fluorescent label.  
     
     
         92 . A method for identifying candidate Bax modulatory agents, comprising: 
 combining in aqueous binding conditions an agent, a Bax polypeptide capable of binding to a Bax polypeptide forming a Bax/Bax homodimer under the aqueous binding conditions;    determining whether the agent inhibits formation of Bax/Bax homodimers;    combining in aqueous binding conditions an agent, a Bax polypeptide capable of binding to a Bcl-2 polypeptide under the aqueous binding conditions, a Bcl-2 polypeptide capable of binding to a Bax polypeptide under the aqueous binding conditions;    determining whether the agent inhibits or augments the formation of Bax/Bcl-2 heterodimers; and    identifying agents which preferentially inhibit formation of Bax/Bcl-2 heterodimers relative to Bax/Bax homodimers as candidate Bax/Bcl-2 modulatory agents.    
     
     
         93 . The method of  claim 92 , wherein the binding of Bcl-2 to Bax requires domain I or domain II.  
     
     
         94 . The Bax/Bcl-2 modulatory agent identified by a method of  claim 92 .  
     
     
         95 . The Bax/Bcl-2 modulatory agent of  claim 94 , wherein the Bax/Bcl-2 modulatory agent, when administered to a cell stimulated to undergo apoptosis and expressing Bcl-2, inhibits the death repressing activity of Bcl-2 and thereby induces apoptosis.  
     
     
         96 . A method for treating a disease comprising administering an agent that modulates apoptosis, said method comprising the step of administering an efficacious dose of an agent that modulates Bax/Bcl-2 heterodimer formation.  
     
     
         97 . The method of  claim 96 , wherein said disease is neurodegencrative disease, inflammatory disease, neoplasia, hyperplasia, autoimmune disease, or a lymphoproliferative disease.  
     
     
         98 . The method of  claim 96 , wherein said agent inhibits formation of Bax/Bcl-2 heterodimers.  
     
     
         99 . A method of treating a disease comprising administering an agent that modulates apoptosis, said method comprising the step of administering an efficacious dose of an agent that modulates Bax/Bax homodimer formation.  
     
     
         100 . The method of  claim 99 , wherein said disease is neurodegenerative disease, inflammatory disease, neoplasia, hyperplasia, autoimmune disease, or a lymphoproliferative disease.  
     
     
         101 . The method of  claim 99 , wherein said agent inhibits formation of Bax/Bcl-2 heterodimers.  
     
     
         102 . A method for treating a disease comprising administering an agent that modulates apoptosis, said method comprising the step of administering an efficacious dose of an agent that modulates the ratio of Bax protein to Bcl-2 protein in a cell.  
     
     
         103 . The method of  claim 102 , wherein said disease is neurodegenerative disease, inflammatory disease, neoplasia, hyperplasia, autoimmune disease, or a lymphoproliferative disease.  
     
     
         104 . The method of  claim 102 , wherein said agent inhibits formation of Bax/Bcl-2 heterodimers.  
     
     
         105 . The method of  claim 102 , wherein said agent is an antisense polymucleotide or a plynucleotide encoding a Bax polypeptide.  
     
     
         106 . A method of identifying a Bax-interacting protein, said method comprising the step of immunprecipitating Bax protein from a cell extract and identifying an accessory protein bound to Bax.  
     
     
         107 . The method of  claim 106 , wherein a chemical crosslinking agent is added prior to immunoprecipitating.  
     
     
         108 . A method of identifying a cDNA sequence encoding a Baxinteracting protein, said method comprising the step of expressing in a yeast host a yeast two-hybrid system wherein a first GAL4 hybrid comprises a fusion sequence comprising a Bax sequence comprising domain I or domain II linked to a GAL4 activator domain and a second GAL4 hybrid comprises a fusion sequence comprising a cDNA library member sequence linked to a GAL4 DNA-binding domain.  
     
     
         109 . A method of identifying a cDNA sequence encoding a Baxinteracting protein, said method comprising the step of expressing in a yeast host a yeast two-hybrid system wherein a first GAL4 hybrid comprises a fusion sequence comprising a Bax sequence comprising domain I or domain II linked to a GAL4 DNAbinding domain and a second GAL4 hybrid comprises a fusion sequence comprising a cDNA library member sequence linked to a GAL4 activator domain.  
     
     
         110 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide is complementary to a sequence having at least 85% sequence identity to SEQ ID NOs: 1, 4, or 6.  
     
     
         111 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide is complementary to a sequence comprising SEQ ID NOs: 1, 4, or 6.  
     
     
         112 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide is complementary to a sequence consisting essentially of SEQ ID NOs: 1, 4, or 6.  
     
     
         113 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide is complementary to a sequence consisting of SEQ ID NOs: 1, 4, or 6.  
     
     
         114 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide is SEQ ID NOs: 32, 33, or 34.  
     
     
         115 . (New) The antisense polynucleotide of  claim 70  comprising a polynucleotide that is complementary to a sequence that encodes a polypeptide comprising SEQ ID NOs: 2, 3, 5, 7, 8, or 9.  
     
     
         116 . (New) The antisense polynucleotide of  claim 70 , wherein the polynucleotide inhibits production of a polypeptide comprising SEQ ID NOs: 2, 3, 5, 7, 8, or 9.  
     
     
         117 . (New) An antisense polynucleotide comprising a polynucleotide of at least 25 consecutive nucleotides that specifically hybridizes to a sequence having at least 85% sequence identity to SEQ ID NOs: 1, 4, or 6.  
     
     
         118 . The antisense polynucleotide of  claim 117 , wherein the sequence comprises SEQ ID NOs: 1, 4, or 6.  
     
     
         119 . (New) The antisense polynucleotide of  claim 117 , wherein the sequence consists essentially of SEQ ID NOs: 1, 4, or 6.  
     
     
         120 . (New) The antisense polynucleotide of  claim 117 , wherein the sequence consists of SEQ ID NOs: 1, 4, or 6.  
     
     
         121 . (New) The antisense polynucleotide of  claim 117 , wherein the polynucleotide is SEQ ID NOs: 32, 33, or 34.  
     
     
         122 . (New) The antisense polynucleotide of  claim 117 , wherein the polynucleotide is complementary to a sequence that encodes a polypeptide comprising SEQ ID NOs: 2, 3, 5, 7, 8, or 9.  
     
     
         123 . (New) The antisense polynucleotide of  claim 117 , wherein the polynucleotide inhibits production of a polypeptide comprising SEQ ID NOs: 2, 3, 5, 7, 8, or 9.  
     
     
         124 . (New) A composition comprising the antisense polynucleotide of  claim 70  and a pharmaceutical acceptable carrier.  
     
     
         125 . (New) A composition comprising the antisense polynucleotide of  claim 117  and a pharmaceutical acceptable carrier.