Cell death regulator
AssigneeWashington University
InventorStanley J. Korsmeyer
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Abstract
The invention provides a bcl-2 related protein, bcl-2 muteins, and uses thereof.
Claims21
A method for producing a mutant bcl-2 protein that substantially lacks death repressor activity and/or substantially lacks binding to Bax, said method comprising the steps of;
- expressing in a cell or translation reaction a polynucleotide encoding a mutant bcl-2 protein comprising a polypeptide sequence which is substantially identical to a naturally-occurring bcl-2 protein and which has an amino acid substitution or deletion in a BH1 or BH2 domain, whereby the mutant bcl-2 protein is substantially incapable of binding to Bax and/or is substantially incapable of death repressor activity.
A method of identifying candidate bcl-2-modulating agents, comprising:
- performing a heterodimerization assay which includes a bcl-2 polypeptide comprising a BH1 and/or BH2 domain with a Bax polypeptide and an agent;
- determining whether the agent inhibits heterodimerization of the bcl-2 polypeptide to the Bax polypeptide;
- identifying agents which inhibit said heterodimerization as candidate bcl-2 modulating agents which inhibit death repressor activity of bcl-2.
A mutant bcl-2 polypeptide comprising an amino acid substitution or deletion in a BH-1 or BH-2 domain, wherein amino acid substitution or deletion is in an amino acid postion corresponding to amino acid residues 136-155 or 187-202 of SEQ ID NO:10, as aligned in FIG. 22(a-c), whereby the mutant bcl-2 protein is substantially incapable of binding to Bax and/or is substantially incapable of death repressor activity.
A method for inhibiting the cell death repressor activity of bcl-2, said method comprising the steps of:
- introducing a mutation into a bcl-2 polynucleotide encoding a bcl-2 polypeptide to produce a mutated bcl-2 polynucleotide, whereby said mutated bcl-2 polynucleotide encodes a mutant bcl-2 polypeptide comprising an amino acid substitution or deletion in a BH1 or BH2 domain, and wherein said mutant bcl-2 polypeptide substantially lacks binding to Bax and/or substantially lacks death repressor activity.
A method for identifying a mutant bcl-2 comprising an amino acid substitution or deletion at an amino acid position of a BH1 or BH2 domain, said method comprising obtaining a sample containing a bcl-2 protein and detecting whether the bcl-2 protein has a substitution at a position in a BH1 or BH2 domain relative to naturally-occurring bcl-2 protein.
A method for identifying mutant bcl-2 proteins which substantially lack binding to Bax and/or substantially lack death repressor activity, said method comprising the steps of:
- introducing a mutation into a bcl-2 polynucleotide encoding a bcl-2 polypeptide to produce a mutated bcl-2 polynucleotide, whereby said mutated bcl-2 polynucleotide encodes a mutant bcl-2 polypeptide comprising an amino acid substitution or deletion in a BH1 or BH2 domain;
- expressing said mutant bcl-2 polypeptide in a mammalian cell which expresses Bax and which is capable of undergoing bcl-2-sensitive apoptosis;
- determining whether expression of the mutant bcl-2 polypeptide inhibits death repressor activity of a bcl-2 protein endogenous to said mammalian cell and/or whether said mutant bcl-2 protein itself lacks death repressor activity and/or are incapable of providing inhibition of said bcl-2-sensitive apoptosis;
- and
- identifying mutant bcl-2 proteins which inhibit endogenous bcl-2 death repressor activity and/or which are incapable of providing inhibition of said bcl-2-sensitive apoptosis as being bcl-2 proteins which lack binding to Bax and/or substantially lack death repressor activity.
Dependencies are inferred from the claim text.