Conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO for mediating apoptosis
AssigneeThomas Jefferson University
InventorEmad S. Alnemri
Life of this patent
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Abstract
The invention provides caspase-9-related peptides and polypeptides capable of binding to an Inhibitor of Apoptosis Protein (IAP), as well as caspase-9 mutant that fail to undergo normal processing and fail to bind to an IAP. Nucleic acid molecules, including expression vectors, encoding such peptides and polypeptides are also provided. Such peptides and polypeptides, are useful for inducing apoptosis and identifying inhibitors and enhancer of apoptosis.
Claims94
An isolated nucleic acid molecule comprising a polynucleotide having a sequence encoding a peptide or polypeptide comprising at least an amino acid sequence set forth in SEQ ID NO:13, or a variant thereof, wherein said peptide or polypeptide specifically binds to at least a portion of an Inhibitor of Apoptosis Protein (IAP).
An isolated nucleic acid molecule consisting essentially of a polynucleotide having a sequence encoding a peptide or polypeptide comprising at least an N-terminus amino acid sequence set forth in SEQ ID NO:11.
An isolated nucleic acid molecule consisting essentially of a polynucleotide having a sequence encoding a peptide or polypeptide comprising at least an N-terminus amino acid sequence of Ala—Val—Pro—Tyr, as set forth in SEQ ID NO:15.
An isolated nucleic acid molecule consisting essentially of a polynucleotide having a sequence encoding a peptide or polypeptide comprising at least an N-terminus amino acid sequence set forth in SEQ ID NO:12.
An isolated nucleic acid molecule comprising a polynucleotide having a sequence encoding a peptide or polypeptide comprising a first portion of a procaspase-9 that specifically binds at least a portion of an IAP and a second portion of a procaspase-9 containing a mutated active site, wherein said peptide or polypeptide specifically binds at least a portion of an IAP and lacks cysteine protease activity.
An isolated nucleic acid molecule comprising a polynucleotide having a sequence encoding a peptide or polypeptide comprising an amino acid sequence of SEQ ID NO:13, and further comprising at least a portion of a caspase-3, wherein said peptide or polypeptide exhibits caspase-3 enzymatic activity that is inhibited by an IAP or an IAP BIR3 domain.
An isolated nucleic acid molecule comprising a polynucleotide having a sequence encoding a peptide or polypeptide comprising at least a portion of a mutated procaspase-9, wherein said portion fails to undergo normal processing and said portion possesses wild type caspase-9 enzymatic activity.
A peptide or polypeptide comprising at least an amino acid sequence set forth in SEQ ID NO:13, wherein said peptide or polypeptide specifically binds to at least a portion of an Inhibitor of Apoptosis Protein (IAP).
A peptide or polypeptide comprising the amino acid residues set forth in SEQ ID NO:11 or a variant thereof, wherein said peptide or polypeptide specifically binds to at least a portion of an IAP.
A peptide or polypeptide comprising the amino acid residues set forth in SEQ ID NO:15 or a variant thereof, wherein said peptide or polypeptide specifically binds to at least a portion of an IAP.
A peptide or polypeptide comprising the amino acid residues set forth in SEQ ID NO:12 or a variant thereof, wherein said peptide or polypeptide specifically binds to at least a portion of an IAP.
A peptide or polypeptide comprising a first portion of a procaspase-9, or a variant thereof, that specifically binds at least a portion of an IAP and a second portion of a procaspase-9, or a variant thereof, containing a mutated active site, wherein said peptide or polypeptide specifically binds to at least a portion of an IAP and lacks cysteine protease activity.
A peptide or polypeptide comprising an amino acid sequence of SEQ ID NO:13, and further comprising at least a portion of a caspase-3, or a variant thereof, wherein said peptide or polypeptide exhibits caspase-3 enzymatic activity that is inhibited by an IAP BIR3 domain.
A peptide or polypeptide comprising at least a portion of a mutated procaspase-9 or a variant thereof, wherein said portion fails to undergo normal processing and said portion possesses wild type caspase-9 enzymatic activity.
An antibody that specifically binds to a peptide or polypeptide set forth in SEQ ID NO:13 that specifically binds to at least a portion of an IAP.
An antibody that specifically binds to an epitope located on the N-terminus of a caspase-9-p12.
A method of identifying an inhibitor or enhancer of a caspase-mediated apoptosis comprising:
- (a) contacting a cell containing a vector expressing a peptide or polypeptide comprising at least an amino acid sequence set forth in SEQ ID NO:13 that is capable of specifically binding to at least a portion of an IAP with a candidate inhibitor or candidate enhancer;
- and
- (b) detecting cell viability,
- wherein an increase in cell viability as compared to a control indicates the presence of an inhibitor and a decrease in cell viability as compared to a control indicates the presence of an enhancer.
A method of identifying an inhibitor or enhancer of a caspase-mediated apoptosis comprising:
- (a) contacting a cell containing a vector expressing a peptide or polypeptide comprising at least an amino acid sequence corresponding to SEQ ID NO:13 that is capable of specifically binding to at least a portion of an IAP with a candidate inhibitor or candidate enhancer;
- and
- (b) detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing activity,
- wherein a decrease in processing as compared to a control indicates the presence of an inhibitor and an increase in processing indicates the presence of an enhancer.
A method of identifying an inhibitor or enhancer of a caspase-mediated apoptosis comprising:
- (a) contacting a cell containing a vector expressing a peptide or polypeptide comprising at least an amino acid sequence corresponding to SEQ ID NO:13 that is capable of specifically binding to at least a portion of an IAP with a candidate inhibitor or candidate enhancer;
- and
- (b) detecting caspase enzymatic activity,
- wherein a decrease in enzymatic activity as compared to a control indicates the presence of an inhibitor and an increase in enzymatic activity indicates the presence of an enhancer.
A method for identifying a compound that inhibits a peptide or polypeptide comprising an amino acid sequence set forth in SEQ ID NO:13 that specifically binds at least a portion of an IAP from binding to said portion of an IAP, comprising:
- (a) contacting a candidate compound with said peptide or polypeptide in the presence of said portion of an IAP;
- and
- (b) detecting displacement or inhibition of binding of said portion of an IAP from said peptide or polypeptide.
A method for identifying a compound that inhibits a peptide or polypeptide comprising an amino acid sequence set forth in SEQ ID NO:13 that specifically binds at least a portion of an IAP from binding to said portion of an IAP, comprising:
- (a) contacting a candidate compound with said peptide or polypeptide in the presence of said portion of an IAP;
- and
- (b) performing a functional assay that confirms displacement of said portion of an IAP from said peptide or polypeptide.
Dependencies are inferred from the claim text.