Abstract
ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing / chemistry
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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Amino Acid Sequence
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Apoptosis Regulatory Proteins
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Apoptosis*
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Binding Sites
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Caspase 9
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Caspase Inhibitors*
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Caspases / metabolism
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Cell Line
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Cell Line, Tumor
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Crystallography, X-Ray
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Cysteine Proteinase Inhibitors / chemistry*
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Cysteine Proteinase Inhibitors / genetics
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Cysteine Proteinase Inhibitors / metabolism*
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Electron Spin Resonance Spectroscopy
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Humans
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Inhibitor of Apoptosis Proteins
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Intracellular Signaling Peptides and Proteins
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Kinetics
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Mitochondrial Proteins / chemistry
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Mitochondrial Proteins / genetics
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Mitochondrial Proteins / metabolism*
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Models, Molecular
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Molecular Sequence Data
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Neoplasm Proteins / chemistry
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism*
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Protein Binding
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Protein Engineering*
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Protein Structure, Tertiary
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Proteins / chemistry
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Proteins / genetics
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Proteins / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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X-Linked Inhibitor of Apoptosis Protein
Substances
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Adaptor Proteins, Signal Transducing
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Apoptosis Regulatory Proteins
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BIRC7 protein, human
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Carrier Proteins
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Caspase Inhibitors
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Cysteine Proteinase Inhibitors
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DIABLO protein, human
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Inhibitor of Apoptosis Proteins
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Intracellular Signaling Peptides and Proteins
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Mitochondrial Proteins
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Neoplasm Proteins
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Proteins
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Recombinant Fusion Proteins
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X-Linked Inhibitor of Apoptosis Protein
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XIAP protein, human
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CASP9 protein, human
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Caspase 9
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Caspases