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.
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Research Article|
December 14 2004
Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP
Domagoj VUCIC;
Domagoj VUCIC
1
*Department of Molecular Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Matthew C. FRANKLIN;
Matthew C. FRANKLIN
1
†Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Heidi J. A. WALLWEBER;
Heidi J. A. WALLWEBER
†Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Kanad DAS;
Kanad DAS
†Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Brendan P. ECKELMAN;
Brendan P. ECKELMAN
‡Graduate Program in Molecular Pathology, University of California San Diego, La Jolla, CA 92037, U.S.A.
§Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Hwain SHIN;
Hwain SHIN
2
‡Graduate Program in Molecular Pathology, University of California San Diego, La Jolla, CA 92037, U.S.A.
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Linda O. ELLIOTT;
Linda O. ELLIOTT
∥Department of Medicinal Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Saloumeh KADKHODAYAN;
Saloumeh KADKHODAYAN
¶Department of Bioanalytical Research and Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Kurt DESHAYES;
Kurt DESHAYES
†Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
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Guy S. SALVESEN;
Guy S. SALVESEN
§Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Wayne J. FAIRBROTHER
Wayne J. FAIRBROTHER
3
*Department of Molecular Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
†Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
3To whom correspondence should be addressed (email fairbro@gene.com).
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Publisher: Portland Press Ltd
Received:
June 29 2004
Revision Received:
September 22 2004
Accepted:
October 15 2004
Accepted Manuscript online:
October 15 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 11–20.
Article history
Received:
June 29 2004
Revision Received:
September 22 2004
Accepted:
October 15 2004
Accepted Manuscript online:
October 15 2004
Connected Content
A commentary has been published:
IAP proteins: sticking it to Smac
Citation
Domagoj VUCIC, Matthew C. FRANKLIN, Heidi J. A. WALLWEBER, Kanad DAS, Brendan P. ECKELMAN, Hwain SHIN, Linda O. ELLIOTT, Saloumeh KADKHODAYAN, Kurt DESHAYES, Guy S. SALVESEN, Wayne J. FAIRBROTHER; Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP. Biochem J 1 January 2005; 385 (1): 11–20. doi: https://doi.org/10.1042/BJ20041108
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