Cited by 152Cell death mechanisms and regulationCancer-related Molecular PathwaysUbiquitin and proteasome pathways

Requirement of Both the Second and Third BIR Domains for the Relief of X-linked Inhibitor of Apoptosis Protein (XIAP)-mediated Caspase Inhibition by Smac

Open access
Huang, Yihua, Rich, Rebecca L., Myszka, David G., Wu, Hao·2003·Journal of Biological Chemistry

Abstract

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.

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  • Upon release from mitochondria, the N-terminal amino acid alanine-valine-proline-isoeuceucine (AVPI) of Smac binds to the domain of XIAP, depriving XIAP of its ability to bind to caspase, thereby promoting apoptosis.

    “Upon release from mitochondria, the N-terminal amino acid alanine-valine-proline-isoeuceucine (AVPI) of Smac binds to the domain of XIAP, depriving XIAP of its ability to bind to caspase, thereby promoting apoptosis.458”

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Authors
Huang, Y., Rich, R. L., Myszka, D. G., & Wu, H.
Affiliations
  • Cornell University (US)
  • University of Utah (US)
Corresponding author
Wu, Hao
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2003-12
Volume
278
Issue
49
Pages
49517-49522
ISSN
0021-9258 (ISSN-L)
OpenAlex
W2160029849
Cited by
152
References
34
Citations by year
2026: 1 · 2025: 3 · 2024: 3 · 2023: 5 · 2022: 5 · 2021: 2 · 2020: 7 · 2019: 6 · 2018: 3 · 2017: 5 · 2016: 5 · 2015: 3 · 2014: 9 · 2013: 10 · 2012: 4
FWCI
3.22
Type
journal article
Language
en
Open access
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Institute of Allergy and Infectious Diseases (AI50872)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
XIAP, Smac/DIABLO, caspase inhibition, caspase-7, caspase-9, effector caspases, initiator caspases, BIR3 domain, ternary complex, apoptosis, protein-protein interaction affinity
MeSH terms
Carrier Proteins, Humans, Protein Binding, Proteins, Dimerization, Caspases, Mitochondrial Proteins, Intracellular Signaling Peptides and Proteins, Apoptosis Regulatory Proteins, X-Linked Inhibitor of Apoptosis Protein, Caspase Inhibitors

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