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c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment
R J Clem1, T T Sheu, B W Richter
1Department of Molecular Microbiology and Immunology, Johns Hopkins Schools of Public Health and Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.
Insights
Full-length cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/c-IAP2) do not prevent cell death. However, removing their C-terminal RING domains restores antiapoptotic function, revealing a novel regulatory mechanism.
Area of Science:
- Cellular Biology
- Apoptosis Regulation
- Protein Function
Background:
- Human cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/c-IAP2) are known to have antiapoptotic roles.
- Previous studies suggested c-IAP1/c-IAP2 protect cells from various apoptotic stimuli.
Purpose of the Study:
- To investigate the antiapoptotic activity of full-length c-IAP1 and c-IAP2.
- To determine the role of the C-terminal RING domains in regulating c-IAP1/c-IAP2 function.
- To elucidate the mechanism by which c-IAP1/c-IAP2 regulate apoptosis.
Main Methods:
- Transfection of mammalian cells with constructs encoding full-length or truncated c-IAP1/c-IAP2.
- Induction of apoptosis via Bax overexpression, TNF-alpha treatment, and Sindbis virus infection.
- In vitro cleavage assays using apoptotic cell extracts and purified caspase-3.
- Analysis of protein fragments and their functional consequences.
Main Results:
- Full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by various stimuli.
- Deletion of the C-terminal RING domains restored the antiapoptotic activity of c-IAP1 and c-IAP2.
- c-IAP1 was cleaved during apoptosis into 52- and 35-kDa fragments containing the RING domain.
- The spacer-RING domain alone induced apoptosis, with optimal activity requiring both regions but not the caspase recruitment domain.
Conclusions:
- The C-terminal RING domain negatively regulates the antiapoptotic function of the N-terminal BIR domain in c-IAP1/c-IAP2.
- Cleavage of c-IAP1 by caspases during apoptosis releases fragments that may contribute to cell death.
- The spacer-RING domain possesses proapoptotic activity, highlighting a dual role for c-IAP1/c-IAP2 in apoptosis regulation.