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The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases
N Roy1, Q L Deveraux, R Takahashi
1The Burnham Institute, Program on Apoptosis and Cell Death Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.
Insights
Inhibitor of apoptosis proteins (IAPs) regulate cell death. This study shows c-IAP-1 and c-IAP-2 inhibit caspases-3 and -7, similar to XIAP, while NAIP does not directly inhibit these caspases.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The inhibitor of apoptosis (IAP) protein family is crucial for regulating programmed cell death.
- Mechanisms of IAP-mediated apoptosis inhibition have been largely unclear.
- XIAP, a known IAP, inhibits caspases-3 and -7, key proteases in apoptosis.
Purpose of the Study:
- To investigate the apoptotic regulatory functions of c-IAP-1, c-IAP-2, and NAIP.
- To determine if c-IAP-1, c-IAP-2, and NAIP bind to and inhibit caspases.
- To elucidate the specific caspases targeted by these IAP family members.
Main Methods:
- In vitro binding assays to test IAP-caspase interactions.
- Enzyme activity assays to measure caspase inhibition.
- Cell-free systems with cytosolic extracts to study caspase activation.
- Gene transfection in intact cells to assess IAP function during apoptosis induction.
Main Results:
- c-IAP-1 and c-IAP-2 specifically bind and inhibit caspases-3 and -7, but not caspase-8, -1, or -6.
- NAIP did not show significant binding or inhibition of tested caspases.
- Overexpression of c-IAP-1 and c-IAP-2 suppressed apoptosis induced by etoposide in intact cells.
- c-IAP-1 and c-IAP-2 inhibition of caspases differs from p35, as they are not cleaved.
Conclusions:
- c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting distal caspases-3 and -7.
- NAIP likely inhibits apoptosis through alternative mechanisms distinct from direct caspase inhibition.
- These findings clarify the roles of different IAP family members in apoptosis regulation.