Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro.
During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, P. O. Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom.
Marion
M
MacFarlane
Wendy
W
Merrison
Shawn B
SB
Bratton
Gerald M
GM
Cohen
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death; D003546:Cysteine Endopeptidases; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; D011506:Proteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex