Mechanism Of Release Of Mitochondrial Apoptotic Proteins

Apoptosis is of great importance to development and tissue homeostasis in metazoans. There are two known pathways of apoptosis:extrinsic pathway of apoptosis and intrinsic pathway of apoptosis that is also called mitochondrial pathway of apoptosis. During mitochondrial pathway of apoptosis, mitochondria undergo changes including the release of apoptotic proteins from mitochondria into cytosol such as cytochrome c and SMAC, mitochondrial fragmentation and mitochondrial dysfunction, etc. Albeit breath-taking progresses in apoptosis reseach in the past, the mechanism underlying those mitochondrial events during apoptosis remains elusive.Among those apoptotic changes, the release of cytochrome c into cytosol is the most important and most extensively studied one. Bax/Bak oligomerization and OPA1complex disassembly are reported to control this process through mediating mitochondrial outer membrane permeabilization and mitochondrial cristae remodeling respectively. However, how important of those two events are for cytochrome c release and the relationship between them are controversial.Here using inducible tBid and Bim cell systems as mitochondrial apoptosis models, we identified that the mitochondrial metalloprotease OMA1is a crucial regulator of mitochondrial pathway of apoptosis. Knockdown or knockout of OMA1prevented Bim/tBid induced OPA1cleavage, OPA1complex disassembly, cytochrome c and SMAC release, mitochondrial fragmentation, cristae remodeling, respiration rate drop in the cell, and cell death. Knockdown OPA1or re-expression of wild type OMA1but not its protease dead mutant OMA1H331A restored Bim induced apoptosis in OMA1knockdown cells. Hence the proapoptotic function of OMA1depends on its proteolytic activity and OPAl. During mitochondrial apoptosis,OMA1cleaves OPA1and causes OPA1complex disassembly, which further leads to cytochrome c and SMAC release, mitochondrial fragmentation and mitochondrial dysfunction. In healthy cells,OMA1is self-cleaved into a40KD protein S-OMA1which is the proteolytic active form and maintains a basal activity. Upon apoptotic stimuli or other mitochondrial stress treatment, the60KD full length protein L-OMA1can be stabilized while S-OMA1is further activated and cleaves itself and other substrates such as OPAl. Double knockdown of Bax and Bak significantly blocked Bim induced OMA1activation, OPA1cleavage and OPA1complex disassembly. Moreover knockdown of Bax and Bak blocked Bim induced mitochondrial membrane potential loss and ADP-stimulated respiration rate drop while knockdown of OMA1did not show similar effect. Knockdown of OMA1also did not affect tBid/Bim induced Bax and Bak oligomerization. Dissipation of mitochondrial membrane potential is a known trigger of OMA1activation. Therefore the activation of OMA1is downstream of Bax/Bak and is probably triggered by Bax/Bak dependent mitochondrial membrane potential dissipation during apoptosis. In summary our results defined OMA1as a central regulator of mitochondrial apoptosis. The detailed molecular mechanism of OMA1activation and Bax/Bak mediated mitochondrial membrane potential loss would be very interesting research subjects in the future.In the meantime, we carried out a high throughput compound screen to identify small molecule inhibitors of tBid/Bim induced apoptosis. We picked out8efficient inhibitors and did thorough characterization of4of them, numbered#11,#12,#13and#18. Those4compounds inhibited tBid/Bim induced cytochrome c release but not SMAC release nor mitochondrial fragmentation.#11and#12further blocked OPA1complex disassembly during apoptosis. None of the4compounds blocked Bax/Bak oligomerization. Furthermore, knockdown of OPA1can impair#11’s and#12’s activity. This infers that OPA1or its associated protein is at least one of the target(s) of#11and#12. Those compounds may offer new drug candidates for treating diseases associated with excess apoptosis and identification of the exact functional targets of those inhibitors would give us novel insights into the regulation of mitochondrial pathway of apoptosis and offer new therapeutic target(s) as well.