Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis.

2014Oct
Proc. Natl. Acad. Sci. U.S.A.
Proc Natl Acad Sci U S A 2014 Oct 1;111(41):14782-7. Epub 2014 Oct 1.
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Intrinsic apoptotic stimuli initiate mammalian cells' apoptotic program by first activating the proteins that have only Bcl-2 homology domain 3 (BH3), such as Bcl-2 interacting mediator of cell death (Bim) and truncated BH3 interacting death domain agonist (tBid), which in turn trigger conformational changes in BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak) proteins that enable oligomer formation on the mitochondria, causing cytochrome c and other apoptogenic proteins in the intermembrane space to leak out. Leaked cytochrome c then initiates apoptotic caspase activation through a well-defined biochemical pathway. However, how oligomerized Bax and Bak cause cytochrome c release from mitochondria remains unknown. We report here the establishment of cell lines in which Bim or tBid can be inducibly expressed to initiate apoptosis in a controlled, quantitative manner. We used these cell lines to examine apoptotic events after Bax and Bak oligomerization but before cytochrome c release. The mitochondrial metalloprotease OMA1 was activated in this system in a Bax- and Bak-dependent fashion. Activated OMA1 cleaved the dynamin-like GTPase, optical nerve atrophy 1, an event that is critical for remodeling of mitochondrial cristae. Knockdown or knockout of OMA1 in these cells attenuated cytochrome c release. Thus it is clear that oligomerized Bax and Bak trigger apoptosis by causing both the permeabilization of the mitochondrial outer membrane and activation OMA1.

Affiliation

National Institute of Biological Sciences, Beijing 102206, China; Graduate School of Peking Union Medical College, Beijing 100730, China; and Chinese Academy of Medical Sciences, Beijing 100730, China

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2014Oct
Proc. Natl. Acad. Sci. U.S.A.
Proc Natl Acad Sci U S A 2014 Oct 1;111(41):14782-7. Epub 2014 Oct 1.

Intrinsic apoptotic stimuli initiate mammalian cells' apoptotic program by first activating the proteins that have only Bcl-2 homology domain 3 (BH3), such as Bcl-2 interacting mediator of cell death (Bim) and truncated BH3 interacting death domain agonist (tBid), which in turn trigger conformational changes in BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak) proteins that enable oligomer formation on the mitochondria, causing cytochrome c and other apoptogenic proteins in the intermembrane space to leak out. Leaked cytochrome c then initiates apoptotic caspase activation through a well-defined biochemical pathway. However, how oligomerized Bax and Bak cause cytochrome c release from mitochondria remains unknown. We report here the establishment of cell lines in which Bim or tBid can be inducibly expressed to initiate apoptosis in a controlled, quantitative manner. We used these cell lines to examine apoptotic events after Bax and Bak oligomerization but before cytochrome c release. The mitochondrial metalloprotease OMA1 was activated in this system in a Bax- and Bak-dependent fashion. Activated OMA1 cleaved the dynamin-like GTPase, optical nerve atrophy 1, an event that is critical for remodeling of mitochondrial cristae. Knockdown or knockout of OMA1 in these cells attenuated cytochrome c release. Thus it is clear that oligomerized Bax and Bak trigger apoptosis by causing both the permeabilization of the mitochondrial outer membrane and activation OMA1.

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.

Bax and Bak promote apoptosis by perturbing the permeability of the mitochondrial outer membrane and facilitating the release of cytochrome c by a mechanism that is still poorly defined. During apoptosis, Bax and Bak also promote fragmentation of the mitochondrial network, possibly by activating the mitochondrial fission machinery. It has been proposed that Bax/Bak-induced mitochondrial fission may be required for release of cytochrome c from the mitochondrial intermembrane space, although this has been a subject of debate. Here we show that Bcl-xL, as well as other members of the apoptosis-inhibitory subset of the Bcl-2 family, antagonized Bax and/or Bak-induced cytochrome c release but failed to block mitochondrial fragmentation associated with Bax/Bak activation. These data suggest that Bax/Bak-initiated remodeling of mitochondrial networks and cytochrome c release are separable events and that Bcl-2 family proteins can influence mitochondrial fission-fusion dynamics independent of apoptosis.

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Affiliation Details

  • National Institute of Biological Sciences, Beijing 102206, China; Graduate School of Peking Union Medical College, Beijing 100730, China; and Chinese Academy of Medical Sciences, Beijing 100730, China
  • National Institute of Biological Sciences
Affiliation National Institute of Biological Sciences, Beijing 102206, China; Graduate School of Peking Union Medical College, Beijing 100730, China; and Chinese Academy of Medical Sciences, Beijing 100730, China