Abstract
Apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity anchored to the mitochondrial inner membrane, is known to be involved in complex I maintenance. During apoptosis, AIF can be released from mitochondria and translocate to the nucleus, where it participates in chromatin condensation and large-scale DNA fragmentation. The mechanism of AIF release is not fully understood. Here, we show that a prolonged (∼10 min) increase in intracellular Ca2+ level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca2+ increase, followed by rapid normalization of the Ca2+ level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca2+ into staurosporine-treated cells caused the activation of a calpain, located in the intermembrane space of mitochondria. The activated calpain, in turn, cleaved membrane-bound AIF, and the soluble fragment was released from the mitochondria upon outer membrane permeabilization through Bax/Bak-mediated pores or by the induction of Ca2+-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca2+, but not the suppression of caspase activity, prevented processing and release of AIF. Combined, these results provide novel insights into the mechanism of AIF release during cell death.
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Abbreviations
- AIF:
-
apoptosis-inducing factor
- Atg5:
-
autophagy-related gene 5
- IMS:
-
intermembrane space
- IP3:
-
inositol 1,4,5-trisphosphate
- MEF:
-
mouse embryonic fibroblast
- MPT:
-
mitochondrial permeability transition
- NSCLC:
-
non-small-cell lung carcinoma
- OMM:
-
outer mitochondrial membrane
- PKC 412:
-
protein kinase C inhibitor
- STS:
-
staurosporine
- tBid:
-
truncated Bid
References
Thompson CB . Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456–1462.
Arends MJ, Wyllie AH . Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 1991; 32: 223–254.
Cande C, Vahsen N, Kouranti I, Schmitt E, Daugas E, Spahr C et al. AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene 2004; 23: 1514–1521.
Du C, Fang M, Li Y, Li L, Wang X . Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000; 102: 33–42.
Liu X, Kim CN, Yang J, Jemmerson R, Wang X . Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996; 86: 147–157.
Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L et al. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein–caspase interaction. J Biol Chem 2002; 277: 432–438.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397: 441–446.
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102: 43–53.
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R . A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 2001; 8: 613–621.
Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol 2001; 3: 839–843.
Joseph B, Ekedahl J, Lewensohn R, Marchetti P, Formstecher P, Zhivotovsky B . Defective caspase-3 relocalization in non-small cell lung carcinoma. Oncogene 2001; 20: 2877–2888.
Gallego MA, Joseph B, Hemstrom TH, Tamiji S, Mortier L, Kroemer G et al. Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas. Oncogene 2004; 23: 6282–6291.
Hemstrom TH, Joseph B, Schulte G, Lewensohn R, Zhivotovsky B . PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage. Exp Cell Res 2005; 305: 200–213.
Miramar MD, Costantini P, Ravagnan L, Saraiva LM, Haouzi D, Brothers G et al. NADH oxidase activity of mitochondrial apoptosis-inducing factor. J Biol Chem 2001; 276: 16391–16398.
Klein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN et al. The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature 2002; 419: 367–374.
Vahsen N, Cande C, Briere JJ, Benit P, Joza N, Larochette N et al. AIF deficiency compromises oxidative phosphorylation. EMBO J 2004; 23: 4679–4689.
Loeffler M, Daugas E, Susin SA, Zamzami N, Metivier D, Nieminen AL et al. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 2001; 15: 758–767.
Otera H, Ohsakaya S, Nagaura Z, Ishihara N, Mihara K . Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. EMBO J 2005; 24: 1375–1386.
Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 2001; 410: 549–554.
Polster BM, Basanez G, Etxebarria A, Hardwick JM, Nicholls DG . Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria. J Biol Chem 2005; 280: 6447–6454.
Yuste VJ, Moubarak RS, Delettre C, Bras M, Sancho P, Robert N et al. Cysteine protease inhibition prevents mitochondrial apoptosis-inducing factor (AIF) release. Cell Death Differ 2005; 12: 1445–1448.
Cao G, Xing J, Xiao X, Liou AK, Gao Y, Yin XM et al. Critical role of calpain I in mitochondrial release of apoptosis-inducing factor in ischemic neuronal injury. J Neurosci 2007; 27: 9278–9293.
Badugu R, Garcia M, Bondada V, Joshi A, Geddes JW . N terminus of calpain 1 is a mitochondrial targeting sequence. J Biol Chem 2008; 283: 3409–3417.
McConkey DJ, Orrenius S . The role of calcium in the regulation of apoptosis. J Leukoc Biol 1996; 59: 775–783.
Nicotera P, Zhivotovsky B, Orrenius S . Nuclear calcium transport and the role of calcium in apoptosis. Cell Calcium 1994; 16: 279–288.
Orrenius S, Zhivotovsky B, Nicotera P . Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003; 4: 552–565.
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 2006; 8: 1124–1132.
Hood JL, Logan BB, Sinai AP, Brooks WH, Roszman TL . Association of the calpain/calpastatin network with subcellular organelles. Biochem Biophys Res Commun 2003; 310: 1200–1212.
Hood JL, Brooks WH, Roszman TL . Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus. J Biol Chem 2004; 279: 43126–43135.
Molinari M, Carafoli E . Calpain: a cytosolic proteinase active at the membranes. J Membr Biol 1997; 156: 1–8.
Goll DE, Thompson VF, Li H, Wei W, Cong J . The calpain system. Physiol Rev 2003; 83: 731–801.
Arrington DD, Van Vleet TR, Schnellmann RG . Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am J Physiol Cell Physiol 2006; 291: C1159–C1171.
Churbanova IY, Sevrioukova IF . Redox-dependent changes in molecular properties of mitochondrial apoptosis-inducing factor. J Biol Chem 2008; 283: 5622–5631.
Kruman I, Guo Q, Mattson MP . Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells. J Neurosci Res 1998; 51: 293–308.
Murphy RC, Schneider E, Kinnally KW . Overexpression of Bcl-2 suppresses the calcium activation of a mitochondrial megachannel. FEBS Lett 2001; 497: 73–76.
Acknowledgements
This work was supported by grants from the Swedish Research Council, the Swedish and the Stockholm Cancer Societies, the Swedish Childhood Cancer Foundation, the EC FP-6 (Oncodeath and Chemores) as well as the FP7 (Apo-Sys) programs. MO is a post-doctoral fellow of the Wenner-Gren Foundation, Stockholm, Sweden. MH is the recipient of a stipend for academic exchange from the DAAD, Germany. We thank Bruno Antonsson for the generous gift of plasmid pET23tBid and the late Stan Korsmeyer for Bax/Bak DKO MEFs.
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Norberg, E., Gogvadze, V., Ott, M. et al. An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death. Cell Death Differ 15, 1857–1864 (2008). https://doi.org/10.1038/cdd.2008.123
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DOI: https://doi.org/10.1038/cdd.2008.123
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