Abstract
Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.
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Abbreviations
- cyto.c:
-
cytochrome c
- cyto.c1:
-
cytochrome c1
- SCR:
-
succinate-cyto.c reductase
- KD:
-
knock-down
- wt:
-
wild type
- STS:
-
staurosporine
- RCR:
-
respiratory control ratio
- NAC:
-
N-Acetyl Cysteine
- ATP:
-
adenosine-triphosphate
- Apaf-1:
-
apoptotic protease-activating factor 1
- ROS:
-
reactive oxygen species
- casp.3:
-
caspase 3
References
Boveris A, Chance B . The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 1973; 134:707–716.
Mitchell P . Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: protonmotive ubiquinone cycle. FEBS Lett 1975; 56:1–6.
Brand MD, Murphy MP . Control of electron flux through the respiratory chain in mitochondria and cells. Biol Rev Camb Philos Soc 1987; 62:141–193.
Xia T, Jiang C, Li L, Wu C, Chen Q, Liu SS . A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro. FEBS Lett 2002; 510:62–66.
Crofts AR . The cytochrome bc1 complex: function in the context of structure. Annu Rev Physiol 2004; 66:689–733.
Zhao Y, Wang ZB, Xu JX . Effect of cytochrome c on the generation and elimination of O2*- and H2O2 in mitochondria. J Biol Chem 2003; 278:2356–2360.
Zhang L, Yu L, Yu CA . Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria. J Biol Chem 1998; 273:33972–33976.
Cape JL, Bowman MK, Kramer DM . A semiquinone intermediate generated at the Qo site of the cytochrome bc1 complex: importance for the Q-cycle and superoxide production. Proc Natl Acad Sci USA 2007; 104:7887–7892.
Wallace DC . A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 2005; 39:359–407.
Wallace DC . Mitochondrial diseases in man and mouse. Science 1999; 283:1482–1488.
Jin S, DiPaola RS, Mathew R, White E . Metabolic catastrophe as a means to cancer cell death. J Cell Sci 2007; 120:379–383.
Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275:1129–1132.
Green DR, Reed JC . Mitochondria and apoptosis. Science 1998; 281:1309–1312.
Shi Y . Apoptosome: the cellular engine for the activation of caspase-9. Structure 2002; 10:285–288.
Riedl SJ, Salvesen GS . The apoptosome: signalling platform of cell death. Nat Rev Mol Cell Biol 2007; 8:405–413.
Riedl SJ, Li W, Chao Y, Schwarzenbacher R, Shi Y . Structure of the apoptotic protease-activating factor 1 bound to ADP. Nature 2005; 434:926–933.
Bao Q, Riedl SJ, Shi Y . Structure of Apaf-1 in the auto-inhibited form: a critical role for ADP. Cell Cycle 2005; 4:1001–1003.
Marzo I, Susin SA, Petit PX, et al. Caspases disrupt mitochondrial membrane barrier function. FEBS Lett 1998; 427:198–202.
Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR . The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2000; 2:156–162.
Cepero E, King AM, Coffey LM, Perez RG, Boise LH . Caspase-9 and effector caspases have sequential and distinct effects on mitochondria. Oncogene 2005; 24:6354–6366.
Bossy-Wetzel E, Green DR . Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. J Biol Chem 1999; 274:17484–17490.
Annis MG, Soucie EL, Dlugosz PJ, et al. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 2005; 24:2096–2103.
Ott M, Gogvadze V, Orrenius S, Zhivotovsky B . Mitochondria, oxidative stress and cell death. Apoptosis 2007; 12:913–922.
Orrenius S, Gogvadze V, Zhivotovsky B . Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 2007; 47:143–183.
Chandra D, Tang DG . Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria. J Biol Chem 2003; 278:17408–17420.
Ricci JE, Waterhouse N, Green DR . Mitochondrial functions during cell death, a complex (I-V) dilemma. Cell Death Differ 2003; 10:488–492.
Ricci JE, Gottlieb RA, Green DR . Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol 2003; 160:65–75.
Chen Q, Gong B, Almasan A . Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ 2000; 7:227–233.
Chen Q, Chai YC, Mazumder S, et al. The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction. Cell Death Differ 2003; 10:323–334.
Sun F, Huo X, Zhai Y, et al. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 2005; 121:1043–1057.
Poot M, Zhang YZ, Kramer JA, et al. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem 1996; 44:1363–1372.
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.
Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y . Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 2000; 406:855–862.
Xia D, Yu CA, Kim H, et al. Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 1997; 277:60–66.
Pletjushkina OY, Lyamzaev KG, Popova EN, et al. Effect of oxidative stress on dynamics of mitochondrial reticulum. Biochim Biophys Acta 2006; 1757:518–524.
Kagan VE, Tyurin VA, Jiang J, et al. Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors. Nat Chem Biol 2005; 1:223–232.
Suto D, Sato K, Ohba Y, Yoshimura T, Fujii J . Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism. Biochem J 2005; 392:399–406.
Abdullaev Z, Bodrova ME, Chernyak BV, et al. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect. Biochem J 2002; 362:749–754.
Jiang S, Cai J, Wallace DC, Jones DP . Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved. J Biol Chem 1999; 274:29905–29911.
Marchetti P, Susin SA, Decaudin D, et al. Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 1996; 56:2033–2038.
Ricci JE, Munoz-Pinedo C, Fitzgerald P, et al. Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain. Cell 2004; 117:773–786.
Weng C, Li Y, Xu D, Shi Y, Tang H . Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem 2005; 280:10491–10500.
Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 1997; 278:1966–1968.
Mancuso M, Filosto M, Stevens JC, et al. Mitochondrial myopathy and complex III deficiency in a patient with a new stop-codon mutation (G339X) in the cytochrome b gene. J Neurol Sci 2003; 209:61–63.
Borisov VB . Defects in mitochondrial respiratory complexes III and IV, and human pathologies. Mol Aspects Med 2002; 23:385–412.
Acin-Perez R, Bayona-Bafaluy MP, Fernandez-Silva P, et al. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol Cell 2004; 13:805–815.
Keilin D, Hartree EF . Activity of the cytochrome system in heart muscle preparations. Biochem J 1947; 41:500–502.
Yu CA, Yu L, King TE . Soluble cytochrome b-c1 complex and the reconstitution of succinate-cytochrome c reductase. J Biol Chem 1974; 249:4905–4910.
Poe M, Gutfreund H, Estabrook RW . Kinetic studies of temperature changes and oxygen uptake in a differential calorimeter: the heat of oxidation of NADH and succinate. Arch Biochem Biophys 1967; 122:204–211.
Acknowledgements
This work was supported by grants from the National Basic Research Program (973 program project, No 2009CB521800 and 2011CB910900), the National Natural Science Foundation of China (No 30910103910) to Q Chen and 973 program project (2010CB912200) to Y Zhu. We thank Prof Richard Flavell (Yale School of Medicine, USA) for his kind supply of caspase 3-deficient MEF cells; Prof Douglas Green (St. Jude Children's Research Hospital, USA) for providing the cyto.c-GFP constructs; Prof Xiaodong Wang (University of Texas Southwestern Medical center, USA), Dr Aimin Zhou (Cleveland State University, USA), Dr Wei-Xing Zong (Stony Brook University, USA), Dr Honggang Wang (H. Lee Moffitt Cancer Center, USA) and Prof Jun Zhou (Nankai University, China) for their critical reading of the manuscript and valuable comments.
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(Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Cyto.c1 was cleaved in MEF cells, but not in caspase 3-knockout cells. (PDF 117 kb)
Supplementary information, Figure S2
Confocal microscopic images caspase activation and mitochondrial morphology following staurosporine treatment. (PDF 104 kb)
Supplementary information, Figure S3
NAC prevents the production and mitochondrial fragmentation. (PDF 39 kb)
Supplementary information, Figure S4
Cyto.c1 cleavage and caspase activation in rho 206 cells. (PDF 100 kb)
Supplementary information, Data S1
Materials and Methods (PDF 12 kb)
Supplementary information, Video S1C
Video S1 Caspase activation in wild type and D106A cyto.c1 cells. Time lapse imaging of caspase activation and mitochondrial morphology following staurosporine (0.1 μM) treatment. The simultaneous recording of mitochondrial morphology (red) and fluorescent images of activated caspases (green) was carried out by a ZEISS LSM510 laser scanning confocal microscope system as described in Supplementary information, Data S1 and Figure S3. Confocal images were taken every 3 min for 4-12 h. The time was chosen as we previously showed that caspase is activated at this particular time. Video S1A: cells expressing wild type cyto.c1; Video S1B: cells expressing mutant cyto.c1 D106A; Video S1C: mock cells in the presence of z-VAD (100 μM) (GIF 8637 kb)
Supplementary information, Video S2C
Video S2 GFP-cyto.c release in wild type and D106A cyto.c1 cells. Cell suspensions were transferred to a thermostated chamber with a glass cover-slip bottom, allowed adhering for 24 h, then cells were co-transfected with pEGFP-C3-cyto.c and MitoDsRed (DNA, 10:1) for 36 h before treatment with staurosporine. Cells with GFP-labeled cyto.c correctly located at mitochondria were chosen to perform the time lapse imaging. The simultaneous recording of mitochondrial morphology (mitoDsRed) and GFP-cyto.c (green) were carried out by a ZEISS LSM510 laser scanning confocal microscope system as described in Supplementary information, Data S1. Confocal images were taken every 2 min for 1 h after the addition of staurosporine. Video S2A: cells expressing wild type cyto.c1; Video S2B: cells expressing mutant cyto.c1 D106A; Video S2C: mock cells in the presence of z-VAD (100 μM). (GIF 14660 kb)
Supplementary information, Video S3B
Video S3 z-VAD-fmk can inhibit cyto.c release when added 3 or 6 h after staurosporine treatment. Single clonally cell expressing GFP-cyto.c (pEGFP-C3-cyto.c) and MitoDsRed located in mitochondria was acquired for assay. Cells were treated with 0.1 μM staurosporine for 3 h (A) or 6 h (B) before supplemented with z-VAD (100 μM). The simultaneous recording of mitochondrial morphology (mitoDsred) and GFP-cyto.c (green) were carried out by a ZEISS LSM510 laser scanning confocal microscope system as described in Supplementary information, Data S1. Confocal images were taken every 2 min. Video S3A: z-VAD added at 3 h after staurosporine treatment; Video S3B: z-VAD added at 6 h after staurosporine treatment. (GIF 32014 kb)
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Zhu, Y., Li, M., Wang, X. et al. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release. Cell Res 22, 127–141 (2012). https://doi.org/10.1038/cr.2011.82
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DOI: https://doi.org/10.1038/cr.2011.82
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