Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors

Abstract

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.

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Figure 1: Lipidomics and oxidative lipidomics of apoptosis.
Figure 2: Characterization of peroxidase activity of CL–cyt c complexes in model systems.
Figure 3: MS analysis of CL oxidation in HL-60 cells.
Figure 4: Characterization of peroxidase activity of CL–cyt c complex in mitochondria.
Figure 5: Cyt c–catalyzed oxidation of CL is required for release of proapoptotic factors from mitochondria into the cytosol of cells during apoptosis.
Figure 6: Oxidized cardiolipin (TLCLox) effectively releases Smac/Diablo from mitochondria isolated from cyt c−/− mouse embryonic cells as well as cyt c and Smac/Diablo from mitochondria of cyt c+/+ cells.
Figure 7: Distribution of CL in mitochondrial membranes and effects of CL unsaturation on STS-triggered apoptosis in HL-60.

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Acknowledgements

This work was supported by the US National Institutes of Health (grants 1RO1 HL70755 and PO1 HL070807), the US National Insitute for Occupational Safety and Health (grant 1RO1 OH008282) and the International Human Frontier Science Program.

Author information

Correspondence to Valerian E Kagan.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Effects of a phenolic lipid antioxidant, EPE, on phospholipid peroxidation in lipsomes and HL-60 cells. (PDF 23 kb)

Supplementary Fig. 2

Assessments of competitive binding of CL with cyt c. (PDF 14 kb)

Supplementary Fig. 3

Characterization of peroxidase activity of CL/cyt c complexes in model systems. (PDF 47 kb)

Supplementary Fig. 4

MS analysis of CL oxidation in model systems and mitochondria. (PDF 46 kb)

Supplementary Fig. 5

Characterization of peroxidase activity of CL/cyt c complex in mitochondria. (PDF 39 kb)

Supplementary Fig. 6

Western blots of cyt c, Smac/Diablo (a) and Bax (b) in cyt c+/+ and cyt c−/− cells. (PDF 31 kb)

Supplementary Fig. 7

Assessments of competitive interactions between CL/cyt c and tBid. (PDF 23 kb)

Supplementary Methods (PDF 149 kb)

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