Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state.
Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.
Borutaite
Vilmante
V
Institute for Biomedical Research, Kaunas University of Medicine, Eiveniu 4, Kaunas LT 50009, Lithuania. vilbor@vector.kmu.lt
Brown
Guy C
GC
D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D019311:Staurosporine / Q000494:pharmacology; D013772:Tetramethylphenylenediamine / Q000494:pharmacology*; D013997:Time Factors
D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009842:Oligopeptides; D011994:Recombinant Proteins; C108637:aspartyl-glutamyl-valyl-aspartal; D007770:L-Lactate Dehydrogenase; D020169:Caspases; D019311:Staurosporine; D013772:Tetramethylphenylenediamine