Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine (TMPD) 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. 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 TMPD (tetramethylphenylenediamine) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporin. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporininduced 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 were 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. Introduction The intrinsic pathway of apoptosis is initiated by various stimuli that cause the release of cytochrome c from mitochondria into the cytoplasm (1). Once released, cytochrome c binds to the cytosolic adaptor protein Apaf-1 causing the formation of the so-called ‘apoptosome’ leading to activation of caspase-9, which then cleaves downstream caspases such as caspase-3 (2). The release of cytochrome c from mitochondria has been thought to be an irreversible step for the apoptotic process (3). However, recent evidence suggests that the execution phase of apoptosis is highly regulated even after cytochrome c is released (4;5). And one of possible levels of such regulation could be the redox state of cytochrome c. Since the discovery by Xiadong Wang that cytochrome c release from mitochondria was central to apoptosis (6), there has been some question as to whether the two redox states of cytochrome c are equally effective in promoting caspase activation (7-10). But shortly after that discovery there were two papers apparently showing that cytochrome c redox state had no effect on apoptosis (9;11). Furthermore, Hampton et al (9) showed that cytochrome c added to cytosol was rapidly reduced, and this reduction was enhanced by addition of dithiothreitol (DTT). DTT is routinely added when assaying caspase activation because caspases are inactivated by oxidation (12), http://www.jbc.org/cgi/doi/10.1074/jbc.M700322200 The latest version is at JBC Papers in Press. Published on August 9, 2007 as Manuscript M700322200 Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from