ROS production by adrenodoxin does not cause apoptosis in fission yeast.

We previously showed that production of reactive oxygen species (ROS) caused by overexpression of the mitochondrial electron transfer protein adrenodoxin (Adx) induces apoptosis in mammalian cells. In the fission yeast Schizosaccharomyces pombe, ROS are also produced in cells that undergo an apoptotic-like cell death, but it is not yet clear whether they are actually causative for this phenomenon or whether they are merely produced as a by-product. Therefore, the purpose of this study was to trigger mitochondrial ROS production in fission yeast by overexpression of either wildtype Adx (Adx-WT) or of several activated Adx mutants and to investigate its consequences. It was found that strong expression of either Adx-WT or Adx-S112W did not produce any ROS, while Adx-D113Y caused a twofold and Adx1-108 a threefold increase in ROS formation as compared to basal levels. However, no typical apoptotic markers or decreased viability could be observed in these strains. Since we previously observed that an increase in mitochondrial ROS formation of about 60% above basal levels is sufficient to strongly induce apoptosis in mammalian cells, we conclude that S. pombe is either very robust to mitochondrial ROS production or does not undergo apoptotic cell death in response to mitochondrial ROS at all.

Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria.

Mitochondrial cytochrome P450 systems are an indispensable component of mammalian steroid biosynthesis; they catalyze regio- and stereo-specific steroid hydroxylations and consist of three protein entities: adrenodoxin reductase (AdR), adrenodoxin (Adx), and a mitochondrial cytochrome P450 enzyme, e.g., CYP11A1 (P450 side chain cleavage, P450scc). It is known that the latter two are able to generate reactive oxygen species (ROS) in vitro . In this study, we investigated whether this ROS generation also occurs in vivo and, if so, whether it leads to the induction of apoptosis. We found that overexpression of either human or bovine Adx causes a significant loss of viability in 11 different cell lines. This loss of viability does not depend on the presence of the tumor suppressor protein p53. Transient overexpression of human Adx in HCT116 cells leads to ROS production, to a disruption of the mitochondrial transmembrane potential (DeltaPsi), to cytochrome c release from the mitochondria, and to caspase activation. In contrast, the effect of transient overexpression of human CYP11A1 on cell viability varies in different cell lines, with some being sensitive and others not. We conclude that mitochondrial cytochrome P450 systems are a source of mitochondrial ROS production and can play a role in the induction of mitochondrial apoptosis.