Structural and Functional Analysis of Novel Human Cytochrome cTargets in Apoptosis*

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivoand in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome cduring apoptosis is well established, relatively little is known about its participation in signaling pathways in vivodue to its essential role during respiration. To obtain a better understanding of the role of cytochrome cin the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome cas bait in this study. In this approach, novel cytochrome cinteraction partners were identified whose in vivointeraction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome cand its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome credox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome cinteracts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome cmay interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.