Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.

Significance: Dysfunction of mitochondria, the powerhouses of living cells, favors the onset of human diseases, namely neurodegenerative diseases, cardiovascular pathologies, and cancer. Actually, respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 during the insulin-induced neuroprotection response following a brain ischemic injury. Here, we report that the decrease in neuronal death could be directly ascribed to changes in mitochondrial metabolism—including lower production of reactive oxygen species—and cell homeostasis induced by cytochrome c phosphorylation. Our findings thus provide the basis for understanding the molecular mechanism and potential use of phosphomimetic species of cytochrome c , thereby yielding new opportunities to develop more efficient therapies against acute pathologies.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and 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 c redox 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 c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

Cytochrome c (Cc) is a protein that functions as an electron carrier in the mitochondrial respiratory chain. However, Cc has moonlighting roles outside mitochondria driving the transition of apoptotic cells from life to death. When living cells are damaged, Cc escapes its natural mitochondrial environment and, once in the cytosol, it binds other proteins to form a complex named the apoptosome—a platform that triggers caspase activation and further leads to controlled cell dismantlement. Early released Cc also binds to inositol 1,4,5‐triphosphate receptors on the ER membrane, which stimulates further massive Cc release from mitochondria. Besides the well‐characterized binding proteins contributing to the proapoptotic functions of Cc, many novel protein targets have been recently described. Among them, histone chaperones were identified as key partners of Cc following DNA breaks, indicating that Cc might modulate chromatin dynamics through competitive binding to histone chaperones. In this article, we review the ample set of recently discovered antiapoptotic proteins—involved in DNA damage, transcription, and energetic metabolism—reported to interact with Cc in the cytoplasm and even the nucleus upon DNA breaks.

Significance: Cell response to physiological changes and oxidative stress involves the modulation of mitochondrial metabolism. Its dysfunction favors the development of hypoxia-dependent pathologies, including ischemia and cancer. A key modulator of mitochondrial activity is cytochrome c , whose cell function is regulated by tyrosine phosphorylation. However, how such modification affects cytochrome c structure and function is barely known. Here we report that a phosphomimetic mutant of cytochrome c exhibits enhanced dynamics, which could be responsible for the observed differences in cytochrome c functionality in oxidative stress and cell death. Thus, phosphorylation of cytochrome c becomes a target for further development of robust therapeutic approaches.

Significance: Histone chaperones are key regulators of transcriptional activity in damaged chromatin regions in the DNA damage response. Here we show that cytochromec(Cc) targets the histone chaperone SET/template-activating factor (TAF)-Iβ (SET/TAF-Iβ) in the cell nucleus upon DNA damage, resulting in the blocking of the SET/TAF-Iβ function. Ccis actually translocated into the nuclei of cells treated with specific DNA damage inducers and not upon death-receptor pathway or stress-induced stimuli. Cclocks the domains engaged in histone binding of SET/TAF-Iβ, inhibiting its nucleosome assembly activity. Structural characterization of the complex between Ccand SET/TAF-Iβ provides a valuable template for designing drugs aimed at silencing the oncogenic effect of SET/TAF-Iβ.