Structural and Functional Analysis of Novel Human Cytochrome c Targets in Apoptosis

Abstract

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. Molecular & Cellular

Respiratory complexes III and IV can each bind two molecules of cytochrome c at low ionic strength

“…These residues are surrounding the heme crevice and constitute the interaction surface (Fig. 2c), which is very well-conserved among complexes involving c-type cytochromes [14,29,[46][47][48][49][50][51][52][53][54][55]. In addition, significant CSP are detected (Dd avg -P 0.05 ppm) for Lys7, Lys13, Val20, Ser47, Lys73, Val83, Lys88 and Ala92.…”

“…The organization of the mitochondrial electron transport chain is a subject of intense debate at present, where two different models are being considered: the fluid model, that proposes a random organization for individual respiratory protein components, and the solid model, suggesting a stable association between individual complexes [6][7][8]. Apart their role in respiration, Cc and Cc 1 are clearly involved in the development of programmed cell death [9][10][11][12][13][14][15]. Such a dual role of Cc is regulated by post-translational modifications -namely, phosphorylation and nitration of tyrosine residues -that affect the binding of Cc to its physiological counterparts, either in the mitochondria or in the cytoplasm [16][17][18][19][20][21][22][23].…”

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochromec

“…Recently, SET/TAF-1β was found to be recruited to DNA breaks to modulate the DNA damage response through the retention of certain chromatin proteins, resulting in chromatin compaction (16). We have identified SET/TAF-Iβ as a target protein for cytochrome c (Cc) following the release of the latter from mitochondria in human cells treated with apoptotic agents (17,18). Although the hemeprotein Cc serves as an electron carrier between complexes III and IV in the mitochondrial respiratory chain, its role in nuclei has yet to be convincingly elucidated.…”

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…In mammalian cells, extramitochondrial Cc interacts with apoptosis activating factor 1 (Apaf-1) in the cytoplasm to spark the caspase proteolytic cascade (15). It has recently been shown that Cc can interact with several other proteins outside the mitochondria in humans and plants (18)(19)(20)(21). The similarities between the Cc signaling networks in both organisms suggest that key programmed cell-death pathways are conserved throughout evolution (22).…”

“…In addition, Cc aids in controlling ROS levels by oxidizing superoxide anions (11) and exhibiting peroxidase activity (12); the latter, however, also yields lipid peroxidation (13,14). Furthermore, Cc plays a crucial role in programmed cell death, a process that is only partially understood (15)(16)(17)(18)(19)(20)(21). In this context, a fraction of Cc binds and oxidizes cardiolipin (CL) at the internal mitochondrial membrane, thereby facilitating the release of unbound Cc to the cytoplasm (16,17).…”