The Life Span Determinant p66Shc Localizes to Mitochondria Where It Associates with Mitochondrial Heat Shock Protein 70 and Regulates Trans-membrane Potential

Abstract

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

“…Hence, while p66Shc triggers the mitochondrial pathway of apoptosis in T cells, the mechanism responsible for mitochondrial recruitment does not appear to involve ROS-dependent PTP opening, at variance with fibroblasts. 13 Trolox treatment abrogated however the enhanced apoptotic response to A23187 of p66Shc-expressing cells (Figure 4), suggesting a role for p66Shc-dependent ROS production in additional apoptotic pathways independent of mitochondria. Support to this hypothesis comes from the functional analysis of the p66Shc S36 mutant (p66SA).…”

“…6,10 A fraction of p66Shc localizes to mitochondria as a complex with Hsp70, wherefrom it is released upon UV irradiation. 13 Mitochondrial p66Shc increases ROS production and favors opening of the permeability transition pore (PTP), 13 a high-conductance inner membrane channel involved in the apoptotic response to a number of stimuli. 14 The mechanism whereby p66Shc mediates mitochondrial ROS production has been recently clarified with the demonstration that p66Shc is a redox enzyme able to oxidize cytochrome c and produce H 2 O 2 , causing PTP opening and apoptosis.…”

“…The apoptogenic activity of p66Shc in fibroblasts has been ascribed to its capacity to promote production of ROS which, by inducing PTP opening, cause mitochondrial damage, cytochrome c release and activation of the caspase cascade. 10,13,15 To understand the mechanisms underlying T cell priming to apoptosis by p66Shc, we used Jurkat T cell transfectants stably expressing either wild-type p66Shc or a substitution mutant where S36 was mutated to alanine (p66SA) (Figure 1a). Apoptosis was induced using the Ca 2 þ ionophore A23187.…”

“…p66Shc induces apoptosis in fibroblasts by triggering PTP opening. 13 Opening of the PTP in response to a number of stimuli, including Ca 2 þ , arachidonic acid and ROS, results in osmotic swelling of the mitochondria followed by outer membrane rupture, cytochrome c release, ATP depletion and cell death. 18 To investigate the potential role of PTP opening in mitochondrial membrane permeabilization and cytochrome c release in T cells, p66Shc-expressing cells were treated with cyclosporin A (CsA), which desensitizes the PTP by interacting with cyclophilin D. 18 Neither the kinetics nor the extent of mitochondrial depolarization were modified by CsA (Figure 2e).…”

Evidence for only two independent pathways for decreasing senescence in Caenorhabditis elegans

“…In addition, mice lacking p66shc live 30% longer than wild type (Migliaccio et al 1999). Recent work has shown that p66shc is associated with mitochondrial membranes and has been shown to regulate mitochondrial transmembrane potential (Orsini et al 2004). …”