Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.     

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

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.     

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.     

丝氨酸蛋白酶HtrA2/Omi可参与调节线粒体中的适配蛋白p66Shc

目的:p66Shc在线粒体内积累和HtrA2/Omi的功能缺陷都能导致线粒体损伤,诱导细胞凋亡.探讨在线粒体中HtrA2对p66Shc的调控作用.方法:构建p66Shc和成熟型HtrA2的真核表达质粒,共转染HEK293T细胞,免疫印迹法(Western blot)检测p66Shc蛋白;构建原核表达质粒,大肠杆菌纯化蛋白,体外切割实验,SDS-PAGE分离后考马斯亮蓝染色检测;提取HtrA2功能缺陷小鼠( mnd2)大脑组织的线粒体,检测线粒体内p66Shc的蛋白水平.结果:细胞实验和体外实验证明HtrA2可以切割p66Shc,且在mnd2小鼠大脑中,线粒体内p66Shc的蛋白水平明显升高(P＜0.05).结论:p66Shc是HtrA2的直接底物,且HtrA2参与调节线粒体中p66Shc的蛋白水平,揭示了HtrA2发挥神经保护功能新的可能机制.     

Cell death: insights into the ultrastructure of mitochondria

An essential step in many forms of cell death is the release from mitochondria of “death effectors” which once in the cytoplasm activate signalling pathways leading to cellular demise. In this context mitochondria are known as regulators of cell death functioning as a node where signals are integrated. The discovery that alterations and remodelling of ultrastructural architecture of mitochondria are required to trigger the complete release of cytochrome c in the cytoplasm and the notion that mitochondrial architecture determines/influences the function of this organelle has fostered investigations on mitochondrial dynamics and on the machinery that regulates this process during cell death. In this review I shall summarize the current knowledge of mitochondrial inner membrane remodelling during cell death and discuss the role of mitochondrial proteins in governing structural alterations. I shall then discuss the role of the adaptor protein p66Shc as a regulator of mitochondrial metabolism during apoptosis.     

The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.     

Prolonged swimming promotes cellular oxidative stress and p66Shc phosphorylation,but does not induce oxidative stress in mitochondria in the rat heart

Abstract

Exercise-induced changes in p66Shc-dependent signaling pathway are still not fully understood. The p66Shc protein is one of the key players in cell signaling, particularly in response to oxidative stress. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the phosphorylation of p66Shc as well as the induction of mitochondrial and cellular oxidative stress in rat hearts.

Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their hearts were removed immediately for experiments.

The exercise protocol caused increased levels of the following oxidative stress parameters in cardiac cells: DNA damage, protein carbonyls, and lipid dienes. There was also increased phosphorylation of p66Shc without any alterations in Akt and extracellular signal-regulated kinases. Changes in the ferritin L levels and the L to H subunit ratio were also observed in the exercised hearts compared with the control hearts. Despite increased phosphorylation of p66Shc, no significant increase was observed in either mitochondrial H₂O₂ release or mitochondrial oxidative stress markers. Regardless of the changes in phosphorylation of p66Shc, the antioxidant enzyme activities (superoxide dismutase and catalase) and anti-apoptotic (Bcl2), and pro-apoptotic (Bax) protein levels were not affected by prolonged swimming. Further studies are required to investigate whether p66Shc phosphorylation is beneficial or detrimental to cardiac cells after exercise cessation.     

A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria

The human Src homology and collagen (Shc) gene encodes three protein isoforms of 46, 52, and 66 kDa that belong to a family of molecular adapters involved in several signal transduction pathways. Recently, the 66-kDa isoform has been shown to play a central role in controlling reactive oxygen species metabolism and life span in mammals. Despite the large amount of information available on the biology and biochemistry of Shc proteins, very little is known regarding the regulation of their subcellular localization. Here we demonstrate the specific and selective localization of p46Shc to the mitochondrial matrix. Through deletion mapping experiments, we show that targeting of p46Shc to mitochondria is mediated by its first 32 amino acids, which behave as a bona fide mitochondrial targeting sequence. We further demonstrate that the N-terminal location of the signal peptide is critical for its function. This accounts for the observation that p52Shc and p66Shc, containing the same sequence but more internally located, display a remarkably different subcellular localization. These findings indicate that p46Shc may exert a non-redundant biological function in signal transduction pathways involving mitochondria.     

Mitochondrial DNA copy number is regulated by cellular proliferation: a role for Ras and p66(Shc)

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.     

氧化应激下p66Shc的调控作用

氧化应激产生的过量活性氧簇（reactive oxygen species,ROS)可通过分子毒性作用或相关信号通路影响相关病理生理学过程. p66Shc是Shc蛋白家族的重要成员之一. 氧化应激下p66Shc能被蛋白激酶Cβ（protein kinase Cβ,PKCβ）、Jun氨基末端激酶（Jun N terminal kinase,JNK）和p53等激活,促进线粒体产生ROS.本文将对氧化应激下p66Shc的作用以及调控其作用的信号转导机制做一综述.     

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.     

Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex

The tumour suppressor activity of the p53 protein has been explained by its ability to induce apoptosis in response to a variety of cellular stresses. Thus, understanding the mechanism by which p53 functions in the execution of cell death pathways is of considerable importance in cancer biology. Recent studies have indicated that p53 has a direct signalling role at mitochondria in the induction of apoptosis, although the mechanisms involved are not completely understood. Here we show that, after cell stress, p53 interacts with the pro-apoptotic mitochondrial membrane protein Bak. Interaction of p53 with Bak causes oligomerization of Bak and release of cytochrome c from mitochondria. Notably, we show that formation of the p53-Bak complex coincides with loss of an interaction between Bak and the anti-apoptotic Bcl2-family member Mcl1. These results are consistent with a model in which p53 and Mcl1 have opposing effects on mitochondrial apoptosis by interacting with, and modulating the activity of, the death effector Bak.