BACKGROUND & AIMS: :Oxidative stress affects the availability of key-regulators of vascular homeostasis and controls a number of signaling pathways relevant to myocardial and vascular disease. Reactive oxygen species are generated by different intracellular molecular pathways principally located in mitochondria. The notion that mice carrying a targeted mutation of the p66(Shc) gene display prolonged lifespan, reduced production of intracellular oxidants, and increased resistance to oxidative stress-induced apoptosis prompted a series of studies aimed at defining the biochemical function of p66(Shc) and its possible implication in cardiovascular diseases. Indeed, p66(Shc-/-) mice are protected against vascular, cardiac, and renal impairment attributable to hypercholesterolemia, aging, diabetes, and ischemia/reperfusion. The present review focuses on the biochemical and physiological function of the p66(Shc) adaptor protein as well as on the mechanisms linking p66(Shc)-associated generation of free radicals to the pathophysiology of aging and cardiovascular disease. On the whole, the evidence so far reported and here discussed supports the concept that pharmacological modulation of p66(Shc) expression and activity may be a novel and effective target for the treatment of atherosclerotic vascular disease as well as myocardial adaptation to hypertrophic, inflammatory and neuro-hormonal stimuli in the overloaded heart.

背景与目标： 氧化应激影响血管内稳态关键调节剂的可用性，并控制与心肌和血管疾病相关的许多信号通路。活性氧由主要位于线粒体的不同细胞内分子途径产生。携带p66(Shc) 基因靶向突变的小鼠显示出延长的寿命，细胞内氧化剂的产生减少以及对氧化应激诱导的细胞凋亡的抵抗力增强的观点，引发了一系列旨在确定p66(Shc) 的生化功能的研究。) 及其在心血管疾病中的可能意义。实际上，p66(Shc-/-) 小鼠可以防止由于高胆固醇血症，衰老，糖尿病和缺血/再灌注而引起的血管，心脏和肾脏损害。本综述着重于p66(Shc) 衔接蛋白的生化和生理功能，以及将p66(Shc) 相关的自由基产生与衰老和心血管疾病的病理生理联系起来的机制。总体而言，迄今为止报道和此处讨论的证据支持以下概念: p66(Shc) 表达和活性的药理调节可能是治疗动脉粥样硬化性血管疾病以及心肌对肥厚，炎症和神经的适应性的新颖有效靶标。负荷过重的心脏中的激素刺激。

BACKGROUND & AIMS: :Our previous studies have indicated an essential role of p52shc in mediating IGF-I activation of MAPK in smooth muscle cells (SMC). However, the role of the p66 isoform of shc in IGF-I signal transduction is unclear. In the current study, two approaches were employed to investigate the role of p66shc in mediating IGF-I signaling. Knockdown p66shc by small interfering RNA enhanced IGF-I-stimulated p52shc tyrosine phosphorylation and growth factor receptor-bound protein-2 (Grb2) association, resulting in increased IGF-I-dependent MAPK activation. This was associated with enhanced IGF-I-stimulated cell proliferation. In contrast, knockdown of p66shc did not affect IGF-I-stimulated IGF-I receptor tyrosine phosphorylation. Overexpression of p66shc impaired IGF-I-stimulated p52shc tyrosine phosphorylation and p52shc-Grb2 association. In addition, IGF-I-dependent MAPK activation was also impaired, and SMC proliferation in response to IGF-I was inhibited. IGF-I-dependent cell migration was enhanced by p66shc knockdown and attenuated by p66shc overexpression. Mechanistic studies indicated that p66shc inhibited IGF-I signal transduction via competitively inhibiting the binding of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to SHP substrate-1 (SHPS-1), leading to the disruption of SHPS-1/SHP-2/Src/p52shc complex formation, an event that has been shown previously to be essential for p52shc phosphorylation and Grb2 recruitment. These findings indicate that p66shc functions to negatively regulate the formation of a signaling complex that is required for p52shc activation in response to IGF-I, thus leading to attenuation of IGF-I-stimulated cell proliferation and migration.

背景与目标： : 我们以前的研究表明p52shc在介导平滑肌细胞 (SMC) 中MAPK的igf-i激活中起重要作用。然而，shc的p66亚型在igf-i信号转导中的作用尚不清楚。在本研究中，采用了两种方法来研究p66shc在介导igf-i信号中的作用。通过小干扰RNA敲除p66shc增强了igf-i刺激的p52shc酪氨酸磷酸化和生长因子受体结合蛋白2 (Grb2) 的关联，从而导致igf-i依赖性MAPK激活增加。这与igf-i刺激的细胞增殖增强有关。相反，敲除p66shc不会影响igf-i刺激的igf-i受体酪氨酸磷酸化。p66shc的过表达损害了igf-i刺激的p52shc酪氨酸磷酸化和p52shc-Grb2缔合。此外，igf-i依赖性MAPK激活也受到损害，并且抑制了响应igf-i的SMC增殖。p66shc敲低增强了igf-i依赖性细胞迁移，p66shc过表达减弱了igf-i依赖性细胞迁移。机理研究表明，p66shc通过竞争性抑制含Src同源结构域的蛋白酪氨酸磷酸酶-2 (SHP-2) 与SHP底物-1 (SHPS-1) 的结合来抑制igf-i信号转导，导致SHPS-1/SHP-2/Src/p52shc复合物形成的破坏，先前已证明对p52shc磷酸化和Grb2募集至关重要的事件。这些发现表明，p66shc对响应igf-i的p52shc激活所需的信号复合物的形成产生负调节作用，从而导致igf-i刺激的细胞增殖和迁移的减弱。

BACKGROUND & AIMS: :Intestinal epithelial oxidative stress and apoptosis constitute key pathogenic mechanisms underlying intestinal ischemia/reperfusion (I/R) injury. We previously reported that the adaptor 66 kDa isoform of the adaptor molecule ShcA (p66Shc)-mediated pro-apoptotic pathway was activated after intestinal I/R. However, the upstream regulators of the p66Shc pathway involved in intestinal I/R remain to be fully identified. Here, we focused on the role of a prolyl-isomerase, peptidyl-prolyl cis-trans isomerase (Pin1), in the regulation of p66Shc activity during intestinal I/R. Intestinal I/R was induced in rats by superior mesenteric artery (SMA) occlusion. Juglone (Pin1 inhibitor) or vehicle was injected intraperitoneally before I/R challenge. Caco-2 cells were exposed to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model. We found that p66Shc was significantly up-regulated in the I/R intestine and that this up-regulation resulted in the accumulation of intestinal mitochondrial reactive oxygen species (ROS) and massive epithelial apoptosis. Moreover, intestinal I/R resulted in elevated protein expression and enzyme activity of Pin1 as well as increased interaction between Pin1 and p66Shc. This Pin1 activation was responsible for the translocation of p66Shc to the mitochondria during intestinal I/R, as Pin1 suppression by juglone or siRNA markedly blunted p66Shc mitochondrial translocation and the subsequent ROS generation and cellular apoptosis. Additionally, Pin1 inhibition alleviated gut damage and secondary lung injury, leading to improvement of survival after I/R. Collectively, our findings demonstrate for the first time that Pin1 inhibition protects against intestinal I/R injury, which could be partially attributed to the p66Shc-mediated mitochondrial apoptosis pathway. This may represent a novel prophylactic target for intestinal I/R injury.

背景与目标： 肠上皮氧化应激和细胞凋亡是肠缺血/再灌注 (I/R) 损伤的关键致病机制。我们先前曾报道过，肠I/R后，衔接子分子ShcA (p66Shc) 介导的促凋亡途径的衔接子66 kDa亚型被激活。然而，参与肠道I/R的p66Shc途径的上游调节剂仍有待完全鉴定。在这里，我们专注于脯氨酰异构酶肽酰脯氨酰顺反式异构酶 (Pin1) 在肠道I/R期间调节p66Shc活性中的作用。肠系膜上动脉 (SMA) 闭塞诱导大鼠肠I/R。在I/R激发之前，腹膜内注射Juglone (Pin1抑制剂) 或赋形剂。Caco-2细胞在体外暴露于缺氧/复氧 (H/R) 以模拟体内I/R模型。我们发现p66Shc在I/R肠中显着上调，并且这种上调导致肠线粒体活性氧 (ROS) 的积累和大量上皮细胞凋亡。此外，肠道I/R导致Pin1的蛋白表达和酶活性升高，以及Pin1与p66Shc之间的相互作用增加。这种Pin1激活是肠I/R期间p66Shc向线粒体易位的原因，因为juglone或siRNA对Pin1的抑制显着减弱了p66Shc线粒体易位以及随后的ROS生成和细胞凋亡。此外，Pin1抑制减轻了肠道损伤和继发性肺损伤，从而改善了I/R后的生存率。总的来说，我们的发现首次证明了Pin1抑制可以防止肠I/R损伤，这可能部分归因于p66Shc-mediated的线粒体凋亡途径。这可能代表了肠I/R损伤的新型预防靶标。

BACKGROUND & AIMS: BACKGROUND:The risk of diabetic complications is modified by genetic and epigenetic factors. p66Shc drives the hyperglycaemic cell damage and its deletion prevents experimental diabetic complications. We herein tested whether p66Shc expression in peripheral blood mononuclear cells (PBMCs) predicts adverse outcomes in people with diabetes. METHODS:In a cohort of 100 patients with diabetes (16 type 1 and 84 type 2), we quantified baseline p66Shc expression in PBMCs by quantitative PCR. Patients were extensively characterized for demographics, anthropometrics, biochemical data, prevalence of complications, and medications. With a pseudo-prospective design, we retrieved cardiovascular death, major adverse cardiovascular events (MACE), and new occurrence of micro- or macroangiopathy during follow-up. RESULTS:At baseline, patients were on average 60 year old, with 10-year diabetes duration, and overall poor glycaemic control (HbA1c 7.8%). Patients with high versus low p66Shc expression (based on median value) had very similar baseline characteristics. Average p66Shc expression did not differ by presence/absence of complications. During a median 5.6-year follow-up, the primary endpoint of cardiovascular death or MACE occurred in 22 patients, but no relation was detected between cardiovascular outcomes and p66Shc expression. In patients who developed new complications at follow-up, baseline p66Shc was significantly higher, especially for macroangiopathy. The incidence of new macroangiopathy was > 3-times higher in patients with high versus those with low baseline p66Shc expression. CONCLUSIONS:p66Shc expression in PBMCs was not associated with prevalent diabetic complications but predicted new onset of complications, especially macroangiopathy, although no relation with hard cardiovascular endpoints was detected.

背景与目标：

BACKGROUND & AIMS: :Intrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc(-/-) mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.

背景与目标： : 内在的凋亡缺陷在很大程度上是慢性淋巴细胞白血病 (CLL) 中恶性b细胞延长存活的基础。在这里，我们显示Shc家族适配器p66Shc将b细胞受体 (BCR) 与Erk和Akt依赖性生存途径解偶联，从而增强b细胞凋亡。与正常外周b细胞相比，发现CLL b细胞中p66Shc表达严重受损。此外，根据IGHV基因的突变状态，在预后良好或不利的患者中观察到p66Shc表达的显着差异，在预后不利的组中表达最低。对与CLL凋亡缺陷有关的基因表达的分析表明，CLL患者Bcl-2家族的促凋亡和抗凋亡成员的平衡发生了变化。CLL b细胞的重构实验以及从p66Shc(-/-) 小鼠的b细胞上获得的数据表明，p66Shc表达与Bcl-2家族对促凋亡成员的偏见相关。数据表明p66Shc是b细胞凋亡的新型调节剂，可减弱BCR依赖性存活信号并调节Bcl-2家族表达。此外，他们提供了证据，证明CLL b细胞中的p66Shc表达缺陷可能是导致这些细胞中抗凋亡Bcl-2家族成员失衡的原因。

BACKGROUND & AIMS: BACKGROUND/AIMS:Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear. Previous study demonstrated the central role of mitochondrial ROS (mtROS) in the pathogenesis of cisplatin nephrotoxicity. The purpose of this study was to explore the mechanism of mtROS regulation in cisplatin nephrotoxicity. METHODS:p53, MnSOD and p66shc were detected at mRNA and protein levels by qPCR and western blot in HK2 cells. mtROS levels were determined by DCFDA and MitoSOX staining. Cell viability and cell apoptosis were accessed by CCK-8 assay, TUNEL assay and flow cytometry, respectivesly. siRNAs were used to knock down p53 and p66shc expression and subsequent changes were observed. In vivo assays using a mouse model of cisplatin-induced acute kidney injury were used to validate the in vitro results. RESULTS:In HK2 cells, cisplatin exposure decreased the MnSOD and increased the expression of p53 and p66shc. MnTBAP, a MnSOD mimic, blocked cisplatin-induced the generation of mtROS and cell injury. P66shc and p53 siRNAs rendered renal cells resistant to cisplatin-induced mtROS production and cell death. Furthermore, knockdown of p53 restored MnSOD and inhibiting p66shc. Consistent with these results, we revealed that p53 inhibitor reduced cisplatin-induced oxidative stress and apoptosis by regulating MnSOD and p66shc in the kidney of cisplatin-treated mice. CONCLUSION:Our study identifies activation of p53 signalling as a potential strategy for reducing the nephrotoxicity associated with cisplatin treatments and, as a result, broadens the therapeutic window of this chemotherapeutic agent.

背景与目标：

BACKGROUND & AIMS: :Oxidative stress plays a role in cardiovascular dysfunction. This is of interest in diabetes, a clinical condition characterized by oxidative stress and increased prevalence of cardiovascular disease. The role of p66(shc) in oxidative stress-related response has been demonstrated by resistance to and reduction of oxidative stress and prolonged lifespan in p66(shc-/-) mice. In this study we assess p66(shc) gene expression in peripheral blood mononuclear cells (PBM) from type 2 diabetic patients and healthy subjects. The p66(shc) mRNA level was assessed using RT-PCR with two sets of primers mapping for different p66(shc) regions. p66(shc) is expressed in both monocytes and lymphocytes. The level of p66(shc) mRNA was significantly higher in type 2 diabetic patients compared with controls (0.38 +/- 0.07 densitometric units vs. 0.13 +/- 0.08; P < 0.0001). In addition, total plasma 8-isoprostane levels, a marker of oxidative stress, were higher in type 2 diabetics (0.72 +/- 0.04 ng/ml) than in normal subjects (0.43 +/- 0.04, P < 0.001) and were significantly correlated to the p66(shc) mRNA level in PBM from type 2 diabetics (r(2) = 0.47; P = 0.0284). In conclusion, diabetes induces p66(shc) gene expression in circulating PBM; this up-regulation in expression is significantly associated with markers of oxidative stress. p66(shc) gene expression in PBM may represent a useful tool to investigate the oxidative stress involved in the pathogenesis of long-term diabetic complications.

背景与目标： : 氧化应激在心血管功能障碍中起作用。这在糖尿病中引起了人们的兴趣，糖尿病是一种以氧化应激和心血管疾病患病率增加为特征的临床疾病。p66(shc) 在氧化应激相关反应中的作用已通过对p66(shc-/-) 小鼠的抵抗和减少氧化应激和延长寿命而得到证明。在这项研究中，我们评估了2型糖尿病患者和健康受试者外周血单核细胞 (PBM) 中p66(shc) 基因的表达。使用rt-pcr评估p66(shc) mRNA水平，并使用两组引物定位不同的p66(shc) 区域。p66(shc) 在单核细胞和淋巴细胞中均表达。与对照组相比，2型糖尿病患者的p66(shc) mRNA水平显着更高 (0.38/- 0.07光密度单位vs. 0.13/- 0.08; P <0.0001)。此外，2型糖尿病患者的总血浆8-异前列腺素水平 (氧化应激的标志物) (0.72/- 0.04 ng/ml) 高于正常受试者 (0.43/- 0.04，P <0.001)，并且与2型糖尿病患者PBM中的p66(shc) mRNA水平显着相关 (r(2) = 0.47; P = 0.0284)。总之，糖尿病在循环PBM中诱导p66(shc) 基因表达; 这种表达上调与氧化应激标志物显着相关。PBM中p66(shc) 基因的表达可能是研究与长期糖尿病并发症发病机制有关的氧化应激的有用工具。

BACKGROUND & AIMS: :Reactive oxygen species (ROS), mainly originated from mitochondrial respiration, are critical inducers of oxidative damage and involved in tissue dysfunction. It is not clear, however, whether oxidative stress is the result of an active gene program or it is the by-product of physiological processes. Recent findings demonstrate that ROS are produced by mitochondria in a controlled way through specialized enzymes, including p66Shc, and take part in cellular process aimed to ensure adaptation and fitness. Therefore, genes generating specifically ROS are selected determinants of life span in response to different environmental conditions.

背景与目标： 活性氧 (ROS) 主要来源于线粒体呼吸，是氧化损伤的关键诱导剂，参与组织功能障碍。然而，尚不清楚氧化应激是活性基因程序的结果还是生理过程的副产品。最近的发现表明，ROS是由线粒体通过特殊的酶 (包括p66Shc) 以受控的方式产生的，并参与旨在确保适应性和适应性的细胞过程。因此，特定产生ROS的基因是响应不同环境条件而选择的寿命决定因素。

BACKGROUND & AIMS: :A high incidence of permanent embryo arrest occurs during the first week of in vitro development. We hypothesize that this developmental arrest event is regulated by the stress adaptor protein p66shc, a genetic determinant of life span in mammals, which regulates ROS metabolism, apoptosis, and cellular senescence. The aim of this study was to assess the relationship between intracellular oxidative stress levels with the incidence of embryo arrest and the expression of senescent-associated genes in embryos produced under different oxygen tensions. Embryos cultured under 20% oxygen conditions showed approximately 10-fold increase in oxidative stress, 2-fold increase in the percentage of 2- to 4-cell arrest, and significantly lower developmental capabilities compared to embryos cultured under a 5% oxygen environment. Quantification by real-time PCR and by semiquantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels, respectively, in embryos cultured in 20% versus those cultured in 5% oxygen atmosphere. No significant changes in p53 mRNA and protein levels were detected among embryos derived from both oxygen tensions. Taken together, these results demonstrate that p66shc, but not p53, is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ROS. These results further substantiate that p66shc and oxidative stress are associated with a p53-independent embryonic arrest event for in vitro-produced embryos.

背景与目标： : 在体外发育的第一周内，永久性胚胎停滞的发生率很高。我们假设这种发育停滞事件是由应激衔接蛋白p66shc调节的，p66shc是哺乳动物寿命的遗传决定因素，它调节ROS代谢，凋亡和细胞衰老。这项研究的目的是评估细胞内氧化应激水平与胚胎停滞发生率之间的关系，以及在不同氧气张力下产生的胚胎中衰老相关基因的表达。与在20% 氧环境下培养的胚胎相比，在5% 氧环境下培养的胚胎显示氧化应激增加约10倍，在2-4-细胞阻滞百分比增加2倍，以及显著更低的发育能力。通过实时PCR和半定量免疫荧光定量显示，在20% 培养的胚胎中，与在5% 氧气气氛中培养的胚胎相比，p66shc mRNA和蛋白质水平分别显著更高。在来自两种氧张力的胚胎中，未检测到p53 mRNA和蛋白质水平的显着变化。总之，这些结果表明，在表现出更高的胚胎停滞频率和细胞内ROS数量的胚胎群体中，p66shc而非p53明显更丰富。这些结果进一步证实了p66shc和氧化应激与体外产生的胚胎的p53-independent胚胎停滞事件有关。

BACKGROUND & AIMS: :The adaptor protein p66shc promotes cellular oxidative stress and apoptosis. Here, we demonstrate a novel mechanistic relationship between p66shc and the kruppel like factor-2 (KLF2) transcription factor and show that this relationship has biological relevance to p66shc-regulated cellular oxidant level, as well as KLF2-induced target gene expression. Genetic knockout of p66shc in mouse embryonic fibroblasts (MEFs) stimulates activity of the core KLF2 promoter and increases KLF2 mRNA and protein expression. Similarly, shRNA-induced knockdown of p66shc increases KLF2-promoter activity in HeLa cells. The increase in KLF2-promoter activity in p66shc-knockout MEFs is dependent on a myocyte enhancing factor-2A (MEF2A)-binding sequence in the core KLF2 promoter. Short-hairpin RNA-induced knockdown of p66shc in endothelial cells also stimulates KLF2 mRNA and protein expression, as well as expression of the endothelial KLF2 target gene thrombomodulin. MEF2A protein and mRNA are more abundant in p66shc-knockout MEFs, resulting in greater occupancy of the KLF2 promoter by MEF2A. In endothelial cells, the increase in KLF2 and thrombomodulin protein by shRNA-induced decrease in p66shc expression is partly abrogated by knockdown of MEF2A. Finally, knockdown of KLF2 abolishes the decrease in the cellular reactive oxygen species hydrogen peroxide observed with knockdown of p66shc, and KLF2 overexpression suppresses cellular hydrogen peroxide levels, independent of p66shc expression. These findings illustrate a novel mechanism by which p66shc promotes cellular oxidative stress, through suppression of MEF2A expression and consequent repression of KLF2 transcription.

背景与目标： : 衔接蛋白p66shc促进细胞氧化应激和凋亡。在这里，我们证明了p66shc和kruppel样因子2 (KLF2) 转录因子之间的一种新的机制关系，并表明这种关系与p66shc-regulated细胞氧化剂水平以及KLF2-induced靶基因表达具有生物学相关性。小鼠胚胎成纤维细胞 (mef) 中p66shc的基因敲除刺激核心KLF2启动子的活性并增加KLF2 mRNA和蛋白质的表达。类似地，shRNA诱导的p66shc敲除增加HeLa细胞中的KLF2-promoter活性。p66shc-knockout MEFs中KLF2-promoter活性的增加取决于核心KLF2启动子中的肌细胞增强factor-2A (MEF2A) 结合序列。短发夹RNA诱导的内皮细胞中p66shc的敲低也刺激KLF2 mRNA和蛋白的表达，以及内皮KLF2靶基因血栓调节蛋白的表达。MEF2A蛋白和mRNA在p66shc-knockout mef中更丰富，导致MEF2A对KLF2启动子的占有更大。在内皮细胞中，shRNA诱导的p66shc表达降低引起的KLF2和血栓调节蛋白的增加被MEF2A的敲除部分消除。最后，KLF2的敲低消除了通过敲低p66shc观察到的细胞活性氧过氧化氢的减少，而KLF2的过表达抑制了细胞过氧化氢的水平，而与p66shc的表达无关。这些发现说明了p66shc通过抑制MEF2A表达和随后抑制KLF2转录来促进细胞氧化应激的新机制。

BACKGROUND & AIMS: :Accumulation of oxidative stress is considered to be a causative mediator of kidney disease, and oxidative stress can affect some key regulators of kidney homeostasis and control a number of signaling pathways that are relevant to kidney disease. The p66Shc adaptor protein was discovered more than two decades ago as a pivotal regulator of oxidative stress. Given the importance of oxidative stress in kidney homeostasis, several molecular and cellular studies using a p66Shc antagonist have depicted a role for p66Shc in renal pathophysiology. The specificity of p66Shc functions may depend upon their intracellular localization and expression in the kidney. This review focuses on the biochemical functions of the p66Shc adaptor protein, as well as its potential implications in the pathophysiology of kidney disease. In addition, the concept that pharmacologic modulation of p66Shc expression and activity may serve as a novel and effective target for the treatment of kidney disease is discussed.

背景与目标： : 氧化应激的积累被认为是肾脏疾病的致病介质，氧化应激可以影响肾脏稳态的一些关键调节因子，并控制许多与肾脏疾病相关的信号通路。p66Shc衔接蛋白是二十多年前发现的氧化应激的关键调节剂。鉴于氧化应激在肾脏稳态中的重要性，一些使用p66Shc拮抗剂的分子和细胞研究已经描述了p66Shc在肾脏病理生理学中的作用。p66Shc功能的特异性可能取决于其在肾脏中的细胞内定位和表达。这篇综述着重于p66Shc衔接蛋白的生化功能，以及其在肾脏疾病病理生理学中的潜在意义。此外，还讨论了p66Shc表达和活性的药理调节可能是治疗肾脏疾病的新的有效靶标的概念。

BACKGROUND & AIMS: :Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

背景与目标： : 巨自噬/自噬已成为淋巴细胞稳态，激活和分化的中心过程。基于我们的发现，SHC1 (p66SHC) 促凋亡ROS升高的SHC家族衔接子的p66亚型抑制了这些细胞中的MTOR信号传导，在此我们研究了p66SHC在b细胞自噬中的作用。我们显示p66SHC通过其CYCS (细胞色素c，体细胞) 结合域破坏线粒体功能，从而损害ATP的产生，从而导致AMPK活化和自噬通量增强。虽然p66SHC与CYCS的结合足以触发细胞凋亡，但p66SHC-mediated自噬还取决于其通过其N末端内的特异性结合基序与膜相关LC3-II相互作用的能力。重要的是，p66SHC还通过诱导线粒体去极化，线粒体外膜的蛋白质泛素化以及活性AMPK的局部募集而对线粒体稳态产生影响。这些事件引发线粒体吞噬，其完全执行依赖于p66SHC作为LC3-II受体的作用，该受体将吞噬膜带入线粒体。重要的是，p66SHC还促进了缺氧诱导的b细胞线粒体吞噬。此外，p66SHC缺乏症可增强b细胞向浆细胞的分化，这受细胞内ROS水平和低氧生发中心环境的控制。结果表明，线粒体p66SHC是b细胞自噬和线粒体吞噬的新型调节剂，并p66SHC-mediated自噬和凋亡在b细胞存活和分化中的协调作用。缩写: ACTB: actin beta; AMPK: AMP活化蛋白激酶; ATP: 三磷酸腺苷; ATG: 自噬相关; CYCS: 细胞色素c，体细胞; CLQ: 氯喹; COX: 环氧合酶; CTR: 对照; GFP: 绿色荧光蛋白; HIFIA/Hif α: 缺氧诱导因子1亚基 α; IMS: 膜间空间; LIR: LC3相互作用区; MAP1LC3B/LC3B: 微管相关蛋白1轻链3β; MTOR/mTOR: 雷帕霉素激酶的机制靶标; OA: 寡霉素和抗霉素A; OMM: 线粒体外膜; PHB: 禁止蛋白; PBS: 磷酸盐缓冲盐水; PINK1: PTEN诱导的推定激酶1; RFP: 红色荧光蛋白; ROS: 活性氧; SHC: 含src同源2结构域的转化蛋白; TMRM: 四甲基罗丹明，甲酯; TOMM: 线粒体外膜的转位酶; ULK1: unc-51样自噬激活激酶1; WT: 野生型。

BACKGROUND & AIMS: :p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.

背景与目标： : p66Shc是一种长寿衔接蛋白，被证明是与衰老和心血管疾病有关的活性氧 (ROS) 代谢的关键调节剂。血管内皮生长因子 (VEGF) 主要通过VEGF受体2 (VEGFR2) 刺激内皮细胞 (EC) 的迁移和增殖。我们已经证明，源自Rac1-dependent NADPH氧化酶的ROS参与了ECs中的VEGFR2自磷酸化和血管生成相关反应。然而，p66Shc在ECs中VEGF信号传导和生理反应中的作用尚不清楚。在这里，我们显示VEGF通过JNK/ERK或PKC途径促进Ser36上的p66Shc磷酸化，以及Rac1与ECs中p66Shc的非磷酸化形式结合。用短干扰RNA耗尽内源性p66Shc会抑制VEGF诱导的Rac1活性和ROS产生。富集小窝蛋白的脂质筏的分级分离表明，p66Shc在小窝/脂质筏中的VEGFR2磷酸化以及下游p38MAP激酶激活中起关键作用。这反过来又刺激了VEGF诱导的EC迁移，增殖和毛细血管样管的形成。这些研究揭示了p66Shc作为ROS依赖性VEGFR2信号与ECs中血管生成相关的阳性调节剂的新作用，并建议p66Shc作为各种血管生成依赖性疾病的潜在治疗靶标。

BACKGROUND & AIMS: Aims:Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66Shc contributes to obesity-related vascular disease. Methods and results:ROS-driven endothelial dysfunction was observed in visceral fat arteries (VFAs) isolated from obese subjects when compared with normal weight controls. Gene profiling of chromatin-modifying enzymes in VFA revealed a significant dysregulation of methyltransferase SUV39H1 (fold change, -6.9, P < 0.01), demethylase JMJD2C (fold change, 3.2, P < 0.01), and acetyltransferase SRC-1 (fold change, 5.8, P < 0.01) in obese vs. control VFA. These changes were associated with reduced di-(H3K9me2) and trimethylation (H3K9me3) as well as acetylation (H3K9ac) of histone 3 lysine 9 (H3K9) on p66Shc promoter. Reprogramming SUV39H1, JMJD2C, and SRC-1 in isolated endothelial cells as well as in aortas from obese mice (LepOb/Ob) suppressed p66Shc-derived ROS, restored nitric oxide levels, and rescued endothelial dysfunction. Consistently, in vivo editing of chromatin remodellers blunted obesity-related vascular p66Shc expression. We show that SUV39H1 is the upstream effector orchestrating JMJD2C/SRC-1 recruitment to p66Shc promoter. Indeed, SUV39H1 overexpression in obese mice erased H3K9-related changes on p66Shc promoter, while SUV39H1 genetic deletion in lean mice recapitulated obesity-induced H3K9 remodelling and p66Shc transcription. Conclusion:These results uncover a novel epigenetic mechanism underlying endothelial dysfunction in obesity. Targeting SUV39H1 may attenuate oxidative transcriptional programmes and thus prevent vascular disease in obese individuals.

背景与目标：

BACKGROUND & AIMS: :Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

背景与目标： : 转移性去势抵抗 (CR) 前列腺癌 (PCa) 是一种致命疾病，目前尚无有效的治疗方法。p66Shc是先前显示的氧化酶，可通过产生活性氧 (ROS) 来促进雄激素非依赖性细胞生长，并且在临床PCa和多个CR PCa细胞系中升高。我们假设p66Shc还通过ROS增加PCa细胞的迁移活性，并研究相关机制。使用tranwell测定法，我们的研究揭示了p66Shc蛋白的水平与几种PCa细胞系的细胞迁移能力相关。此外，我们显示过氧化氢处理以剂量依赖性方式诱导表达低水平p66Shc的PCa细胞迁移，而抗氧化剂则抑制迁移。相反，表达高水平内源性p66Shc或通过cDNA转染的PCa细胞具有增加的细胞迁移，这在p66Shc shRNA转染或表达氧化酶缺陷的显性阴性p66Shc W134F突变体后会减轻。蛋白质微阵列和免疫印迹分析显示多种蛋白质，包括ErbB-2、AKT、mTOR、ERK、FOXM1、PYK2和Rac1，在p66Shc-elevated细胞中被激活。使用各自的小分子抑制剂检查了它们在PCa迁移中的参与。使用cDNA转染进一步验证了Rac1的作用，并且显着地，发现p66Shc通过Rac1激活促进了片状脂蛋白的形成。总而言之，我们目前的研究结果清楚地表明，p66Shc还通过ROS介导的迁移相关蛋白 (尤其是rac1) 的激活来调节PCa细胞的迁移。