心脏特异性高度表达的抗凋亡蛋白——ARC

与凋亡(apoptosis)相关的许多心脏疾病如心肌梗死、心肌病及心衰等严重威胁着人类的健康和生命.寻找有效手段防治这些心脏病是当前医学研究的热点.ARC(带有caspase富集功能域的凋亡抑制因子)是新近发现的唯一在心脏大量且特异表达的抗凋亡蛋白,其全称是带有caspase富集功能域的凋亡抑制因子.ARC可被持续性磷酸化并参与阻断凋亡发生途径的多个层面.因此,ARC是一种强大的抗心肌凋亡蛋白.     

ARC蛋白调控心肌细胞肥大和凋亡

中国科学院动物研究所以大鼠乳鼠的原代心肌细胞为模型,系统研究了ARC蛋白在心肌细胞肥大和心肌细胞凋亡中的调控作用。ARC是一个在心肌细胞中高表达的抗细胞凋亡蛋白,对于心肌细胞具有保护作用。     

癌变组织特异高表达抗凋亡蛋白——ARC

带有caspase富集功能域的凋亡抑制因子（ apoptosis repressor with a caspase recuitment domain, ARC ）是新近发现的重要抗凋亡蛋白。在正常组织中,ARC高度特异地表达在终末分化组织如心肌、骨骼肌和大脑,而在非终末分化组织不表达或微量表达。但是,当非终末分化组织发生癌变时表达大量的ARC。ARC过表达可以抑制阿霉素或射线诱导的癌细胞死亡,提示ARC在癌细胞中具有抗趋化或抗放射线损伤的作用。因此,ARC可能成为一种新的肿瘤标记物,也可能是决定肿瘤对各种治疗方法反应性的一个重要因素。     

心脏瓣膜病中线粒体凋亡调控因子与microRNA的相互作用

为了揭示心脏瓣膜病中线粒体凋亡调控因子与microRNA的相互作用,本研究检测了心脏瓣膜病患者瓣膜组织病变部位和周围健康组织中的线粒体凋亡调控因子PUMA、Drp1和ARC的表达,并分析了病变部位的ROS水平和SOD活性,同时考察了miR-214和miR-15a对凋亡因子PUMA和ARC的影响。研究显示,PUMA和Drp1在病变组织中表达水平显著升高,而ARC表达显著降低(p0.05)。病变组织中ROS水平显著升高,而SOD活性显著降低(p0.05)。瓣膜组织病变部位的miR-214表达水平显著升高,而miR-15a显著降低(p0.05)。应用miRNA抑制剂下调miR-214和miR-15a后,PUMA和ARC的表达水平被显著上调(p0.05)。本研究表明在心脏瓣膜病患者中,miR-214和miR-15a分别靶向ARC和PUMA。     

HAX-1研究进展

HAX-1为凋亡调节蛋白,其结构与Bcl-2家族相近,含有BH1、BH2结构域。HAX-1在成年人心肌细胞中通过抑制caspase9的活化从而起到抗凋亡作用,同时也是促凋亡蛋白Omi/HtrA2的底物,在凋亡过程中被其降解。另外,HAX-1能够与蛋白的3＇非翻译区结合,可能参与了对mRNA的调节。HAX-1在机体细胞内广泛表达,与多种蛋白存在相互作用,具有多种生物学功能。简要综述了近年来有关HAX-1蛋白的抗细胞凋亡、参与细胞迁移,以及在病毒感染、疾病中的作用。     

ARC的生理与病理生理意义

ARC全称是带有caspase富集功能域的凋亡抑制因子(apoptosis repressor with a caspase recruitment domain)。在正常状态下,ARC高度特异地表达是在终末分化组织如心肌、骨骼肌和大脑,而在非终末分化组织不表达或微量表达。在病理状态下,ARC在心肌、骨骼肌和神经系统损伤中发挥重要作用;在非终末分化组织来源的癌细胞系中高度表达,并呈组织差异性,因此ARC是一个新的肿瘤标记物。     

IAP家族分子与肿瘤靶向治疗

凋亡抑制因子(inhibitor of apoptosis proteins,IAPs)是一类高度保守的内源性抗细胞凋亡因子家族,主要通过抑制Caspase活性和参与调节核因子NF-κB的作用而抑制细胞凋亡。细胞抗凋亡机制在肿瘤发生、发展以及肿瘤耐药性形成中发挥重要作用。肿瘤细胞高表达IAPs是导致肿瘤细胞抵抗凋亡的关键。细胞凋亡调控异常与肿瘤细胞耐药密切相关,增强肿瘤细胞对化疗药物的敏感性成为近年来肿瘤治疗的重要策略之一。该文综述了IAP家族蛋白的结构、生物学特性及其作为肿瘤治疗靶点的研究进展。     

一种抗凋亡蛋白——存活素的研究

存活素(survivin)是一种新近发现的16．5kD细胞内蛋白,属于抗细胞凋亡蛋白家族。在胚胎和各类肿瘤中均表达,但在除胸腺和睾丸以外的成人组织中未被发现。阐明survivin在细胞凋亡和增生中的分子机制及其作用,在抗癌治疗和抑制对机体有害的细胞凋亡方面具有重要意义。     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

XIAP在胰腺癌化疗耐药及治疗中的研究进展

X连锁的凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)是凋亡抑制蛋白家族中的一员,具有抗凋亡作用.研究发现XIAP在胰腺癌中呈高表达,并且能诱导胰腺癌细胞及组织对化疗耐药.通过在基因水平及蛋白水平降低XIAP的表达对胰腺癌的治疗具有重要意义.AEG 35156是针对XIAP的反义寡核苷酸分子,能够抑制胰腺癌细胞及组织生长.RNAi能够稳定下调胰腺癌细胞中XIAP水平,从而加强TRAIL诱导的细胞凋亡,并能提高胰腺癌细胞对化疗的敏感性.针对XIAP的小分化合物能够抑制XIAP的功能,释放被XIAP抑制的凋亡起始和效应分子以及XIAP抑制的其他促凋亡蛋白,提高多种肿瘤细胞的凋亡指数及对放化疗的敏感性.XAFl能抑制XIAP的抗凋亡作用.本文就XIAP在胰腺癌化疗耐药及治疗中的研究进展做一综述.     

Apoptosis repressor with caspase recruitment domain is dramatically reduced in cardiac, skeletal, and vascular smooth muscle during hypertension

Apoptosis repressor with caspase recruitment domain (ARC) is a unique anti-apoptotic protein with a distinct tissue distribution. In addition, unlike most anti-apoptotic proteins which act on one pathway, ARC can inhibit apoptosis mediated by both the death-receptor and mitochondrial signaling pathways. In this study, we confirm previous reports showing high levels of ARC protein in rat heart and skeletal muscle, but demonstrate for the first time that ARC is also expressed in rat aorta. Immunoblot analysis on endothelium-denuded aorta as well as immunohistochemical analysis on intact aorta demonstrated that ARC was highly expressed in smooth muscle. Immunoblot analysis also found that ARC protein was severely downregulated in skeletal muscle (−82%; P < 0.001), heart (−80%; P < 0.001), and aorta (−71%; P < 0.001) of spontaneously hypertensive rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats. Decreased ARC levels were also confirmed in tissues of hypertensive animals by immunohistochemical analysis. Collectively, this data suggests that ARC protein is expressed in vascular smooth muscle and is significantly reduced in several target tissues during hypertension.     

The cardioprotective effect of postconditioning is mediated by ARC through inhibiting mitochondrial apoptotic pathway

Objective  Postconditioning protects the heart against ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis. However, the molecular mechanism by which postconditioning suppresses apoptosis remains to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) has been demonstrated to possess the ability to protect cardiomyocytes from apoptosis induced by ischemia/reperfusion. It is not yet clear as to whether ARC contributes to the inhibitory effect of postconditioning against cardiomyocyte apoptosis. Methods  The cultured cardiomyocytes from 1-day old male Sprague–Dawley rats were exposed to 3 h hypoxia followed by 3 h of reoxygenation. Cells were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia before 3 h of reoxygenation. Results  Hypoxia/reoxygenation led to a decrease of endogenous ARC protein levels. In contrast, postconditioning could block the reduction of endogenous ARC protein levels. Interestingly, inhibition of endogenous ARC expression by ARC antisense oligodeoxynucleotides reduced the inhibitory effect of postconditioning against apoptosis. Furthermore, our data showed that postconditioning suppressed the loss of mitochondrial membrane potential, Bax activation and the release of mitochondrial cytochrome c to cytosol. However, these inhibitory effects of postconditioning disappeared upon knockdown of endogenous ARC. Conclusion  Our data for the first time demonstrate that ARC plays an essential role in mediating the cardioprotective effect of postconditioning against apoptosis initiated by the mitochondrial pathway.     

Apoptosis repressor with caspase recruitment domain (ARC) inhibits myogenic differentiation

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, is highly expressed in differentiated heart and skeletal muscle. Apoptosis and differentiation share numerous common pathways; therefore, we examined the impact of ARC on H9c2-myoblast differentiation. We demonstrate that ARC expression levels increase and stabilize upon differentiation. ARC-overexpression in pre-differentiated H9c2-cells suppresses differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. In summary, we show a novel role for ARC in the regulation of muscle differentiation.     

Evidence for a pro-apoptotic phenotype in skeletal muscle of hypertensive rats

In this report, we demonstrate that soleus muscle of spontaneously hypertensive rats (SHR) had significantly lower protein levels of apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) as well as significantly higher protein levels of second mitochondria-derived activator of caspase (Smac) and procaspase-8 compared to normotensive Wistar-Kyoto (WKY) rats. In addition, soleus muscle from hypertensive rats had significantly increased caspase-8 proteolytic enzyme activity as well as significantly elevated reactive oxygen species (ROS) generation and higher hydrogen peroxide (H₂O₂) content. There was no change in the protein levels of the antioxidant enzymes, catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD). Interestingly, ARC protein migrated at approximately 32 kDa in SHR but at 30 kDa in WKY rat muscle; possibly indicating a post-translational modification. These results demonstrate that soleus muscle of hypertensive rats display a pro-apoptotic phenotype and augmented ROS generation.     

ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis

Inhibition of cardiomyocyte apoptosis plays a key role in preconditioning-triggered cardioprotection. However, the molecular mechanism(s) by which preconditioning inhibits apoptosis is not fully understood. Apoptosis repressor with caspase recruitment domain (ARC) possesses the ability to block hypoxia-induced cardiomyocyte apoptosis. We tested whether ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis. Cardiomyocytes from 1-day-old male Sprague-Dawley rats were preconditioned by exposing to 10 min of hypoxia, followed by 30 min of reoxygenation. Then, the preconditioned and non-preconditioned cardiomyocytes were exposed to 90 min of hypoxia followed by 120 min of reoxygenation. The results showed that preconditioning inhibited cell death induced by hypoxia and reoxygenation. Hypoxia and reoxygenation could induce a decrease of ARC protein levels. Intriguingly, preconditioning could maintain ARC protein levels. Inhibition of endogenous ARC expression by ARC antisense oligonucleotides reduced the inhibitory effect of preconditioning on apoptosis. Furthermore, preconditioning-induced suppression of the release of mitochondrial cytochrome c to cytosol and caspase-3 activation could be abolished by the inhibition of endogenous ARC expression using ARC antisense oligonucleotides. Conclusion: These data indicate that ARC participates in preconditioning-triggered cardioprotection by interfering with cytochrome c release and caspase-3 activation.     

Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12

The mechanisms of apoptosis in the loss of myocytes in skeletal muscle with age and the role of mitochondrial and sarcoplasmic reticulum-mediated pathways of apoptosis are unknown. Moreover, it is unknown whether lifelong calorie restriction prevents apoptosis in skeletal muscle and reverses age-related alterations in apoptosis signaling. We investigated key apoptotic regulatory proteins in the gastrocnemius muscle of 12 and 26 month old ad libitum fed and 26 month old calorie-restricted male Fischer-344 rats. We found that apoptosis increased with age and that calorie-restricted rats showed less apoptosis compared with their age-matched cohorts. Moreover, pro- and cleaved caspase-3 levels increased significantly with age and calorie-restricted rats had significantly lower levels than the aged ad libitum group. Neither age nor calorie restriction had any effect on muscle caspase-3 enzyme activity, but the levels of X-linked inhibitor of apoptosis, particularly an inhibitor of caspase-3, increased with age and were reduced significantly in the 26 month old calorie-restricted cohort. The apoptotic inhibitor apoptosis repressor with a caspase recruitment domain (ARC), which inhibits cytochrome c release, underwent an age-associated decline in the cytosol but increased with calorie restriction. In contrast, mitochondrial ARC levels increased with age and were lower in calorie-restricted rats than in age-matched controls, suggesting a translocation of this protein to attenuate oxidative stress. The translocation of ARC may explain the reduction in cytosolic cytochrome c levels observed with age and calorie restriction. Moreover, we found a striking approximately 350% increase in the expression of procaspase-12 (caspase located at the sarcoplasmic reticulum) with age which was significantly lower in the 26 month old calorie-restricted group. The total protein level of apoptosis-inducing factor in the plantaris muscle increased with age and was reduced calorie-restricted rats compared with age-matched controls, but there were no significant changes in this pro-apoptotic protein in the isolated nuclei. Calorie restriction is able to lower the apoptotic potential in aged skeletal muscle by altering several key apoptotic proteins toward cellular survival, thereby reducing the potential for sarcopenia.