Beclin 1对凋亡和自噬的调控作用

Beclin 1是自噬关键调控蛋白之一,参与自噬体膜形成.近期,大量研究结果指出, Beclin 1是caspase家族蛋白酶的全新底物,可被caspase剪切.剪切后的Beclin 1失去自噬调节功能,转而加剧凋亡进程.因而,Beclin 1对细胞凋亡和自噬起着重要的调控作用. 本文主要对细胞凋亡和自噬的相关性,以及Beclin 1在两通路中的调控作用进行了回顾与总结.在此基础上,进一步讨论了Beclin 1与人类疾病如肿瘤、神经系统退行性疾病的关联.最后,简要介绍了实验室常用于Beclin 1研究的工具.     

谷氧还蛋白1调节高糖诱导的心肌细胞自噬

谷氧还蛋白1（Grx1）在体内具有广泛的抗氧化、抗凋亡作用,与氧化应激损伤导致的糖尿病和心肌病等多种疾病的发病机制密切相关. 研究表明,糖尿病心血管病与自噬调节异常密切相关,但糖尿病心血管病变时自噬水平如何调节才能够保护受损的心肌还尚未定论.为研究自噬在高糖诱导心肌细胞凋亡中的作用及其与Grx1的关系,以明确Grx1对高糖诱导的心肌细胞凋亡的抑制作用及相关机制,本研究以高糖诱导大鼠心肌细胞H9c2建立高糖损伤模型,采用氧化还原蛋白免疫印迹法检测蛋白质的氧化水平.免疫印迹检测活性caspase 3蛋白和自噬蛋白Beclin1和LC3以及抗凋亡蛋白Bcl 2的表达水平.研究发现,高糖可诱导蛋白质的氧化水平增加,而Grx1可拮抗高糖诱导的H9c2细胞中蛋白质的氧化.并且含血清的高糖（25和50 mmol/L）作用H9c2心肌细胞后,自噬蛋白Beclin 1表达水平在6～48 h显著上调.同时发现,活性caspase 3水平也呈时间依赖性表达上调,caspase 3和自噬蛋白表达水平的同趋势增加,说明升高的自噬水平与心肌细胞凋亡的调节有关.Grx1保护组的自噬蛋白及活性caspase 3表达水平均显著下调,Grx1抑制剂镉组可拮抗Grx1调节的自噬蛋白和凋亡蛋白水平,说明Grx 1通过抑制自噬及caspase 3水平抑制高糖诱导的心肌细胞凋亡.以上研究结果提示,通过提高Grx1/GSH抗氧化系统功能,调节氧化还原稳态,可以有效减少高糖诱导的心肌损伤,保护糖尿病心脏功能.     

Autophagy regulation: RNF2 targets AMBRA1

The phosphatidylinositol 3-kinase complex I (PI3K complex I) is a crucial regulator of autophagy, which contains Beclin 1 (or ATG6), ATG14L, VPS34 (or the class III phosphatidylinositol 3-kinase and its adaptor VPS15) and AMBRA1, and controls autophagosome formation. In a paper recently published in Cell Research, Xia et al. report that during nutrient deprivation the ubiquitin E3 ligase RNF2 is recruited to the PI3K complex I, and ubiquitinates AMBRA1 to trigger its degradation and downregulate autophagy.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components¹. The ubiquitin-proteasome pathway is also critical during the process of autophagy. The formation of autophagosomes (double-membrane bound vacuoles that sequester cargo in bulk or in a selective manner before their delivery to the lysosomal compartment) depends on ubiquitination-like activity². The selective removal of cargo (e.g., protein aggregates, organelles) by autophagy is dependent in many cases on the ubiquitination of the cargo³. Recent studies also indicate that ubiquitination regulates the activity of autophagy proteins that comprise autophagosomes⁴.Autophagosome formation is dependent on evolutionarily conserved ATG (autophagy-related) proteins initially identified in yeast². These proteins function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via stages of initiation, elongation, and sealing. Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homolog of yeast ATG6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34² (Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P), is crucial to recruitment of ATG, which is required for the elongation and sealing of the autophagosomal membrane.Open in a separate windowFigure 1Role of RNF2 and WASH during autophagosome formation. Upon autophagy stimulation by nutrient deprivation, Beclin 1 forms a complex with VPS34 and its adaptor VPS15, ATG14L, and AMBRA1. In this complex, Beclin 1 is K63-polyubiquitylated (green circles) by the E3 ligase AMBRA1 to activate the production of PI3P by VPS34. The production of PI3P at the phagophore recruits WIPI proteins to trigger the ATG machinery to elongate and seal the membrane to form an autophagosome. After the induction of autophagy, WASH and RNF2 are recruited to downregulate the autophagy pathway by inhibiting the VPS34 activity. WASH negatively regulates autophagy through suppression of Beclin 1 K63-linked polyubiquitination whereas RNF2 is recruited to the complex via the Beclin 1 interactor WASH. RNF2 catalyzes K48-linked polyubiquitination (orange circles) of AMBRA1 to mediate its proteasomal degradation.Recently, Xia and colleagues⁵ demonstrate that RNF2 (also called Ring1B) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitination of AMBRA1. RNF2 is a member of the RING-domain ubiquitin E3 ligase family⁶. In a series of experiments involving two-hybrid screens with RNF2 as bait, Xia and colleagues⁵ showed that RNF2 interacts with AMBRA1 and that this interaction is enhanced upon autophagy stimulation in cells cultured in the absence of serum and amino acids. Deletion of RNF2 robustly stimulates autophagy in response to starvation whereas restoration of RNF2 in RNF2^−/− mouse embryo fibroblasts (MEFs) reduces autophagy. An RNF2 mutant with no E3 ligase activity does not impair autophagy in RNF2^−/− MEFs. The authors further showed that RNF2 downregulates autophagy by promoting the degradation of AMBRA1. RNF2 catalyzes the K48-linked polyubiquitination of AMBRA1 which mediates its proteasomal degradation. The crucial site for AMBRA1 K48-linked polyubiquitination is lysine 45, and a K45R AMBRA1 mutant is not sensitive to RNF2-mediated ubiquitination and is able to sustain VPS34 activity and autophagy. Beclin 1 exists in different complexes involved in different steps of the autophagic pathway². In the current study, the authors showed that RNF2 is associated with the PI3K complex 1 with Beclin 1 ATG14 and AMBRA1, a complex involved in early stage of autophagosome formation. Interestingly, in the absence of RNF2, the association of Beclin 1 with VPS34 in the PI3K complex 1 is enhanced.Recently, Fan''s group reported that AMBRA1-DDB1-CUL4A is the E3 ligase that mediates K63-linked ubiquitination of Beclin 1 to enhance its binding to VPS34 in response to starvation⁷. In the present study⁵, the group showed that the K63-linked ubiquitination of Beclin 1 is inhibited by RNF2. A screen identified WASH as an interactor of RNF2. WASH is part of a complex that promotes actin polymerization to facilitate endosomal protein sorting⁸ and has been recently shown to interact with Beclin 1 and to be associated with autophagosomal membrane⁷. This interaction impairs the AMBRA1-mediated K63 polyubiquitination of Beclin 1. WASH recruits RNF2 to AMBRA1 and the PI3K complex 1 in response to starvation. The absence of WASH abolishes the K48-linked polyubiquitination of AMBRA1. In addition, WASH overexpression partially impairs the interaction of AMBRA1 with Beclin1 to block its K63-linked polyubiquitination⁷. The study by Xia et al. reveals a novel layer of regulation: WASH recruits RNF2 to promote AMBRA1 degradation to impair Beclin 1 ubiquitination⁵. This work points to the complex role of ubiquitination in the regulation of the early stage of autophagy with a balance between activating K63-linked polyubiquitination of Beclin 1 and inhibitory K48-linked polyubiquitination of AMBRA1. These post-translational modifications depend on the activity of two E3 ligases, AMBRA1 and RNF2. This study also stresses the importance of AMBRA1 in stabilization of proteins engaged in the early stage of autophagosome formation via its E3 ligase activity with DDB1-CUL4A to enhance Beclin 1 association with VPS34⁷ and with TRAF6 to stabilize ULK1, which acts in a complex upstream of the PI3K complex 1 in autophagy⁹. Finally, the study by Xia et al. emphasizes that autophagy must be tightly regulated to avoid deleterious effects on cell homeostasis.The present study raises several questions regarding how and when WASH and RNF2 are recruited to downregulate autophagy in response to starvation. Recently it has been shown that WASH is a positive modulator of autophagosome biogenesis in mammalian cells through regulation of endosomal trafficking of ATG9A¹⁰. Altogether, these findings suggest that the role of WASH in autophagy is dependent on its subcellular localization and its partners in intracellular membranes.     

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis

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Autophagy

Multidisciplinary approaches are increasingly being used to elucidate the role of autophagy in health and disease and to harness it for therapeutic purposes. The broad range of topics included in the program of the Vancouver Autophagy Symposium (VAS) 2013 illustrated this multidisciplinarity: structural biology of Atg proteins, mechanisms of selective autophagy, in silico drug design targeting ATG proteins, strategies for drug screening, autophagy-metabolism interplay, and therapeutic approaches to modulate autophagy. VAS 2013 took place at the British Columbia Cancer Research Centre, and was hosted by the CIHR Team in Investigating Autophagy Proteins as Molecular Targets for Cancer Treatment. The program was designed as a day of research exchanges, featuring two invited keynote speakers, internationally recognized for their groundbreaking contributions in autophagy, Dr Ana Maria Cuervo (Albert Einstein College of Medicine, Bronx, NY) and Dr Jayanta Debnath (University of California, San Francisco). By bringing together international and local experts in cell biology, drug discovery, and clinical translation, the symposium facilitated rich interdisciplinary discussions focused on multiple forms of autophagy and their regulation and modulation in the context of cancer.     

Autophagy

Autophagy describes the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. Autophagy is essentially required to prevent accumulation of cellular damage and to ensure cellular homeostasis. Indeed, impaired autophagy has been implicated in a variety of different diseases. We examined the role of autophagy in inflammatory bone loss. We demonstrated that autophagy is activated by the pro-inflammatory cytokine tumor necrosis factor (TNF/TNFα) in osteoclasts of patients with rheumatoid arthritis (RA). Autophagy induces osteoclast differentiation and stimulates osteoclast-mediated bone resorption in vitro and in vivo, thereby highlighting autophagy as a novel mediator of TNF-induced bone resorption.     

Autophagy

Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models.     

Autophagy

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.     

Autophagy

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.     

Autophagy interaction with herpes simplex virus type-1 infection

More than 50% of the U.S. population is infected with herpes simplex virus type-I (HSV-1) and global infectious estimates are nearly 90%. HSV-1 is normally seen as a harmless virus but debilitating diseases can arise, including encephalitis and ocular diseases. HSV-1 is unique in that it can undermine host defenses and establish lifelong infection in neurons. Viral reactivation from latency may allow HSV-1 to lay siege to the brain (Herpes encephalitis). Recent advances maintain that HSV-1 proteins act to suppress and/or control the lysosome-dependent degradation pathway of macroautophagy (hereafter autophagy) and consequently, in neurons, may be coupled with the advancement of HSV-1-associated pathogenesis. Furthermore, increasing evidence suggests that HSV-1 infection may constitute a gradual risk factor for neurodegenerative disorders. The relationship between HSV-1 infection and autophagy manipulation combined with neuropathogenesis may be intimately intertwined demanding further investigation.     

肿瘤细胞的自噬和窖蛋白-1

自噬作用是一种细胞通过溶酶体自我降解的过程,在肿瘤的形成和发展中起着双重作用。窖蛋白-1(Caveolin-1,Cav-1)作为胞膜窖的标志蛋白,介导许多生理和病理过程,包括胞膜窖的形成、膜泡运输、维持细胞胆固醇稳态、信号转导和肿瘤的发生。近年来,许多研究表明肿瘤细胞自噬和Cav-1存在一定关系。文中就近年来肿瘤细胞的自噬与Cav-1的关系及其在肿瘤发生和发展过程中的作用进行综述。     

Autophagy regulates spermatid differentiation via degradation of PDLIM1

Spermiogenesis is a complex and highly ordered spermatid differentiation process that requires reorganization of cellular structures. We have previously found that Atg7 is required for acrosome biogenesis. Here, we show that autophagy regulates the round and elongating spermatids. Specifically, we found that Atg7 is required for spermatozoa flagella biogenesis and cytoplasm removal during spermiogenesis. Spermatozoa motility of atg7-null mice dropped significantly with some extra-cytoplasm retained on the mature sperm head. These defects are associated with an impairment of the cytoskeleton organization. Functional screening revealed that the negative cytoskeleton organization regulator, PDLIM1 (PDZ and LIM domain 1 [elfin]), needs to be degraded by the autophagy-lysosome-dependent pathway to facilitate the proper organization of the cytoskeleton. Our results thus provide a novel mechanism showing that autophagy regulates cytoskeleton organization mainly via degradation of PDLIM1 to facilitate the differentiation of spermatids.