自噬在晶状体发育及白内障的研究进展

自噬,或细胞自我消化,是参与蛋白质和细胞器降解细胞途径。晶状体是由一层晶状体上皮细胞及一系列正在分化的纤维细胞组成,纤维细胞发育通过线粒体、细胞核等细胞器退化完成的。在晶状体中,自噬在晶状体纤维细胞成熟,构成无细胞器区(organell-free zone,OFZ)的过程中,起着重要在作用。很多的研究证明对Atg5,Vsp34,FYCO1等基因在维持晶状体透明性中的作用进行了讨论,并且在晶状体上皮细胞,未成熟纤维细胞中证实自噬小泡的存在。本文旨在概括目前自噬的研究进展,重点介绍自噬与人晶状体发育及其与白内障形成机制的研究进展。     

Ctk1蛋白在自噬过程中的作用

细胞自噬是一种在真核生物中十分保守的溶酶体依赖性胞内降解途径,通过形成双层膜结构包裹细胞质内的生物大分子或者细胞器运送到溶酶体进行降解。在实验中发现,Ctk1蛋白对自噬过程有重要调节作用。Ctk1的缺失或者失活都会导致自噬过程不能正常发生,同时也会影响酵母CVT途径。自噬相关蛋白Atg3与Atg8的结合受到Ctk1的调控影响。因此,Ctk1在自噬过程中自噬体的形成中发挥了重要作用,揭示了Ctk1的新的功能作用。     

线粒体自噬

细胞自噬(autophagy)是细胞依赖溶酶体对蛋白和细胞器进行降解的一条重要途径.目前,将通过细胞自噬降解线粒体的途径称为线粒体自噬(mitophagy).最近几年的证据表明,线粒体自噬是一个特异性的选择过程,并受到各种因子的精密调节,是细胞清除体内损伤线粒体和维持自身稳态的一种重要调节机制.自噬相关分子,如“核心”Atg 复合物,酵母线粒体外膜分子Atg32、Atg33、Uth1和Aup1,哺乳细胞线粒体外膜蛋白PINK1、NIX和胞质的Parkin等,在线粒体自噬中起关键的作用. 线粒体自噬异常与神经退行性疾病如帕金森氏病（Parkinson’s disease,PD）的发生密切相关. 本文就线粒体自噬的研究进展做简要的介绍.     

自噬体膜的来源

细胞自噬是指细胞通过自噬-溶酶体(autolysosome)降解变性蛋白聚集物和受损细胞器的过程. 自噬对于细胞内环境的稳态、物质的平衡、胚胎发育以及疾病的发生发挥重要作用. 在电镜下观察,自噬体膜是一个双层脂质膜结构. 细胞中因缺乏除了自噬相关蛋白9 (autophagy-related protein 9,ATG9)以外的自噬体膜相关蛋白,故难以确定自噬体膜的来源. 自噬体膜的来源也因此成为目前自噬研究领域的热点问题. 关于自噬体膜的来源,学术界存在两种观点：一种认为自噬体膜是细胞在自噬体组装位点(pre-autophagosomal structure, PAS)重新合成的;另一种观点则认为自噬体膜来源于细胞已有的某些细胞器(如内质网、高尔基体、内吞体、质膜和线粒体). 该文综述了近年有关自噬体膜来源于细胞已有的某些细胞器的研究进展,旨在为相关领域的研究提供参考.     

鳞翅目昆虫中自噬相关分子Atg8的研究进展

自噬是细胞通过溶酶体自主降解以实现细胞内物质循环利用的过程,在昆虫细胞分化和个体发育中起着重要作用。鳞翅目昆虫属于完全变态昆虫,会通过自噬和凋亡完成蜕变重建过程,是研究自噬机制的模式生物。自噬相关蛋白Atg8是哺乳动物微管相关蛋白1轻链3的同系物,是自噬相关蛋白的核心蛋白家族,对自噬小体形成、膜的延伸、特定物质识别等具有重要意义。文中就鳞翅目昆虫Atg8在自噬信号通路中的作用、Atg8结构特点、Atg8表达分布及Atg8-PE/Atg8水平与自噬活性关系进行了综述。Atg8-PE是自噬信号通路中两个类泛素结合系统之一,在自噬中起着关键作用。序列分析表明,鳞翅目昆虫Atg8与其他真核生物同源蛋白的整体结构相似,尤其与其他昆虫同源蛋白的氨基酸序列高度一致,体现了Atg8的高度保守性。鳞翅目昆虫发育不同阶段,Atg8在中肠、唾液腺、卵巢、脂肪体、丝腺等器官中的表达分布各不相同。并且,Atg8在核质中分布也存在差异,Atg8在细胞核与细胞质之间的穿梭可能存在蛹化前阶段的某些细胞中。通过检测Atg8-PE在细胞内的表达水平或Atg8含量的变化,可以评价细胞自噬的发生程度。     

细胞自噬对乙肝病毒感染细胞干扰素表达的影响

[目的]细胞自噬(Autophagy)是真核细胞用于清除胞内聚集物、损伤细胞器而维持其稳态平衡的一种溶酶体降解途径.细胞自噬不仅在细胞生长发育、成熟、分化等过程中起重要作用,且与病毒感染、细胞免疫密切相关.通过研究细胞自噬对乙肝病毒感染的Ⅰ型干扰素的影响,为进一步阐明乙肝病毒感染对机体天然免疫反应研究奠定基础.[方法]通过siRNA干扰Beclin1和Atg7表达,检测自噬小体形成,Real-TimePCR检测干扰素因子表达,分析了细胞自噬对乙肝病毒感染细胞中干扰素形成的影响.[结果]干扰Beclin1和Atg7均可抑制细胞自噬发生,抑制细胞自噬可降低干扰素因子的表达,而对细胞活力和细胞凋亡无明显影响.[结论]抑制细胞自噬,可降低HBV感染细胞中IFNβ和IFI27的表达,这在一定程度上意味着,HBV诱导的自噬具有增强感染细胞天然免疫反应的作用.     

溶酶体途径在细胞自噬过程中的功能意义

溶酶体具有高度保守的异质性,是细胞自噬的关键细胞器。细胞质中的蛋白质和细胞器最终在溶酶体降解,故溶酶体在维持细胞结构和功能的平衡方面起着重要生理作用。通过自噬溶酶体途径,细胞可清除某些病原体并参与抗原呈递。细胞自噬与异噬经溶酶体密切联系。自噬过程中溶酶体功能障碍与某些疾病和衰老等相关。对细胞自噬的溶酶体途径及其功能意义作了概述。     

自噬过程的晚期阶段

自噬是高度保守的细胞内降解途径.在此过程中,部分细胞质和细胞器被双层膜的囊泡包裹形成自噬体,随后与溶酶体融合并降解被吞噬的物质.降解产物被释放到细胞质中重新用于必需的物质和能量合成.本文主要关注自噬的晚期阶段,即从自噬体合成结束到溶酶体再生过程.通过对这一过程相关基因及蛋白产物的研究,初步揭示了此过程的分子机制.     

自噬的相关分子机制北大核心CSCD

细胞自噬是真核生物中高度保守的一类生物学途径,它通过降解细胞浆内不同组分,维持细胞自身平衡并帮助细胞在应激情况下生存。自噬在生物体生长发育、免疫防御、肿瘤抑制及神经退行性疾病中都有重大的意义。哺乳动物细胞中,自噬过程主要由自噬相关蛋白(Atg)所形成的一系列复合物所调控,这些蛋白质分别在自噬的启动、自噬泡的形成、延伸及成熟和降解过程中发挥重要的作用。在此,本文针对一些重要的自噬相关蛋白质对近年来自噬分子机制的研究进展做一总结。     

自噬在发育及干细胞中的作用

自噬是亚细胞膜结构发生动态变化并经溶酶体介导的细胞内蛋白质和细胞器降解的过程。通过平衡细胞内的合成和分解代谢,自噬可以维持细胞内环境稳态。干细胞是具有自我更新能力和多向分化潜能的细胞,对组织器官再生和维持组织稳态有重要作用。近年的研究表明,自噬在维持干细胞功能方面有非常重要的作用,本文综述了自噬的形成过程和分子机制及其在发育及干细胞中的作用。     

Mechanism of Atg9 recruitment by Atg11 in the cytoplasm-to-vacuole targeting pathway

Autophagy is a lysosomal degradation pathway for the removal of damaged and superfluous cytoplasmic material. This is achieved by the sequestration of this cargo material within double-membrane vesicles termed autophagosomes. Autophagosome formation is mediated by the conserved autophagy machinery. In selective autophagy, this machinery including the transmembrane protein Atg9 is recruited to specific cargo material via cargo receptors and the Atg11/FIP200 scaffold protein. The molecular details of the interaction between Atg11 and Atg9 are unclear, and it is still unknown how the recruitment of Atg9 is regulated. Here we employ NMR spectroscopy of the N-terminal disordered domain of Atg9 (Atg9-NTD) to map its interaction with Atg11 revealing that it involves two short peptides both containing a PLF motif. We show that the Atg9-NTD binds to Atg11 with an affinity of about 1 μM and that both PLF motifs contribute to the interaction. Mutation of the PLF motifs abolishes the interaction of the Atg9-NTD with Atg11, reduces the recruitment of Atg9 to the precursor aminopeptidase 1 (prApe1) cargo, and blocks prApe1 transport into the vacuole by the selective autophagy-like cytoplasm-to-vacuole (Cvt) targeting pathway while not affecting bulk autophagy. Our results provide mechanistic insights into the interaction of the Atg11 scaffold with the Atg9 transmembrane protein in selective autophagy and suggest a model where only clustered Atg11 when bound to the prApe1 cargo is able to efficiently recruit Atg9 vesicles.     

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.     

Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane. We name this protein Atg36 as its absence blocks pexophagy, and its overexpression induces pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae.     

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Pex3 and Atg37 compete to regulate the interaction between the pexophagy receptor,Atg30, and the Hrr25 kinase

Macroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.     

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17- Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.     

The role of Atg29 phosphorylation in PAS assembly

Macroautophagy (hereafter autophagy) initiates at the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. As the first protein complex targeted to the PAS, the Atg17-Atg31-Atg29 complex serves as the scaffold for other Atg proteins and plays a critical role for the organization of the PAS, and in autophagy initiation. We recently showed that this complex is constitutively formed and activated by the phosphorylation of Atg29 when autophagy is induced. Phosphorylation of Atg29 is required for its interaction with Atg11, another scaffold protein, and its function for promoting the proper assembly of the PAS. Single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends. This structural arrangement allows Atg29 to interact with Atg11, and is critical in the organization of the intact Atg1 complex.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.