自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

自噬与泛素化蛋白降解途径的分子机制及其功能

细胞内所有的蛋白质和大多数的细胞外蛋白都在不断的进行更新,即它们在不断地被降解,并被新合成的蛋白质取代。细胞内蛋白的降解主要通过两个途径,即自噬和泛素蛋白酶体系统。自噬是一种由溶酶体介导的细胞内过多或异常蛋白质的降解机制。在细胞内主要有3种类型的自噬,即分子伴侣介导的自噬、微自噬和巨自噬。泛素蛋白酶体系统是由泛素介导的一种高度复杂的蛋白降解机制,它参与降解细胞内许多蛋白质并且这个过程具有高度特异性。细胞内蛋白质的降解参与调节许多细胞过程,包括细胞周期、DNA修复、细胞生长和分化、细胞质量的控制、病原生物的感染反应和细胞凋亡等。许多严重的人类疾病被认为是由于蛋白质降解系统的紊乱而引起的。文章综述了自噬和泛素化途径及其分子机制,以及蛋白质降解系统紊乱的病理学意义。     

泛素信号途径、自噬受体与细胞选择性自噬

蛋白质泛素化是真核生物细胞内蛋白质合成后最重要和最普遍的修饰方式之一。发生在蛋白质底物上的泛素化,由于其泛素化方式及形成泛素链的连接形式的多样性,又统称为泛素信号途径。研究表明,泛素信号途径对蛋白质的调节作用分为降解相关和非相关的两种。细胞内蛋白质的降解主要通过泛素-蛋白酶体或溶酶体-自噬途径来完成。一般认为,通过泛素-蛋白酶体降解的蛋白质具有很强的选择性,而通过溶酶体-自噬途径降解的蛋白质一般选择性较差。然而,近年来,细胞自噬受体如p62等的发现则表明细胞自噬同样具有很强的选择性,这一类由细胞自噬受体介导的细胞自噬被称为细胞选择性自噬(Selective autophagy)。蛋白质泛素化及降解调控几乎所有类型的细胞活动;与之对应的是,蛋白质泛素化及降解异常与包括肿瘤在内的多种人类疾病的发生发展密切相关。本文综述了泛素信号途径调控蛋白质通过蛋白酶体或自噬途径降解的基本过程和部分最新进展,并结合本实验室的研究成果介绍泛素化修饰细胞自噬受体调控细胞选择性自噬的新机制。     

泛素-蛋白酶体途径在病毒感染中的作用

泛素-蛋白酶体途径——降解溶酶体外蛋白的主要细胞内系统,在许多细胞功能中发挥重要作用。为自身利益如病毒出芽、凋亡抑制和免疫逃避,许多病毒已经进化出了利用泛素-蛋白酶体途径的不同策略。深入理解泛素-蛋白酶体途径在病毒感染中的作用有助于揭示一些病毒病的致病机理和发现新的分子靶标以开发抗病毒药物。因此,将泛素-蛋白酶体途径在病毒感染中的作用方面的最新进展作一综述。     

泛素化介导的非蛋白质降解功能

泛素因标记被26 S蛋白酶体降解的蛋白质而著名．然而近几年发现,泛素作用远不止此,不仅具有参与蛋白质降解这一重要“传统作用”,还起着比先前想象更多变的、更精美的细胞调控作用,是非常重要的细胞过程的多层面调节因子,具有许多重要的非蛋白质降解功能,包括DNA损伤修复、DNA复制、信号传导、转录调节、膜运输、胞吞、蛋白激酶活化、染色质重塑和病毒芽殖．这些功能涉及多聚泛素化和单泛素化及多泛素化．因此,泛素化异常可能涉及疾病的发生和发展．对这些功能的了解可以拓展人们对泛素的认识,有助于对多种细胞过程的深入理解,也有助于相关新药的研发．     

自噬相关去泛素化酶及其小分子抑制剂的研究进展

自噬是进化上高度保守并受到多途径严密调控的细胞生物学过程,其向溶酶体递送多种细胞质组分以进行细胞内物质的降解以及再循环.这一过程涉及到细胞器的更新、错误折叠蛋白质和蛋白质聚集体以及细胞内病原体的清除.因此,自噬对于细胞稳态的维持至关重要,与许多人类疾病的发生发展密切相关.随着细胞自噬调节机制研究的不断深入,越来越多的去泛素化酶被证明在自噬相关的泛素信号调控系统中发挥了重要的作用.这些去泛素化酶作用于细胞自噬的不同阶段,靶向调节不同的泛素化自噬功能元件或自噬底物.去泛素化酶作为包括神经退行性疾病以及肿瘤在内的细胞自噬相关疾病的治疗靶点受到了广泛的关注,其中各类小分子抑制剂的发现为进一步研究去泛素化酶的自噬调节活性及相关疾病的治疗提供了可能.     

病毒利用UPP逃避抗病毒反应的研究进展

泛素-蛋白酶体途径是溶酶体外蛋白降解的主要系统,在许多细胞功能中发挥重要作用。越来越多的证据表明病毒参与泛素-蛋白酶体途径,干扰IFN信号通路和免疫受体表达、凋亡抑制及介导病毒潜伏。深入理解病毒利用泛素-蛋白酶体途径逃避宿主抗病毒反应的策略,有助于揭示病毒的致病机理和鉴定抗病毒药物新靶标。     

泛素-蛋白酶体途径的组成和功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的重要途径,泛素分子主要通过泛素活化酶、泛素结合酶和泛素-蛋白连接酶与靶蛋白结合形成一条多泛素链,最后被26S蛋白酶体识别和降解。泛素-蛋白酶体途径参与细胞内的多种活动过程,包括细胞凋亡、MHCI类抗原的递呈、细胞周期以及细胞内信号转导,与细胞的一些生理功能和病理状态有着密切的联系。本文主要对组成泛素-蛋白酶体途径的各成分作一综述。     

蛋白质SUMO化和泛素化修饰的关系

泛素化和SUMO化是蛋白质翻译后修饰的重要方式,广泛参与调节蛋白质功能和细胞生命活动各个环节。多聚泛素化降解蛋白质,而SUMO化主要调节蛋白质的相互作用和定位等。在不同情况下,SUMO化和泛素化既可协同调节蛋白质功能,也可相互拮抗。最近研究发现,某些底物的SUMO化能够激活体内一类新发现的SUMO依赖的泛素连接酶,启动泛素-蛋白酶体途径降解底物,导致蛋白质SUMO化和汔素化的关系进一步精细化和复杂化。     

Interplay between autophagy and proteasome during protein turnover

Protein homeostasis is epitomized by an equilibrium between protein biosynthesis and degradation: the ‘life and death’ of proteins. Approximately one-third of newly synthesized proteins are degraded. As such, protein turnover is required to maintain cellular integrity and survival. Autophagy and the ubiquitin–proteasome system (UPS) are the two principal degradation pathways in eukaryotes. Both pathways orchestrate many cellular processes during development and upon environmental stimuli. Ubiquitination of degradation targets is used as a ‘death’ signal by both processes. Recent findings revealed a direct functional link between both pathways. Here, we summarize key findings in the field of protein homeostasis, with an emphasis on the newly revealed crosstalk between both degradation machineries and how it is decided which pathway facilitates target degradation.     

Ubiquitin: same molecule, different degradation pathways

Ubiquitin is a common demoninator in the targeting of substrates to all three major protein degradation pathways in mammalian cells: the proteasome, the lysosome, and the autophagosome. The factors that direct a substrate toward a particular route of degradation likely include ubiquitin chain length and linkage type, which may favor interaction with particular receptors or confer differential susceptibility to deubiquitinase activities associated with each pathway.     

Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy

Ubiquitin‐dependent processes can be antagonized by substrate‐specific deubiquitination enzymes involved in many cellular functions. In this study, we show that the yeast Ubp3–Bre5 deubiquitination complex interacts with both the chaperone‐like Cdc48, a major actor of the ubiquitin and proteasome system, and Ufd3, a ubiquitin‐binding cofactor of Cdc48. We observed that these partners are required for the Ubp3–Bre5‐dependent and starvation‐induced selective degradation of yeast mature ribosomes, also called ribophagy. By contrast, proteasome‐dependent degradation does not participate in this process. Our data favour the idea that these factors cooperate to recognize and deubiquitinate specific substrates of ribophagy before their vacuolar degradation.     

Mechanism and function of deubiquitinating enzymes

Attachment of ubiquitin to proteins is a crucial step in many cellular regulatory mechanisms and contributes to numerous biological processes, including embryonic development, the cell cycle, growth control, and prevention of neurodegeneration. In these diverse regulatory settings, the most widespread mechanism of ubiquitin action is probably in the context of protein degradation. Polyubiquitin attachment targets many intracellular proteins for degradation by the proteasome, and (mono)ubiquitination is often required for down-regulating plasma membrane proteins by targeting them to the vacuole (lysosome). Ubiquitin-protein conjugates are highly dynamic structures. While an array of enzymes directs the conjugation of ubiquitin to substrates, there are also dozens of deubiquitinating enzymes (DUBs) that can reverse the process. Several lines of evidence indicate that DUBs are important regulators of the ubiquitin system. These enzymes are responsible for processing inactive ubiquitin precursors, proofreading ubiquitin-protein conjugates, removing ubiquitin from cellular adducts, and keeping the 26S proteasome free of inhibitory ubiquitin chains. The present review focuses on recent discoveries that have led to a better understanding the mechanisms and physiological roles of this diverse and still poorly understood group of enzymes. We also discuss briefly some of the proteases that act on ubiquitin-like protein (UBL) conjugates and compare them to DUBs.     

Substrate receptors of proteasomes

Proteasomes are responsible for the turnover of most cellular proteins, and thus are critical to almost all cellular activities. A substrate entering the proteasome must first bind to a substrate receptor. Substrate receptors can be classified as ubiquitin receptors and non‐ubiquitin receptors. The intrinsic ubiquitin receptors, including proteasome regulatory particle base subunits 1, 10 and 13 (Rpn1, Rpn10, and Rpn13), determine the capability of the proteasome to recognize a ubiquitin chain, and thus provide selectivity for the 26S proteasome. However, the non‐ubiquitin receptors, including proteasome activator 200 (PA200) and PA28γ, have received great attention due to their remarkable compensatory roles relative to canonical ubiquitin‐mediated proteasomal degradation. Herein we review recent advances in understanding the contributions of these substrate receptors to proteasomal degradation, and introduce their substrates and interacting factors. We also provide insights into their biological functions related to spermatogenesis, immune responses, cellular homeostasis, and tumour development. Finally, we summarize advances in developing small‐molecule inhibitors of these substrate receptors and discuss their potential as drug targets.     

To degrade or release: ubiquitin-chain remodeling

The proteasome controls many cellular processes by degrading a large number of regulatory proteins. Most proteins are targeted to the proteasome through covalent tagging by a chain consisting of several copies of the small protein ubiquitin. Finley and coworkers have now discovered two proteins, Hul5 and Ubp6, which regulate degradation further, when bound to the proteasome. Hul5 promotes degradation by extending the number of ubiquitin moieties in the tag on substrates, whereas Ubp6 antagonizes degradation by trimming ubiquitin from the tag. The balance between these two opposing activities might control the substrate specificity of the proteasome and adjusting the balance would provide a new level of degradation control.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin-proteasome pathway

Ubiquitination of proteins is now recognized to target proteins for degradation by the proteasome and for internalization into the lysosomal system, as well as to modify functions of some target proteins. Although much progress has been made in characterizing enzymes that link ubiquitin to proteins, our understanding of deubiquitinating enzymes is less developed. These enzymes are involved in processing the products of ubiquitin genes which all encode fusion proteins, in negatively regulating the functions of ubiquitination (editing), in regenerating free ubiquitin after proteins have been targeted to the proteasome or lysosome (recycling) and in salvaging ubiquitin from possible adducts formed with small molecule nucleophiles in the cell. A large number of genes encode deubiquitinating enzymes suggesting that many have highly specific and regulated functions. Indeed, recent findings provide strong support for the concept that ubiquitination is regulated by both specific pathways of ubiquitination and deubiquitination. Interestingly, many of these enzymes are localized to subcellular structures or to molecular complexes. These localizations play important roles in determining specificity of function and can have major influences on their catalytic activities. Future studies, particularly aimed at characterizing the interacting partners and potential substrates in these complexes as well as at determining the effects of loss of function of specific deubiquitinating enzymes will rapidly advance our understanding of the important roles of these enzymes as biological regulators.