Mechanisms orchestrating the enzymatic activity and cellular functions of deubiquitinases

Deubiquitinases (DUBs) are required for the reverse reaction of ubiquitination and act as major regulators of ubiquitin signaling processes. Emerging evidence suggests that these enzymes are regulated at multiple levels in order to ensure proper and timely substrate targeting and to prevent the adverse consequences of promiscuous deubiquitination. The importance of DUB regulation is highlighted by disease-associated mutations that inhibit or activate DUBs, deregulating their ability to coordinate cellular processes. Here, we describe the diverse mechanisms governing protein stability, enzymatic activity, and function of DUBs. In particular, we outline how DUBs are regulated by their protein domains and interacting partners. Intramolecular interactions can promote protein stability of DUBs, influence their subcellular localization, and/or modulate their enzymatic activity. Remarkably, these intramolecular interactions can induce self-deubiquitination to counteract DUB ubiquitination by cognate E3 ubiquitin ligases. In addition to intramolecular interactions, DUBs can also oligomerize and interact with a wide variety of cellular proteins, thereby forming obligate or facultative complexes that regulate their enzymatic activity and function. The importance of signaling and post-translational modifications in the integrated control of DUB function will also be discussed. While several DUBs are described with respect to the multiple layers of their regulation, the tumor suppressor BAP1 will be outlined as a model enzyme whose localization, stability, enzymatic activity, and substrate recognition are highly orchestrated by interacting partners and post-translational modifications.     

Protein partners of deubiquitinating enzymes

Protein modification by ubiquitin and ubiquitin-like molecules is a critical regulatory process. Like most regulated protein modifications, ubiquitination is reversible. Deubiquitination, the reversal of ubiquitination, is quickly being recognized as an important regulatory strategy. Nearly one hundred human DUBs (deubiquitinating enzymes) in five different gene families oppose the action of several hundred ubiquitin ligases, suggesting that both ubiquitination and its reversal are highly regulated and specific processes. It has long been recognized that ubiquitin ligases are modular enzyme systems that often depend on scaffolds and adaptors to deliver substrates to the catalytically active macromolecular complex. Although many DUBs bind ubiquitin with reasonable affinities (in the nM to microM range), a larger number have little affinity but exhibit robust catalytic capability. Thus it is apparent that these DUBs must acquire their substrates by binding the target protein in a conjugate or by associating with other macromolecular complexes. We would then expect that a study of protein partners of DUBs would reveal a variety of substrates, scaffolds, adaptors and ubiquitin receptors. In the present review we suggest that, like ligases, much of the regulation and specificity of deubiquitination arises from the association of DUBs with these protein partners.     

Ubiquitination Regulates the Neuroprotective Function of the Deubiquitinase Ataxin-3 in Vivo

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.     

去泛素化酶与肿瘤

泛素化修饰(ubiquitination modification)广泛存在于真核生物,通过26S蛋白酶体降解途径或信号传递等,改变蛋白质稳定性、定位和活性等功能,参与细胞的周期、转录、炎症、肿瘤和免疫等各项功能,是一类复杂的动态调控系统.泛素化调节是一个可逆过程,被泛素连接酶(ubiquitin ligase,E3)...     

Regulation of proteolysis by human deubiquitinating enzymes

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein–protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USPs) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN +/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

De‐ubiquitinases on the move: an emerging field in plant biology

A balance between the synthesis and degradation of active proteins governs diverse cellular processes in plants, spanning from cell‐cycle progression and circadian rhythm to the outcome of several hormone signalling pathways. Ubiquitin‐mediated post‐translational modification determines the degradative fate of the target proteins, thereby altering the output of cellular processes. An equally important, and perhaps under‐appreciated, aspect of this pathway is the antagonistic process of de‐ubiquitination. De‐ubiquitinases (DUBs), a group of processing enzymes, play an important role in maintaining cellular ubiquitin homeostasis by hydrolyzing ubiquitin poly‐proteins and free poly‐ubiquitin chains into mono‐ubiquitin. Further, DUBs rescue the cellular proteins from 26S proteasome‐mediated degradation to their active form by cleaving the poly‐ubiquitin chain from the target protein. Any perturbation in DUB activity is likely to affect proteostasis and downstream cellular processes. This review illustrates recent findings on the biological significance and mechanisms of action of the DUBs in Arabidopsis thaliana, with an emphasis on ubiquitin‐specific proteases (UBPs), the largest family among the DUBs. We focus on the putative roles of various protein–protein interaction interfaces in DUBs and their generalized function in ubiquitin recycling, along with their pre‐eminent role in plant development.     

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin‐3

Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin‐3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin‐3, but does not alter its preference for K63‐linked Ub chains. In cells, ubiquitination of endogenous ataxin‐3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin‐3 in protein quality control are modulated through ubiquitination. Ataxin‐3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes.     

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.     

Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP

The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating, and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination, ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or?similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In?addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3.     

去泛素化酶在肾细胞癌中的作用

肾细胞癌（renal cell carcinoma,RCC）是成人肾脏的原发性恶性肿瘤。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）对控制蛋白质水平和调节生理病理过程至关重要。去泛素化酶（deubiquitinases,DUBs）是UPS的关键成分,特别是从靶蛋白中去除泛素链,通过严格调节正常生理学中泛素化和去泛素化之间的平衡,对蛋白质稳态和质量控制显示出至关重要的作用。越来越多的研究表明,功能异常的DUBs与RCC的进展和转移有关。根据底物的不同,一些DUB可能会抑制RCC,而另一些则促进。本文综述了RCC相关DUB的最新研究进展,描述了其分类、功能作用,总结了DUB在RCC中的作用和作用机制,并讨论了靶向DUBs用于癌症治疗。     

When the chains do not break: the role of USP10 in physiology and pathology

Deubiquitination is now understood to be as important as its partner ubiquitination for the maintenance of protein half-life, activity, and localization under both normal and pathological conditions. The enzymes that remove ubiquitin from target proteins are called deubiquitinases (DUBs) and they regulate a plethora of cellular processes. DUBs are essential enzymes that maintain intracellular protein homeostasis by recycling ubiquitin. Ubiquitination is a post-translational modification where ubiquitin molecules are added to proteins thus influencing activation, localization, and complex formation. Ubiquitin also acts as a tag for protein degradation, especially by proteasomal or lysosomal degradation systems. With ~100 members, DUBs are a large enzyme family; the ubiquitin-specific peptidases (USPs) being the largest group. USP10, an important member of this family, has enormous significance in diverse cellular processes and many human diseases. In this review, we discuss recent studies that define the roles of USP10 in maintaining cellular function, its involvement in human pathologies, and the molecular mechanisms underlying its association with cancer and neurodegenerative diseases. We also discuss efforts to modulate USPs as therapy in these diseases.Subject terms: Cell biology, Cell signalling     

The emerging role of deubiquitinating enzymes in genomic integrity,diseases, and therapeutics

The addition of mono-ubiquitin or poly-ubiquitin chain to signaling proteins in response to DNA damage signal is thought to be a critical event that facilitates the recognition of DNA damage lesion site, the activation of checkpoint function, termination and checkpoint response and the recruitment of DNA repair proteins. Despite the ubiquitin modifiers, removal of ubiquitin from the functional proteins by the deubiquitinating enzymes (DUBs) plays an important role in orchestrating DNA damage response as well as DNA repair processes. Deregulated ubiquitination and deubiquitination could lead to genome instability that in turn causes tumorigenesis. Recent TCGA study has further revealed the connection between mutations in alteration of DUBs and various types of tumors. In addition, emerging drug design based on DUBs provides a new avenue for anti-cancer therapy. In this review, we will summarize the role of deubiquitination and specificity of DUBs, and highlight the recent discoveries of DUBs in the modulation of ubiquitin-mediated DNA damage response and DNA damage repair. We will furthermore discuss the DUBs involved in the tumorigenesis as well as interception of deubiquitination as a novel strategy for anti-cancer therapy.     

基于活性和亲和性的泛素探针的发展与合成

泛素（ubiquitin,Ub）作为一种重要的翻译后修饰,参与调控细胞内几乎所有的生命活动。泛素化通常由E1s、E2s、E3s以及去泛素化酶（deubiquitinating enzyme,DUBs）相互协调完成,并在底物蛋白上形成不同链长、不同连接类型的泛素链。这些泛素链可以产生多样的拓扑结构,被含有泛素结合域（Ub binding domain,UBD）的不同识别蛋白结合,进而传递不同的信号。泛素化过程或者识别蛋白的读取一旦发生错误,对细胞来说都可能是灾难性的。为深入了解泛素相关的生理机制,多种泛素探针被设计与合成,用于对目标蛋白酶或识别蛋白进行标记和监测。本综述总结了当前的泛素探针（包括基于活性和基于亲和性的探针）的最新发展,并详细阐述了它们的合成策略。进一步介绍了细胞穿梭型泛素探针在活细胞内的最新应用。     

Substrate properties of ubiquitin carboxyl-terminally derived peptide probes for protein ubiquitination

Protein ubiquitination is a widespread protein posttranslational modification in eukaryotes that regulates essentially every aspect of cellular processes. The attachment of ubiquitin to a protein substrate is accomplished through an enzymatic cascade involving the actions of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are more than 600 E3 ligases estimated to exist in the human genome that regulate the targeting specificity of protein ubiquitination. To understand the dynamic role of protein ubiquitination in biological processes, robust tools need to be developed which can be employed to establish the substrate specificity of each of these E3 ligases. In this report, we show that the ubiquitin carboxyl-terminally derived peptide probes can serve as modest ubiquitin surrogates for the ubiquitination pathway. In the E1-catalyzed probe adenylation assay, peptide probe 3 with a RLRGG recognition sequence exhibited the highest activity, with the k cat/ K 1/2 determined to be 1.1 x 10 (4) M (-1) s (-1), roughly 470-fold lower than that of ubiquitin. The rate of transfer from the E1 peptide probe thioesters to E2 showed clear sequence dependency, with peptide probe 4 with an LRLRGG recognition sequence showed the fastest rate ( t 1/2 = 0.9 min), essentially identical to that of ubiquitin ( t 1/2 = 0.8 min) under our assay conditions. Furthermore, peptide probes 4 and 8 also exhibited the selective, parkin-mediated labeling of tubulins in a semipurified tubulin-parkin complex. Finally, these carboxyl-terminally derived peptide probes were shown to label the ubiquitination substrates in fraction II of the rabbit reticulocyte lysate with an efficiency parallel to their substrate properties. The selective use of these ubiquitin carboxyl-terminally derived peptide probes by the ubiquitination pathway suggests that perhaps more potent peptide ubiquitination probes based on the ubiquitin C-terminal scaffold can be developed through additional structural optimization.     

A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

Structures of proteases for ubiqutin and ubiquitin-like modifiers

Post-translational modifiers can alter the function of proteins in many different ways. The conjugation of ubiquitin (Ub) and ubiqutin-like modifiers (Ubls) to proteins has been shown to be especially crucial in regulating a variety of cellular processes including the cell cycle, growth control, quality control, localization and many more. It is a highly dynamic process and involves a number of enzymes called E1, E2 and E3. Ub and Ubls are removed from the target proteins by deubiquitinating enzymes (DUBs) or Ubl-specific proteases (ULPs), thereby deconjugation can act as an additional level of control over the ubiquitin-conjugation system. In addition, DUBs and ULPs are responsible for activating Ub and Ubls from their inactive corresponding precursor forms. Here we review recent progress in molecular details of these deconjugating enzymes of Ubls.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。