Function and regulation of cullin-RING ubiquitin ligases

Cullin-RING complexes comprise the largest known class of ubiquitin ligases. Owing to the great diversity of their substrate-receptor subunits, it is possible that there are hundreds of distinct cullin-RING ubiquitin ligases in eukaryotic cells, which establishes these enzymes as key mediators of post-translational protein regulation. In this review, we focus on the composition, regulation and function of cullin-RING ligases, and describe how these enzymes can be characterized by a set of general principles.     

Identification and Characterization of Activating and Conjugating Enzymes of the Ubiquitin System from the Unicellular Alga Chiamydomonas reinhardtii

Abstract: Using a biochemical approach we identified families of ubiquitin-activating and ubiquitin-conjugating enzymes in Chiamydomonas reinhardtii . The family of ubiquitin-activating enzymes, characterized by their ability to form thioesters with ubiquitin and eluting off a ubiquitin affinity column by ATP-depletion probably consists of at least four members. Whereas one of these enzymes is active under a broad range of pH values, thioesterof the other UBAs with ubiquitin is restricted to pH 7.5. Two ubiquitin-activating enzymes are metabolically phosphory-lated which is assumed to be an activity control mechanism. Most of the 7 ubiquitin-conjugating enzymes detected in this study were found to bind rather tightly to an anion exchange column, and eluted off the column at specific salt concentra tions. Two of the ubiquitin-conjugating enzymes described here did, however, not bind to this column. These enzymes can, as all other C. reinhardtii ubiquitin-conjugating enzymes, perform thioester formation with ubiquitin regardless of the source (plant/animal) of the ubiquitin-activating enzyme.     

Deubiquitinating enzymes as cellular regulators

Modification of proteins by the covalent attachment of ubiquitin is a key regulatory mechanism of many cellular processes including protein degradation by the 26S proteasome. Deubiquitination, reversal of this modification, must also regulate the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes catalyze the removal of ubiquitin from ubiquitin-conjugated substrate proteins as well as from its precursor proteins. Deubiquitinating enzymes occupy the largest family of enzymes in the ubiquitin system, implying their diverse function in regulation of the ubiquitin-mediated pathways. Here we explore the diversity of deubiquitinating enzymes and their emerging roles as cellular regulators.     

Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) represents the primary means of quality control within the secretory pathway. Critical to this process are ubiquitin protein ligases (E3s) which, together with ubiquitin conjugating enzymes (E2s), mediate the ubiquitylation of proteins targeted for degradation from the ER. In this chapter we review our knowledge of both Saccharomyces cerevisiae and mammalian ERAD ubiquitin ligases. We focus on recent insights into these E3s, their associated proteins and potential mechanisms of action.     

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。     

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。     

Substrate specificity of the ubiquitin and Ubl proteases

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families.     

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。     

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.     

Ubiquitin enzymes in the regulation of immune responses

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.     

The ER‐resident ubiquitin‐specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic‐reticulum‐associated degradation (ERAD). Although the involvement of membrane‐associated ubiquitin‐conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin‐specific protease (USP)19. We show that USP19 is a tail‐anchored ubiquitin‐specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T‐cell receptor‐α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non‐catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane‐anchored DUB involved in the turnover of ERAD substrates.     

Ubiquitin binding proteins protect ubiquitin conjugates from disassembly

As a step in their turnover proteins in eukaryotic cells are coupled to a small protein, ubiquitin, before they are recognised by 26S proteasomes and degraded. However, cells also contain many deubiquitinating enzymes, which can rescue proteins by cleaving off the ubiquitin chains. Here we report that three ubiquitin binding proteins, Rhp23, Dph1 and Pus1, from fission yeast can protect multiubiquitin conjugates against deubiquitination. This protection depends on the ubiquitin binding domains and may promote degradation of ubiquitinated proteins.     

Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing

To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC-MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. This suggests a link between the ubiquitin system and these cellular processes. This map of cellular ubiquitin-associated proteins may be useful for further studies of ubiquitin system function.     

Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer

Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Sub-types of breast cancer defined by the expression of steroid hormones and Her2/Neu oncogene have distinct prognosis and undergo different therapies. Besides differing in their phenotype, sub-types of breast cancer display various molecular lesions that participate in their pathogenesis. BRCA1 is one of the common hereditary cancer predisposition genes and encodes for an ubiquitin ligase. Ubiquitin ligases or E3 enzymes participate together with ubiquitin activating enzyme and ubiquitin conjugating enzymes in the attachment of ubiquitin (ubiquitination) in target proteins. Ubiquitination is a post-translational modification regulating multiple cell functions. It also plays important roles in carcinogenesis in general and in breast carcinogenesis in particular. Ubiquitin conjugating enzymes are a central component of the ubiquitination machinery and are often perturbed in breast cancer. This paper will discuss ubiquitin and ubiquitin-like proteins conjugating enzymes participating in breast cancer pathogenesis, their relationships with other proteins of the ubiquitination machinery and their role in phenotype of breast cancer sub-types.     

Cullin-containing E3 ubiquitin ligases in plant development

In eukaryotes, the ubiquitin-proteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants. Several types of E3 ubiquitin ligases have already been characterized in eukaryotes. Recently, some of these E3 enzymes have been implicated in specific plant signaling pathways.     

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.