针对泛素化与去泛素化酶的化学探针

蛋白质泛素化对真核细胞内几乎所有生理过程都具备调控作用。新的泛素化与去泛素化酶的发现、功能机制研究及相关化学分子干预是该领域的重要科学命题。针对泛素化与去泛素化酶的化学探针开发促进了人们对于蛋白质泛素化的形成、募集、信号传导及脱除过程中所涉及生化事件的理解。本文总结了近年来针对泛素化与去泛素化酶化学探针的发展，归纳了不同类型探针的化学结构及合成方法，并讨论了它们的各类应用，包括筛查泛素依赖的信号传导系统、监控泛素相关酶活性、辅助泛素相关的识别和催化过程的分子机制解析等。     

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 importance of regulatory ubiquitination in cancer and metastasis

Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis.     

Regulation of the TGF-β pathway by deubiquitinases in cancer

The transforming growth factor-β (TGF-β) pathway regulates diverse cellular processes. It signals via serine/threonine kinase receptors and intracellular Smad and non-Smad effector proteins. In cancer cells, aberrant TGF-β signalling can lead to loss of growth inhibition and an increase in invasion, epithelial-to-mesenchymal transition (EMT) and metastasis. Therapeutic targeting of the pro-oncogenic TGF-β responses is currently being explored as a potential therapy against certain invasive and metastatic cancer types. The ubiquitin post-translational regulation system is emerging as a key regulatory mechanism for the control of TGF-β pathway components. In this review, we focus on the role of deubiquitinases (DUBs), which counteract the activity of E3 ubiquitin ligases. We will discuss the mechanisms by which specific DUBs control Smad and non-Smad TGF-β signalling routes, and how perturbation of the expression and function of DUBs contributes to misregulation of TGF-β signalling in cancer.     

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch

Protein ubiquitination is an important means of posttranslational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.     

A new scheme to discover functional associations and regulatory networks of E3 ubiquitin ligases

Background

Protein ubiquitination catalyzed by E3 ubiquitin ligases play important modulatory roles in various biological processes. With the emergence of high-throughput mass spectrometry technology, the proteomics research community embraced the development of numerous experimental methods for the determination of ubiquitination sites. The result is an accumulation of ubiquitinome data, coupled with a lack of available resources for investigating the regulatory networks among E3 ligases and ubiquitinated proteins. In this study, by integrating existing ubiquitinome data, experimentally validated E3 ligases and established protein-protein interactions, we have devised a strategy to construct a comprehensive map of protein ubiquitination networks.

Results

In total, 41,392 experimentally verified ubiquitination sites from 12,786 ubiquitinated proteins of humans have been obtained for this study. Additional 494 E3 ligases along with 1220 functional annotations and 28588 protein domains were manually curated. To characterize the regulatory networks among E3 ligases and ubiquitinated proteins, a well-established network viewer was utilized for the exploration of ubiquitination networks from 40892 protein-protein interactions. The effectiveness of the proposed approach was demonstrated in a case study examining E3 ligases involved in the ubiquitination of tumor suppressor p53. In addition to Mdm2, a known regulator of p53, the investigation also revealed other potential E3 ligases that may participate in the ubiquitination of p53.

Conclusion

Aside from the ability to facilitate comprehensive investigations of protein ubiquitination networks, by integrating information regarding protein-protein interactions and substrate specificities, the proposed method could discover potential E3 ligases for ubiquitinated proteins. Our strategy presents an efficient means for the preliminary screen of ubiquitination networks and overcomes the challenge as a result of limited knowledge about E3 ligase-regulated ubiquitination.

     

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.     

Cellular senescence and protein degradation

Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.     

The emerging role of deubiquitination in nucleotide excision repair

Nucleotide excision repair (NER) protects genome stability by eliminating DNA helix distorting lesions, such as those induced by UV radiation. The addition and removal of ubiquitin, namely, ubiquitination and deubiquitination, have recently been demonstrated as general mechanisms to regulate protein functions. Accumulating evidence shows that several NER factors are subjected to extensive regulation by ubiquitination and deubiquitination. Thus, the balance between E3 ligases and deubiquitinating enzyme activities can dynamically alter the ubiquitin landscape at DNA damage sites, thereby regulating NER efficiency. Current knowledge about XPC ubiquitination by different ubiquitin E3 ligases highlights the importance of ubiquitin linkage types in regulating XPC binding and release from damaged DNA. Here, we discuss the emerging roles of deubiquitinating enzymes and their ubiquitin linkage specificities in NER.     

类泛素化修饰Neddylation的功能和调控机制研究进展

NEDD8 (neural precursor cell-expressed developmentally downregulated 8) 分子是一类结构上与泛素相似的分子,参与蛋白质翻译后修饰,这一过程被称为Neddylation．Neddylation的发生机制与泛素化相似,需要E1、E2、E3介导的一系列酶促反应．Neddylation修饰在Cullin-Roc类泛素连接酶的活性调控中具有至关重要的作用,与泛素化研究相比,在真核细胞内仅发现了很少的能被Neddylation修饰的底物,Neddylation的生理功能也有待深入研究．     

Ubiquitin signaling in immune responses

Ubiquitination has emerged as a crucial mechanism that regulates signal transduction in diverse biological processes, including different aspects of immune functions. Ubiquitination regulates pattern-recognition receptor signaling that mediates both innate immune responses and dendritic cell maturation required for initiation of adaptive immune responses. Ubiquitination also regulates the development, activation, and differentiation of T cells, thereby maintaining efficient adaptive immune responses to pathogens and immunological tolerance to self-tissues. Like phosphorylation, ubiquitination is a reversible reaction tightly controlled by the opposing actions of ubiquitin ligases and deubiquitinases. Deregulated ubiquitination events are associated with immunological disorders, including autoimmune and inflammatory diseases.     

靶向蛋白质泛素修饰及降解在前列腺癌治疗中的机遇与挑战

前列腺癌是中国发病率增长最快的男性肿瘤,抗雄激素治疗耐药是导致前列腺癌患者预后差的主要原因。因此,解决耐药性难题是前列腺癌转化研究的关键问题。哺乳动物细胞利用泛素-蛋白酶体系统实现蛋白质的靶向降解。因此,前列腺癌中关键的癌基因如雄激素受体（AR）的上游泛素化调控因子（如去泛素化酶）是潜在的治疗靶点。然而,这些酶具有较广的底物谱系,存在脱靶的可能性。近来,基于泛素-蛋白酶体系统开发的蛋白质降解靶向嵌合体（proteolysis-targeting chimeras,PROTAC）技术是最具前景和革命性的新型抗癌药物研发技术,能够利用特定E3泛素连接酶对靶蛋白进行降解而不影响其他底物。与传统小分子抑制剂相比,PROTAC分子在克服耐药性以及针对不可成药的靶点方面拥有巨大优势。目前,针对AR的PROTAC降解剂已在II期临床取得了成功,靶向蛋白质泛素化及降解途径的新技术将有望为前列腺癌的临床治疗带来新的突破。     

Conventional and unconventional ubiquitination in plant immunity

Ubiquitination is one of the most abundant types of protein post‐translational modification (PTM) in plant cells. The importance of ubiquitination in the regulation of many aspects of plant immunity has been increasingly appreciated in recent years. Most of the studies linking ubiquitination to the plant immune system, however, have been focused on the E3 ubiquitin ligases and the conventional ubiquitination that leads to the degradation of the substrate proteins by the 26S proteasome. By contrast, our knowledge about the role of unconventional ubiquitination that often serves as non‐degradative, regulatory signal remains a significant gap. We discuss, in this review, the recent advances in our understanding of ubiquitination in the modulation of plant immunity, with a particular focus on the E3 ubiquitin ligases. We approach the topic from a perspective of two broadly defined types of ubiquitination in an attempt to highlight the importance, yet current scarcity, in our knowledge about the regulation of plant immunity by unconventional ubiquitination.     

Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases

The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Here, we show that Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promoted ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. We suggest that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases.     

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.