Roles of various cullin-RING E3 ligases involved in hormonal and stress responses in plants

Post-translational modification plays an important role in the regulation of protein stability, enzyme activity, and the cellular localization of proteins. Ubiquitination is a representative post-translational modification in eukaryotes that is mainly responsible for protein degradation. There have been a number of reports on the role of ubiquitination in various cellular responses in plants, such as regulation of the cell division cycle, stress responses and hormonal signaling. Among the three types of ubiquitination-related enzymes, E3 ubiquitin ligase is critical in determining substrate specificity. The importance of cullin-RING E3 ligase (CRL), a type of E3 ligase, has been emphasized during the recent decade due to its large number and its involvement in various plant cellular processes. Here, we describe how CRL E3 ligase complexes are involved in cellular events mediated by plant hormones and during plant stress adaptation while focusing on their substrate receptors.     

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

病原菌调节宿主细胞泛素化途径的研究进展

泛素化是真核生物特有的蛋白质翻译后修饰,广泛地参与宿主细胞各种信号通路和生理过程.病原菌常通过分泌毒性效应蛋白,对泛素和泛素结合酶进行独特的共价修饰,或者利用泛素连接酶和去泛素化酶的酶学活性,调节宿主泛素化过程,从而干扰宿主细胞的信号转导,促进细菌的感染和生存.本文概述了病原菌效应蛋白调节宿主泛素化途径的主要研究进展和最新发现.     

The ‘ubiquitous’ reality of vector immunology

Ubiquitination (ubiquitylation) is a common protein modification that regulates a multitude of processes within the cell. This modification is typically accomplished through the covalent binding of ubiquitin to a lysine residue onto a target protein and is catalysed by the presence of three enzymes: an activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin‐protein ligase (E3). In recent years, ubiquitination has risen as a major signalling regulator of immunity and microbial pathogenesis in the mammalian system. Still, little is known about how ubiquitin relates specifically to vector immunology. Here, we provide a brief overview of ubiquitin biochemistry and describe how ubiquitination regulates immune responses in arthropods of medical relevance. We also discuss scientific gaps in the literature and suggest that, similar to mammals, ubiquitin is a major regulator of immunity in medically important arthropods.     

RNAi in the regulation of mammalian viral infections

Ubiquitination now ranks with phosphorylation as one of the best-studied post-translational modifications of proteins with broad regulatory roles across all of biology. Ubiquitination usually involves the addition of ubiquitin chains to target protein molecules, and these may be of eight different types, seven of which involve the linkage of one of the seven internal lysine (K) residues in one ubiquitin molecule to the carboxy-terminal diglycine of the next. In the eighth, the so-called linear ubiquitin chains, the linkage is between the amino-terminal amino group of methionine on a ubiquitin that is conjugated with a target protein and the carboxy-terminal carboxy group of the incoming ubiquitin. Physiological roles are well established for K48-linked chains, which are essential for signaling proteasomal degradation of proteins, and for K63-linked chains, which play a part in recruitment of DNA repair enzymes, cell signaling and endocytosis. We focus here on linear ubiquitin chains, how they are assembled, and how three different avenues of research have indicated physiological roles for linear ubiquitination in innate and adaptive immunity and suppression of inflammation.     

Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase

Genetic studies identified Itch, which is a homologous to the E6-associated protein carboxyl terminus (Hect) domain-containing E3 ubiquitin-protein ligase that is disrupted in non-agouti lethal mice or Itchy mice. Itch-deficiency results in abnormal immune responses and constant itching in the skin. Here, Itch was shown to associate with Notch, a protein involved in cell fate decision in many mammalian cell types, including cells in the immune system. Itch binds to the N-terminal portion of the Notch intracellular domain via its WW domains and promotes ubiquitination of Notch through its Hect ubiquitin ligase domain. Thus, Itch may participate in the regulation of immune responses by modifying Notch-mediated signaling.     

A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins

Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13–Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.     

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.     

ERAD‐dependent control of the Wnt secretory factor Evi

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.     

Diversity of bacterial manipulation of the host ubiquitin pathways

Ubiquitination is generally considered as a eukaryotic protein modification, which is catalysed by a three‐enzyme cascade and is reversed by deubiquitinating enzymes. Ubiquitination directs protein degradation and regulates cell signalling, thereby plays key roles in many cellular processes including immune response, vesicle trafficking and cell cycle. Bacterial pathogens inject a series of virulent proteins, named effectors, into the host cells. Increasing evidence suggests that many effectors hijack the host ubiquitin pathways to benefit bacterial infection. This review summarizes the known functions and mechanisms of effectors from human bacterial pathogens including enteropathogenic Escherichia coli, Salmonella, Shigella, Chlamydia and Legionella, highlighting the diversity in their mechanisms for manipulating the host ubiquitin pathways. Many effectors adopt the molecular mimicry strategy to harbour similar structures or functional motifs with those of the host E3 ligases and deubiquitinases. On the other hand, a few of effectors evolve novel structures or new enzymatic activities to modulate various steps of the host ubiquitin pathways. The diversity in the mechanisms enhances the efficient exploitation of the host ubiquitination signalling by bacteria.     

The Met1-linked ubiquitin machinery in inflammation and infection

Ubiquitination is an essential post-translational modification that regulates most cellular processes. The assembly of ubiquitin into polymeric chains by E3 ubiquitin ligases underlies the pleiotropic functions ubiquitin chains regulate. Ubiquitin chains assembled via the N-terminal methionine, termed Met1-linked ubiquitin chains or linear ubiquitin chains, have emerged as essential signalling scaffolds that regulate pro-inflammatory responses, anti-viral interferon responses, cell death and xenophagy of bacterial pathogens downstream of innate immune receptors. Met1-linked ubiquitin chains are exclusively assembled by the linear ubiquitin chain assembly complex, LUBAC, and are disassembled by the deubiquitinases OTULIN and CYLD. Genetic defects that perturb the regulation of Met1-linked ubiquitin chains causes severe immune-related disorders, illustrating their potent signalling capacity. Here, we review the current knowledge about the cellular machinery that conjugates, recognises, and disassembles Met1-linked ubiquitin chains, and discuss the function of this unique posttranslational modification in regulating inflammation, cell death and immunity to pathogens.Subject terms: Signal transduction, Antimicrobial responses, Cell death and immune response

     

Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.     

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.     

The MARCH family E3 ubiquitin ligase K5 alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation

Kaposi's sarcoma (KS) lesions are complex mixtures of KS-associated herpesvirus (KSHV)-infected spindle and inflammatory cells. In order to survive the host immune responses, KSHV encodes a number of immunomodulatory proteins, including the E3 ubiquitin ligase K5. In exploring the role of this viral protein in monocytes, we made the surprising discovery that in addition to a potential role in down regulation of immune responses, K5 also contributes to increased proliferation and alters cellular metabolism. This ubiquitin ligase increases aerobic glycolysis and lactate production through modulation of cellular growth factor-binding receptor tyrosine kinase endocytosis, increasing the sensitivity of cells to autocrine and paracrine factors. This leads to an altered pattern of cellular phosphorylation, increases in Akt activation and a longer duration of Erk1/2 phosphorylation. Overall, we believe this to be the first report of a virally-encoded ubiquitin ligase potentially contributing to oncogenesis through alterations in growth factor signaling cascades and opens a new avenue of research in K5 biology.     

Emerging Roles and Research Tools of Atypical Ubiquitination

Ubiquitination is a posttranslational modification characterized by the covalent attachment of ubiquitin molecules to protein substrates. The ubiquitination modification process is reversible, dynamic, and involved in the regulation of various biological processes, such as autophagy, inflammatory responses, and DNA damage responses. The forms of ubiquitin modification are very diverse, incorporating either a single ubiquitin molecule or a complicated ubiquitin polymer, and different types of ubiquitination usually elicit corresponding cellular responses. The development of research tools and strategies has afforded more detailed insight into atypical ubiquitin signaling pathways that were previously poorly understood. Here, an update on the understanding of atypical ubiquitin chain signaling pathways is provided and the recent development of representative research tools for ubiquitin systems is discussed. In addition, the future challenges in ubiquitin research are reflected on and summarized.     

泛素链的形成机理

泛素化是真核细胞中重要的蛋白质翻译后修饰过程,通过靶向蛋白质降解或其他信号途径参与多种细胞功能.底物蛋白的多聚泛素化修饰是一个持续的过程,其中不仅涉及复杂泛素系统相关酶的参与,而且存在更为复杂的结构上相互作用与泛素链组装机理.不同的泛素链修饰决定了底物蛋白下游的不同命运,泛素结合酶E2在泛素链形成中的重要作用受到越来越多的关注.对泛素链形成机理的深入研究与认识有利于发现与泛素系统相关的疾病靶点和利用泛素化调控方法进行治疗.本综述总结了E2和E3如何决定不同泛素链形成的机制和相关的结构信息,以及两种不同的泛素链组装机制.     

E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers

The mitogen-activated protein kinase (MAPK) signaling pathway is the primary regulatory module of various cellular processes such as cell proliferation, differentiation, and stress responses. This pathway converts external stimuli to cellular responses via three major kinases: mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). Ubiquitination is a post-translational modification of proteins with ubiquitin, which results in the formation of mono- or poly-ubiquitin chains of substrate proteins. Conversely, removal of the ubiquitin by deubiquitinating enzymes (DUBs) is known as deubiquitination. This review summarizes mechanisms of the MAPK signaling pathways (ERK1/2, ERK5, p38, and JNK1/2/3 signaling pathway) in cancers, and of E3 ligases and DUBs that target the MAPK signaling components such as Raf, MEK1/2, ERK1/2, MEKK2/3, MEKK1-4, TAK1, DLK1, MLK1-4, ASK1/2, and MKK3-7.