蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

Ubiquitin and endocytic internalization in yeast and animal cells

Endocytosis is involved in a wide variety of cellular processes, and the internalization step of endocytosis has been extensively studied in both lower and higher eukaryotic cells. Studies in mammalian cells have described several endocytic pathways, with the main emphasis on clathrin-dependent endocytosis. Genetic studies in yeast have underlined the critical role of actin and actin-binding proteins, lipid modification, and the ubiquitin conjugation system. The combined results of studies of endocytosis in higher and lower eukaryotic cells reveal an interesting interplay in the two systems, including a crucial role for ubiquitin-associated events. The ubiquitylation of yeast cell-surface proteins clearly acts as a signal triggering their internalization. Mammalian cells display variations on the common theme of ubiquitin-linked endocytosis, according to the cell-surface protein considered. Many plasma membrane channels, transporters and receptors undergo cell-surface ubiquitylation, required for the internalization or later endocytic steps of some cell-surface proteins, whereas for others, internalization involves interaction with the ubiquitin conjugation system or with ancillary proteins, which are themselves ubiquitylated. Epsins and Eps15 (or Eps15 homologs), are commonly involved in the process of endocytosis in all eukaryotes, their critical role in this process stemming from their capacity to bind ubiquitin, and to undergo ubiquitylation.     

Immunity by ubiquitylation: a reversible process of modification

The conjugation of ubiquitin, a 76-amino-acid peptide, to a protein substrate provides a tag that either marks the labelled protein for degradation or modulates its function. The process of protein ubiquitylation--which is catalysed by coordinated enzymatic reactions that are mediated by enzymes known as E1, E2 and E3--has an important role in the modulation of immune responses. Importantly, protein ubiquitylation is a reversible process, and removal of ubiquitin molecules is mediated by de-ubiquitylating enzymes: for example, A20, which has been implicated in the regulation of immune responses. In addition, the conjugation of ubiquitin-like molecules, such as ISG15 (interferon-stimulated protein of 15 kDa), to proteins is also involved in immune regulation. This Review covers recent progress in our understanding of protein ubiquitylation in the immune system.     

病毒利用泛素系统生存、增殖的方式

真核生物中, 泛素系统是个复杂的体系, 主要包括泛素,26S 蛋白酶体和酶系统E1、E2 、E3。泛素- 蛋白酶体通路是细胞内非溶酶体蛋白降解的主要系统, 在许多细胞功能中发挥重要作用。最近研究发现, 许多病毒利用泛素系统为其自身服务, 这涉及病毒生活史的各个阶段并干扰宿主抗病毒反应的多种方式, 如下调细胞表面免疫分子而实现免疫逃避、调控病毒的基因转录、抑制细胞凋亡、促使病毒出芽和释放等。深入了
解病毒利用泛素系统的机制, 将为研究病毒感染机制提供新的视角, 并为药物研发提供新的靶标。     

When cleavage is not attractive: non-catalytic inhibition of ubiquitin chains at DNA double-strand breaks by OTUB1

Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes.     

Manipulation of the host cell death pathway by Shigella

Host cells deploy multiple defences against microbial infection. One prominent host defence mechanism, the death of infected cells, plays a pivotal role in clearing damaged cells, eliminating pathogens, removing replicative niches, exposing intracellular bacterial pathogens to extracellular immune surveillance and presenting bacteria‐derived antigens to the adaptive immune system. Although cell death can occur under either physiological or pathophysiological conditions, it acts as an innate defence mechanism against bacterial pathogens by limiting their persistent colonization. However, many bacterial pathogens, including Shigella, have evolved mechanisms that manipulate host cell death for their own benefit.     

Bacteria and the ubiquitin pathway

Ubiquitylation participates in a repertoire of reversible post-translational modifications that modulate the function, localization and half-life of proteins by regulating their association with various ubiquitin-binding proteins. In response to pathogen infection, bacterial effectors impact ubiquitin and ubiquitin-like modifications of key proteins in immune and anti-apoptotic signaling cascades. Certain bacteria corrupt the ubiquitylation machinery in order to regulate their virulence factors spatially and temporally or to trigger internalization of bacteria into host cells. Several new examples of how bacterial factors target ubiquitin and ubiquitin-like regulation emphasize the importance of modulating ubiquitin signaling to establish either long-lasting or devastating relationships of bacteria with their hosts.     

Role of Bmi1 in H2A ubiquitylation and Hox gene silencing

Posttranslational histone modifications play a crucial role in the regulation of chromatin structure and gene activity. In previous studies, we identified the histone H2A ubiquitin ligase as Ring2, together in a complex with Ring1, Bmi1, and HPH2 (human polyhomeotic 2). We report here that the oncogene Bmi1 stimulates H2A ubiquitylation both in vitro and in vivo and that Bmi1-regulated H2A ubiquitylation is required for Hox gene silencing and normal cell growth. Our studies indicate that Bmi1 maintains the integrity of the complex through simultaneous interactions with the other subunits. We reconstituted the functional human H2A ubiquitin ligase complex and a panel of subcomplexes of different subunits. Comparisons of the H2A ubiquitin ligase activities of these different complexes revealed that Bmi1 stimulates the H2A ubiquitin ligase activity of Ring2 (and Ring1). Additionally, we demonstrated that the HoxC5 gene is regulated by ubiquitylated H2A in HeLa cells and that ubiquitylated H2A is localized on 5' regulatory regions of the HoxC5 gene. The role of Bmi1 in H2A ubiquitylation and HoxC5 gene expression in vivo was analyzed by RNA interference experiments. Knockdown of Bmi1 causes a global and loci-specific loss of H2A ubiquitylation, up-regulation of the HoxC5 gene, and slower cell growth. Intriguingly, Ring2 binds to its target regions in Bmi1 knockdown cells. Therefore, our studies reveal that Bmi1 is required for H2A ubiquitylation and suggest that H2A ubiquitylation regulates Bmi1-mediated gene expression.     

Ubiquitin in the immune system

Ubiquitination is a post-translational modification process that has been implicated in the regulation of innate and adaptive immune responses. There is increasing evidence that both ubiquitination and its reversal, deubiquitination, play crucial roles not only during the development of the immune system but also in the orchestration of an immune response by ensuring the proper functioning of the different cell types that constitute the immune system. Here, we provide an overview of the latest discoveries in this field and discuss how they impact our understanding of the ubiquitin system in host defence mechanisms as well as self-tolerance.     

Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation

The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIM-dependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.     

Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Proteome-wide catalogs of ubiquitylated proteins reveal a rapid engagement of the ubiquitin–proteasome system in Arabidopsis innate immunity.     

Dynamic regulation of PCNA ubiquitylation/deubiquitylation

Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process.     

Regulatory functions of ubiquitin in diverse DNA damage responses

In recent years there has been intense investigation and rapid progress in our understanding of the cellular responses to various types of endogenous and exogenous DNA damage that ensure genetic stability. These studies have identified numerous roles for ubiquitylation, the post-translational modification of proteins with single ubiquitin or poly-ubiquitin chains. Initially discovered for its role in targeting proteins for degradation in the proteasome, ubiquitylation functions in a variety of regulatory roles to co-ordinate the recruitment and activity of a large number of protein complexes required for recovery from DNA damage. This includes the identification of essential DNA damage response genes that encode proteins directly involved in the ubiquitylation process itself, proteins that are targets for ubiquitylation, proteins that contain ubiquitin binding domains, as well as proteins involved in the de-ubiquitylation process. This review will focus on the regulatory functions of ubiquitylation in three distinct DNA damage responses that involve ubiquitin modification of proliferating cell nuclear antigen (PCNA) in DNA damage tolerance, the core histone H2A and its variant H2AX in double strand break repair (DSBR) and the Fanconi anaemia (FA) proteins FANCD2 and FANCI in cross link repair.     

Regulation of DNA repair by ubiquitylation

The process of ubiquitylation is best known for its role in targeting proteins for degradation by the proteasome. However, recent studies of DNA-repair and DNA-damage-response pathways have significantly broadened the scope of the role of ubiquitylation to include non-proteolytic functions of ubiquitin. These pathways involve the monoubiquitylation of key DNA-repair proteins that have regulatory functions in homologous recombination and translesion DNA synthesis, and involve the polyubiquitylation of nucleotide-excision-repair proteins.     

Lymphocyte subsets as prognostic markers for cancer patients receiving immunomodulative therapy

Immunogenic features of some malignancies have aroused interest in immunotherapy of cancer. Immunotherapy seems most effective in patients with a small tumour burden, and the focus of immunotherapy trials has, thus, lately been on adjuvant treatment. To enable further development of immunotherapy we need to know more about the mechanisms involved in host defence, especially when the system is influenced by extrinsic factors, that is, immunomodulative agents. T lymphocytes play an important role in the host defence against tumour cells trying to escape from immune surveillance. The mechanisms that regulate the host defence systems are complex, and the influence of extrinsic factors such as immunotherapeutic agents is poorly understood. Most data on lymphocyte subsets in malignant disease originate from melanoma or renal cell carcinoma (RCC) studies, although there are scattered data on lymphocyte subsets also in other malignancies. There are several studies implying that the relative amount of CD4+, CD8+, and natural killer (NK) cells may be important and that, by reducing the tumour burden or by using different therapeutic agents, we can stimulate the host defence. However, only some of these studies imply that these changes can have an impact on clinical outcome and prognosis. The findings of the studies reviewed in this paper are mostly encouraging, but whether the lymphocyte subsets have any value as prognostic markers in patients with malignancies receiving immunotherapy is still unclear. Large randomized immunotherapy trials including an observation arm give an ideal opportunity to recognize those immunological changes that are due to therapy, related to the natural host defence, or whether they have any prognostic value.     

Immunological Control of Fish Diseases

All metazoans possess innate immune defence system whereas parameters of the adaptive immune system make their first appearance in the gnathostomata, the jawed vertebrates. Fish are therefore the first animal phyla to possess both an innate and adaptive immune system making them very interesting as regards developmental studies of the immune system. The massive increase in aquaculture in recent decades has also put greater emphasis on studies of the fish immune system and defence against diseases commonly associated with intensive fish rearing. Some of the main components of the innate and adaptive immune system of fish are described. The innate parameters are at the forefront of immune defence in fish and are a crucial factor in disease resistance. The adaptive response of fish is commonly delayed but is essential for lasting immunity and a key factor in successful vaccination. Some of the inherent and external factors that can manipulate the immune system of fish are discussed, the main fish diseases are listed and the pathogenicity and host defence discussed. The main prophylactic measures are covered, including vaccination, probiotics and immunostimulation. A key element in the immunological control of fish diseases is the great variation in disease susceptibility and immune defence of different fish species, a reflection of the extended time the present day teleosts have been separated in evolution. Future research will probably make use of molecular and proteomic tools both to study important elements in immune defence and prophylactic measures and to assist with breeding programmes for disease resistance.