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.     

泛素化修饰调控固有免疫信号通路的研究进展

宿主细胞依赖固有免疫系统识别入侵的病原微生物,经相关细胞信号转导通路,激活促炎症及抗感染的基因表达。泛素化修饰是细胞内广泛存在的蛋白质翻译后修饰机制,全方位调控宿主细胞防御病原微生物的动态过程:一方面,作为多功能的信号调节分子,在时空上精细调节免疫反应的进程,有效地清除入侵的病原体;另一方面,通过降解关键信号转导分子,限制过度免疫反应,避免造成宿主自体损伤。本文总结了泛素化修饰在Toll样受体信号通路(TLR)、RIG-I样受体信号通路(RLR)和STING介导的信号通路中的新功能,以及相关分子调控机制,并对前沿方向进行展望。     

β-拘留蛋白2的活性调控机制

β-拘留蛋白2(β-arrestin2)是arrestins家族的一个成员,广泛表达于全身组织,其不仅可以调节大多数G蛋白偶联受体(G-protein coupled receptors,GPCRs)的脱敏、内化,还能调节多种非GPCRs的内化,或作为支架蛋白质参与MAPK、PI3K/AKT等信号通路。越来越多的研究发现,β-arrestin2在肿瘤、自身免疫性疾病、纤维化疾病、心血管疾病、代谢性疾病等多种疾病进展过程中表达异常,提示其可能在疾病的病理过程中发挥重要的调控作用。β-arrestin2功能的发挥不仅与其在细胞中的表达水平有关,更依赖于对其活性的调控。但对于β-arrestin2的活性如何被调控,以及其活性如何影响其生物学功能的关注较少。近年来,陆续有研究报道了β-arrestin2可发生磷酸化、泛素化、SUMO化、S-亚硝基化等翻译后修饰,探讨了其翻译后修饰的可能位点,并发现翻译后修饰可影响β-arrestin2的细胞定位、调节受体内吞的作用、β-arrestin2与信号分子的相互作用及下游信号通路,对了解β-arrestin2活性调控在细胞中的作用具有重要意义。本文在介绍β-arrestin2的结构特征及其参与的信号转导通路的基础上,对近年来β-arrestin2的翻译后修饰等活性调节机制的研究进展进行综述,以期为以β-arrestin2为可能靶点的药物开发提供参考。     

细菌效应蛋白对宿主细胞Rho小G蛋白信号通路的调节作用

病原体细菌通过自身分泌系统分泌效应蛋白并注入宿主体内,修饰宿主的信号转导系统,破坏宿主细胞中天然免疫有关信号通路,发挥毒性作用使宿主产生疾病。吞噬作用在天然免疫系统中发挥重要作用,这个过程涉及肌动蛋白细胞骨架的重排。Rho(Ras homolog family)小G蛋白家族成员作为细胞骨架结构的重要调控蛋白可调节这一过程,其相关信号通路成为细菌效应蛋白的作用靶点。细菌效应蛋白可以模仿Rho的调节因子破坏信号通路,可以通过剪切Rho C-端的尾部结构使其从细胞膜解离并失去活性,可以直接模仿Rho发挥调控功能,可以影响Rho上游的调控事件影响其活性,也可通过对Rho进行直接的翻译后修饰使其失活,形成有利于细菌生存、繁殖、毒力释放的环境。由此导致的Rho信号通路功能紊乱使宿主产生智力缺陷、免疫功能障碍、癌症等多种疾病。     

细菌类脂A结构与功能研究进展

类脂A分布在革兰氏阴性细菌的外表层,它是脂多糖分子的疏水基团.脂多糖,俗名内毒素,可以引起致命的脓毒症、内毒素血症和多器官功能障碍综合症等疾病.近年来的研究发现:脂多糖分子中只有类脂A部分具有内毒素的活性.当细菌侵入人体后,其表面的类脂A可以刺激宿主细胞表面的Toll样受体-4,在细胞内引起一连串的反应,产生一系列的细胞因子.本文根据近年来内毒素领域的国际研究进展,系统综述了类脂A的结构特征、合成途径和致病机理,并在此基础上分析了内毒素在疫苗开发领域的应用前景.     

蛋白质组学在信号转导研究中的应用

新近发展起来的蛋白质组学高通量技术引入到信号转导通路研究中,产生了一个新的研究领域：信号转导蛋白质组学。其作为功能蛋白质组学的一个重要组成部分,以研究信号转导通路以及其中的信号分子改变的蛋白质组学。克服了传统地针对单条信号转导通路以及其中的单个信号分子研究策略的局限性,能够在一次实验中系统地研究多条信号转导通路中的蛋白质一蛋白质间的相互作用、蛋白质磷酸化等翻译后修饰和下游靶蛋白的改变,有助于全面阐述信号转导通路,已成为一个新的研究热点。     

SUMO化修饰在NF-κB信号通路中的作用

小泛素相关修饰物（smallubiq uitin related modifier,SUMO）修饰是与泛素化修饰类似的蛋白质翻译后修饰,在细胞信号转导、核质运输与转录调控等方面发挥重要作用。核因子-κB（nuclear factor-κB,NF-κB）通路是公认的参与炎症和免疫反应的重要调节通路。近年来研究发现,SUMO通过各种机制广泛参与NF-κB信号通路的调节。研究两者的关系,可能为相关疾病的防治找到新的思路。     

病原菌效应蛋白翻译后修饰宿主免疫防御通路

病原菌效应蛋白破坏宿主细胞的正常信号转导是病原菌和宿主相互作用的重要体现.效应蛋白往往具有独特的生化活性,针对宿主细胞内与抗细菌感染相关的重要通路进行阻断.近年来,在病原菌效应蛋白作用机制的研究中,人们发现了几种由效应蛋白介导的全新的蛋白质翻译后修饰.OspF(outer Shigella protein F)效应蛋白家族具有磷酸化苏氨酸裂合酶活性,通过"消去"修饰和失活宿主MAPK激酶.NleE(non-LEE encoded effector E)效应蛋白则通过半胱氨酸甲基化修饰来抑制感染诱导NF-κB炎症通路的激活.NleB(non-LEEencoded effectorB)蛋白抑制宿主的死亡信号通路,则依赖于其N-乙酰葡萄糖胺转移酶活性介导的对死亡结构域蛋白的精氨酸糖基化修饰.而VopS(Vibrio outer protein S)和IbpA(Immunoglobulin-binding protein A)等含有Fic结构域的蛋白,则可以将AMP基团转移到Rho家族小G蛋白的保守苏氨酸或酪氨酸上,导致小G蛋白的失活和肌动蛋白细胞骨架的紊乱,从而引起细胞毒性.以上效应蛋白作用机制及生化活性的阐明,有助于全方位了解病原菌的致病毒力机制,也开辟了蛋白质翻译后修饰介导病原-宿主相互作用研究的新方向,同时对真核生物的信号转导研究也具有重要指导意义.     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

泛素化和SUMO化对DEC1的拮抗调控作用

分化的胚软骨表达蛋白1(differentiated embryo-chondrocyte expressed gene 1,DEC1)作为一种时钟蛋白,除了在周期节律的调控中发挥转录抑制作用外,还在能量代谢以及多种肿瘤相关的信号通路的调控中发挥重要作用。此外,蛋白质的翻译后修饰是实现蛋白质功能精细调控的一种重要方式。目前发现,DEC1主要可被两种翻译后修饰,即泛素化和SUMO化修饰。尽管泛素化和SUMO化是两种过程非常类似的蛋白质翻译后修饰方式,但是它们对目的蛋白功能的调控却截然不同。由于泛素化和SUMO化与底物的作用靶点都是赖氨酸(Lys),因此在多数情况下,泛素化和SUMO化以拮抗性的方式调控底物蛋白的功能。鉴于此,该文旨在阐述泛素化和SUMO化修饰对DEC1功能的拮抗调节过程,为了解时钟蛋白DEC1对多种信号通路的调控过程中的分子机制提供新的思路。     

内毒素耐受机制的研究进展

内毒素耐受（endotoxin tolerance）早在50多年前就已经引起人们的关注,但其具体的分子机制至今尚不清楚。Toll样受体4（Toll-1ike receptor-4,TLR4）作为脂多糖（LPS）的主要受体,参与LPS信号的跨膜转导,与LPS耐受密切相关。在内毒素耐受过程中,TLR4转导通路中的信号蛋白及下游转录因子在数量、结构和功能上发生改变,可引起炎性因子释放减少、抗炎因子产生增加,并导致特定信号通路（如P13K通路）和负性调节因子（如SHIP1、SOCS、FLN29等）的激活。除此之外,TLR2通路、Gi蛋白、蛋白激酶C（protein kinase C,PKC）以及一些信号分子的剪接异构体等也参与了内毒素耐受现象的发生。总之,内毒素耐受是一个由多种原因引起的、多种生物物质参与的复杂病理生理过程,是机体抵抗G-细菌感染的重要保护机制。因此,探索内毒素耐受的机制,寻求机体内源性的抗炎机制将为败血症等一些致死性感染性疾病的治疗提供新的思路和理论依据。     

The biology of endotoxin

Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity. The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world. However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface. The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.     

Lipopolysaccharide binding to the periplasmic protein LptA

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram‐negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram‐negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG₂, that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin‐labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA–LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N‐terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.     

细胞信号转导与可变剪接调控

大多数真核基因能够发生可变剪接,其调控对于生理和病理状态下细胞功能的实现至关重要,而异常可变剪接则可导致多种疾病。虽然已知可变剪接能够在转录后水平调节基因表达,然而目前仍不清楚特定的可变剪接模式是如何被调控的。越来越多的研究发现细胞信号和外界环境刺激能够调控靶基因的剪接模式,并且已发现一些与可变剪接调控有关的信号转导通路,而后者能够通过修饰剪接因子进而改变剪接因子的亚细胞定位或者活性,从而实现对靶基因可变剪接模式的调控。由细胞信号转导通路所构成的网络能够灵活多样地调控基因剪接,一条信号通路可调控多个基因剪接,而多条信号通路也可调控同一基因剪接,对于理解信号转导过程的分子机制具有重要意义。     

Effect of endotoxin on protein degradation and lipid peroxidation of erythrocytes.

Sepsis has often been associated with infection due to endotoxin (LPS) produced from gram-negative bacteria. Microcirculatory failure is one of the ultimate causes of septic shock. We studied the effect of endotoxin on the protein breakdown and lipid peroxidation of erythrocyte. In vivo (20 ug LPS/100 g) studies in rats showed increased tyrosine production from erythrocyte, as an index of protein degradation in erythrocyte. In vitro studies using 25 microg to 250 microg LPS per ml also showed similar type of increased effect of endotoxin in protein degradation. Washed erythrocyte devoid of plasma and leucocytes did not show any increased effect after endotoxin treatment. Lipid peroxidation was also increased after endotoxin treatment. However, protein degradation was more prominent than lipid peroxidation. We concluded therefore that the protein degradation and lipid peroxidation of erythrocytes caused by endotoxin are probably related to the production of septic shock.     

Detection of the endotoxin of Gram-negative bacteria in human blood

Recent new data on the important role played by lipopolysaccharides (endotoxin) of Gram negative bacteria in physiology and pathogenesis of the most important human infectious and noninfectious diseases testify to the necessity of wide clinical trials of different methods for LPS detection in blood and other physiological fluids. Among presently available diagnostic methods for endotoxinemia detection, the highly sensitive LAL (Limulus Amebocyte Lysate) test in various modifications is most widely used. The LAL test is known to be non-specific, however many drawbacks of this test have been successfully overcome. The results of clinical studies on the determination of the LPS activity in the systemic blood stream and antibody titers to its most common determinants, as well as the reserves of endotoxin binding with granulocytes give grounds for optimistic evaluation of the future studies on the role of LPS in human physiology and pathology. In clinical practice both positive sides and drawbacks of the presently known methods for LPS detection, including the LAL test, must be borne in mind for the complex evaluation of endotoxinemia levels.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.     

Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl‐CoA carboxylase as a PII binding partner

The P_II family comprises a group of widely distributed signal transduction proteins. The archetypal function of P_II is to regulate nitrogen metabolism in bacteria. As P_II can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by P_II may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a P_II protein affinity column was used to identify P_II targets in Azospirillum brasilense. One of the P_II partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl‐CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a P_II target in Arabidopsis thaliana we hypothesized that the P_II–BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the P_II–BCCP interaction is conserved in Escherichia coli. The BCCP–P_II interaction required MgATP and was dissociated by increasing 2‐oxoglutarate. The interaction was modestly affected by the post‐translational uridylylation status of P_II; however, it was completely dependent on the post‐translational biotinylation of BCCP.