嗜肺军团菌调节宿主泛素化途径的效应因子及其分子机制研究进展

嗜肺军团菌是一种胞内寄生菌,其通过特有的Dot/IcmType-IVB分泌系统向胞浆内分泌大量效应因子,其中已知参与宿主泛素化调控的效应因子有十多种.这些效应因子通过对宿主泛素化途径进行调控来达到逃避宿主免疫系统"监视"并大量增殖的目的.参与调控宿主泛素化途径的效应因子包括 AnkB、SidC、LubX、SidH、Le...     

应用IP-2D nano-HPLC-MALDI-TOF-TOF鉴定蛋白质泛素化修饰

近年来,在蛋白质研究中,特别是在蛋白质翻译后修饰(PTM)的研究中,生物质谱技术的应用越来越广泛,与纳升级HPLC的联合应用,使这一技术手段更加有效.针对泛素化在细胞功能调控中发挥关键作用的PTM的特点,将免疫沉淀、2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(IP-2D nano-HPLC-MALDI-TOF-TOF)有机整合,建立了天然状态下蛋白质泛素化位点的鉴定方法,并应用这一方法确定出K562细胞内具有酪氨酸激酶活性的蛋白c-ABL的泛素化位点.为定性鉴定生理和病理状态下内源性蛋白的泛素化修饰提供了借鉴.     

乙酰化修饰在嗜肺军团菌致病过程中的作用

乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。     

糖基化修饰在嗜肺军团菌致病过程中的作用

糖基化修饰是由糖基转移酶催化的广泛存在于生物体内的一种蛋白质翻译后修饰,其与致病菌的毒力密切相关.嗜肺军团菌是一种胞内致病菌,通过其IVB型分泌系统向宿主细胞转运近300种效应蛋白,可介导多种蛋白质翻译后修饰,干扰宿主的细胞进程.其中,Lgt家族、SidI、SetA及LtpM等效应蛋白介导宿主蛋白糖基化修饰,影响宿主蛋...     

嗜肺军团菌效应蛋白之间的调控机制

嗜肺军团菌(Legionella pneumophila)是一种革兰氏阴性致病菌,它可以引起人类军团病。嗜肺军团菌的Dot/Icm分泌系统在其致病过程中至关重要,其向宿主细胞内转运约330种效应蛋白,通过修饰细胞调节因子、抑制细胞凋亡等一系列措施操纵宿主细胞的多种生命活动,以完成自身的增殖与侵染。为避免对宿主生理造成不必要的破坏,嗜肺军团菌已进化出复杂而精细的调控机制来平衡嗜肺军团菌毒力与宿主细胞的稳态,以确保嗜肺军团菌在宿主细胞内的生存。军团菌效应蛋白的功能及分子机制的研究近几年取得突破性进展,嗜肺军团菌效应蛋白之间的作用机理也成为我们进一步研究的热点。该文主要对嗜肺军团菌的致病机制及其效应蛋白间的调控机制进行了综述,为进一步了解嗜肺军团菌致病机制提供了一定的参考。     

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。     

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.     

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。     

去泛素化酶活性的调控

泛素化是一种非常重要的蛋白质翻译后修饰方式,在细胞生命活动的各个方面发挥作用。泛素化修饰是可逆的过程,去泛素化酶通过催化去除底物蛋白质上的泛素从而逆转该过程。去泛素化酶是一类数量众多的蛋白水解酶家族,近年来不断有新的去泛素化酶被发现和报道。鉴于其在细胞功能中的重要作用,去泛素化酶活性受到严格的调控。目前的研究表明,影响去泛素化酶活性的因素很多。本文主要从转录水平的调控、翻译后修饰、蛋白质定位和蛋白质相互作用等调控方式进行论述,以期为研究和利用去泛素化酶治疗疾病提供新思路。     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

Ubiquitin-regulating effector proteins from Legionella

Ubiquitin is relatively modest in size but involves almost entire cellular signaling pathways. The primary role of ubiquitin is maintaining cellular protein homeostasis. Ubiquitination regulates the fate of target proteins using the proteasome- or autophagy-mediated degradation of ubiquitinated substrates, which can be either intracellular or foreign proteins from invading pathogens. Legionella, a gram-negative intracellular pathogen, hinders the host-ubiquitin system by translocating hundreds of effector proteins into the host cell’s cytoplasm. In this review, we describe the current understanding of ubiquitin machinery from Legionella. We summarize structural and biochemical differences between the host-ubiquitin system and ubiquitin-related effectors of Legionella. Some of these effectors act much like canonical host-ubiquitin machinery, whereas others have distinctive structures and accomplish non-canonical ubiquitination via novel biochemical mechanisms.     

Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.     

Structural and functional study of Legionella pneumophila effector RavA

Legionella pneumophila is an intracellular pathogen that causes Legionnaire''s disease in humans. This bacterium can be found in freshwater environments as a free‐living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella‐containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER‐derived secretory vesicles and membranes forming the Legionella‐containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α‐helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C‐terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C‐terminal sequence C⁴⁰⁹WTSFCGLF⁴¹⁷. Yeast‐two‐hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.     

The Legionella pneumophila GTPase Activating Protein LepB Accelerates Rab1 Deactivation by a Non-canonical Hydrolytic Mechanism

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.     

带GFP标记嗜肺军团菌的构建和在细胞感染中的应用

【目的】为能实时直观了解嗜肺军团菌感染细胞的过程,研究细菌在细胞内的变化及其与宿主细胞间的相互作用关系。【方法】通过基因敲除、克隆回补等重组构建绿色荧光蛋白(GFP)稳定高表达的嗜肺军团菌株,利用该菌株建立小鼠巨噬细胞Raw264.7的感染模型。【结果】通过荧光显微镜可实时观察细菌感染细胞的全过程,包括细菌在细胞内的形态变化、增殖和裂解宿主细胞等。【结论】重组菌可替代野生菌株在细胞感染中应用,为直观研究嗜肺军团菌与被感染细胞之间的相互作用关系,以及进行相关药物模型的制备、药物筛选、耐药机制研究等提供了新的手段。     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.