单磷酸腺苷酸化修饰:一种蛋白质翻译后修饰方式

副溶血弧菌(Vibrio parahaemolyticus)能够通过Ⅲ型分泌系统(typeⅢsecretion systems,T3SSs)分泌效应蛋白Vop S,催化三磷酸腺苷(ATP)分子中的单磷酸腺苷(AMP)通过磷酸二酯键共价连接至宿主细胞Rho鸟苷三磷酸激酶(Rho GTPases)成员蛋白Rho A、Rac1和Cdc42的特定的苏氨酸残基上,导致宿主细胞肌动蛋白骨架崩解,细胞变圆。该发现推动了一种蛋白质翻译后修饰方式——单磷酸腺苷酸化(AMPylation)修饰的迅速发展,其中催化AMPylation修饰的蛋白质称为单磷酸腺苷酸化酶(AMPylator)。目前的研究表明,与蛋白质的磷酸化修饰类似,蛋白质AMPylation在真核以及原核生物中都是一种重要的调控蛋白质功能的翻译后共价修饰调节机制。与AMPylation相对应的是去单磷酸腺苷酸化(de-AMPylation),即去单磷酸腺苷酸化酶(de-AMPylase)催化修饰后的蛋白质脱去AMP基团的过程,使底物蛋白重新恢复其原有的生物学功能。现就蛋白质AMPylation修饰的催化过程、AMPylation修饰的研究进展以及AMPylator/de-AMPylase的种类、物种来源、结构、功能和底物等方面的内容进行综合阐述。此外,就目前已有的AMPylation检测方法进行了总结,其中详细阐述了一种化学标记法的原理和过程。     

cAMP信号与动物细胞线粒体功能

线粒体是真核生物细胞内重要的细胞器,主要功能是通过氧化磷酸化作用为细胞生命活动提供能量,并与细胞的生长、发育及衰老等重要生物过程密切相关。许多研究表明,线粒体蛋白质的磷酸化在调控氧化代谢方面发挥了重要作用,而且环腺苷一磷酸(cyclic adenosine monophosphate,c AMP)依赖的蛋白激酶A(protein kinase A,PKA)信号通路参与了该过程的调控,但c AMP/PKA信号通路在调控线粒体代谢方面的作用一直存在争议。因此,该文综述了线粒体内c AMP的来源、线粒体c AMP信号系统及对c AMP对线粒体功能的调控,旨在为全面了解c AMP/PKA信号通路在调控线粒体功能方面的作用提供具体参考。     

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.     

植物蛋白磷酸化的检测方法

蛋白磷酸化是一种重要的蛋白质翻译后修饰方式,几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下,将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸,磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。     

植物蛋白磷酸化的检测方法

蛋白磷酸化是一种重要的蛋白质翻译后修饰方式,几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下,将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸,磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。     

植物同源结构域(PHD结构域)——组蛋白密码的解读器

植物同源结构域(plant homeodomain,PHD结构域),是真核生物中一种进化保守的锌指结构域.多种调控基因转录、细胞周期、凋亡的蛋白质含有PHD结构域.研究表明,PHD结构域涉及多种功能,包括蛋白质相互作用,特别是同核小体组蛋白的作用.目前认为,各种组蛋白修饰(包括甲基化、乙酰化、磷酸化、泛素化等)的模式和组合,调节染色质状态和基因转录活性,并提出了组蛋白密码理论.PHD指结构域能特异性识别组蛋白的甲基化(修饰)密码,可能是组蛋白密码的一种重要解读器.     

腺苷对大鼠心肌钙蛋白Ⅰ及磷脂酰基醇磷酸化的抑制作用

 用差速离心及等密度梯度离心法从大白鼠心肌细胞分离收缩蛋白质及质膜,分别与[γ-~(32)P]ATP保温以观察细胞成分的磷酸化,以及腺苷和腺苷类似物对磷酸化的影响。结果表明,在收缩蛋白质组分,~(32)P主要参入肌钙蛋白I(Troponin I,29000Da);在质膜组分,~(32)P主要参入磷脂酰肌醇-4-一磷酸(PtdIns4P),亦即ATP使磷脂酰肌醇(Ptd Ins)磷酸化。腺苷对此两种磷酸化都有抑制作用,尤以对PtdIns磷酸化的抑制最强烈。cAMP对肌钙蛋白Ⅰ的磷酸化有刺激作用,这与文献报道相符。作者认为,腺苷和cAMP对肌钙蛋白Ⅰ磷酸化的拮抗作用与腺苷和肾上腺素对心肌调节的拮抗作用有明显的相关性。鉴于近年发现,肌醇磷脂转换在调节细胞活动中起重要作用,腺苷对磷脂酰肌醇磷酸化的抑制作用可能有重要的生物学意义。     

接头蛋白Crk与肿瘤

Crk是一种重要的细胞内信号接头蛋白,含有SH2和SH3结构域,在信号传递过程中不仅承上启下,对信号强度还有着调控作用.迄今主要发现了4种Crk分子,即v Crk、CrkⅠ、CrkⅡ和CrkL(Crk Like).CrkⅡ和CrkL在分子结构上具有高度同源性,但对信号分子却具有不同的偏好性,从而体现出功能差异.本文就接头蛋白Crk的结构、Crk与相互作用蛋白质的作用机制以及与肿瘤的发生发展关系进行了阐述.     

含有补体控制蛋白结构域的蛋白质的结构和功能

补体控制蛋白(CCP)结构域分布广泛,含有CCP结构域的蛋白质在补体调节、机体排异和抵御微生物侵袭,甚至肿瘤发生发展等方面具有重要的功能。现在发现的含CCP结构域的蛋白质大约有100多种。我们综述了CCP结构域的基本特征,简要介绍了有代表性的含CCP结构域的蛋白质的功能。     

桩蛋白的结构与功能

桩蛋白(paxillin)是近年来发现的一种信号蛋白,主要定位于黏着斑,包含LD模体、LIM结构域、SH2和SH3结合结构域,在整个分子中还散在着多种磷酸化位点,共同构成了桩蛋白的多结构域性结构。桩蛋白分子本身的酶活性尚不清楚,但很可能作为细胞内的一种接头蛋白与多种功能蛋白质结合。另外．作为黏着斑的重要组成部分,桩蛋白不仅参与了整合蛋白介导的信号转导和黏着斑的组装,在细胞黏附和迁移过程中也发挥了重要作用。     

Fic and non-Fic AMPylases: protein AMPylation in metazoans

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.     

An experimental strategy for the identification of AMPylation targets from complex protein samples

AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS‐based approach utilizing stable isotope‐labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.     

The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins,Translation Elongation Factors and Histones

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.     

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.     

Fido,a Novel AMPylation Domain Common to Fic,Doc, and AvrB

Background

The Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes.

Methodology/Principal Findings

We show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate.

Conclusions/Significance

The demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates.     

Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.     

Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone,BiP/GRP78

Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiP’s Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.     

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.     

Alpha-Synuclein Is a Target of Fic-Mediated Adenylylation/AMPylation: Possible Implications for Parkinson's Disease

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediatedadenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE—alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.