Fic-mediated Adenylylation

Fic介导的腺苷酸化

• 批准号: 8509713
• 负责人: JACK E DIXON
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509713
• 关键词: Actins; Address; Adenosine Monophosphate; Amino Acid Sequence; Amino Acids; Animals; Attention; Bacteria; Bacterial Infections; Biological Sciences; Bite; Bubonic Plague; Catalysis; Cell Signaling Process; Cell membrane; Cells; China; Cleaved cell; Clinic; Complex; Cyclic AMP; Cysteine Protease; Cytoskeleton; Data; Enzymes; Eukaryota; Eukaryotic Cell; Faculty; Family; Genes; Genome; Glutamate-ammonia-ligase adenylyltransferase; Gram-Negative Bacteria; Grant; Guanosine Triphosphate Phosphohydrolases; Hemagglutinin; Histidine; Histophilus; Human; Immune system; Immunoglobulin binding proteins; Individual; Infectious Agent; Institutes; Kinetics; Laboratories; Mammalian Cell; Maps; Mediating; Modification; Organism; Pathogenesis; Peptide Hydrolases; Play; Positioning Attribute; Post-Translational Protein Processing; Postdoctoral Fellow; Process; Prokaryotic Cells; Property; Protein Tyrosine Phosphatase; Proteins; Reporting; Roentgen Rays; Role; Running; Sequence Homology; Serum; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure; Substrate Specificity; Surface Antigens; Toxin; Triad Acrylic Resin; Type III Secretion System Pathway; Tyrosine; Vibrio parahaemolyticus; Virulence; Virulence Factors; Work; Yeasts; Yersinia; Yersinia pestis; analog; career; combat; cytotoxic; cytotoxicity; expectation; human Huntingtin protein; insight; medical schools; novel; pathogenic bacteria; prevent; protein E; protein complex; protein function; public health relevance; rho; rho GTP-Binding Proteins

DESCRIPTION (provided by applicant): Histophilus somni is a major infectious agent worldwide. This bacterium produces a large fibrillar surface antigen called IbpA (immunoglobulin binding protein A). Pasturella multocida, the causative agent of the most common bacterial infection due to an animal bite, also produces a large surface antigen known as PfhB2. PfhB2 shares extensive amino acid sequence identity with IbpA. This suggests that there is a small family of bacteria that harbors these 4000 amino acid toxins. We focused our attention on IbpA since convalescent serum from symptomatic animals infected with H. somni recognizes IbpA, while serum from asymptomatic animals does not. As such, the presence of IbpA directly correlates with H. somni virulence. The COOH-terminus of IbpA is homologous to the Yersinia type III effector protein, YopT, one of several virulence factors used by Yersinia to compromise the host immune system. We previously demonstrated that Yersinia YopT functions as a cysteine protease that cleaves and inactivates Rho GTPases. Our hypothesis was that IbpA's filamentous hemagglutinin-like domains mediate attachment to host cells, while its COOH-terminus containing the YopT homology sequence serves as a cytotoxic effector when internalized into host cells. Contrary to our expectations, we observed that the YopT-like domain of IbpA does not disrupt the actin cytoskeleton despite its conservation of the key catalytic C/H/D triad. Instead, we identified a virulence determinant within IbpA that is localized to a portion of the protein known as the Fic (filamentation induced by c-AMP) domain. Fic domains are found in approximately 1500 proteins encoded by bacteria and are present as single copy genes in many eukaryotic genomes. The function of these Fic domains is unknown. We demonstrated that the Fic domains of IbpA induce cytotoxicity by targeting the host GTPases, RhoA, Rac and Cdc42. The Fic domains of IbpA block signaling of these GTPases by using ATP to catalyze the covalent addition of adenosine monophosphate (AMP) to a tyrosine (Tyr) residue in the GTPase switch I region. This covalent AMP addition leads to a block in downstream signaling of the GTPases, which in turn results in cytotoxicity. The ability to add AMP to the GTPases is dependent on the presence of a conserved histidine (His) in the Fic domain's core motif, HPFxxGNGR. In summary, we have identified a new class of proteins that play an important novel role in bacterial pathogenesis. Our results also suggest that addition of AMP to host proteins may be an underappreciated post-translational modification in both prokaryotes and eukaryotes. The specific aims for this application are: (1) Determine if Fic domains from bacteria and higher eukaryotes all have adenylyl transferase activity. (2A) Study the kinetic properties and catalytic mechanisms of Fic-mediated adenylylation. (2B) Elucidate the cellular substrates of Fic domain containing enzymes. (3) Determine how IbpA enters mammalian cells. (4) Determine the X-ray structures of the IbpA's Fic domain as well as a Fic domain complexed with a non-hydrolyzed analogue of ATP. (5) Determine the X-ray structure of the protein complex containing a Fic domain, a non-hydrolyzed ATP analogue and RhoA. These studies will provide a detailed understanding of the structure and mechanism used by the Fic domain to carry out this novel post-translational modification. This will collectively advance our understanding of how the Fic domain containing proteins function in bacterial pathogenesis. PUBLIC HEALTH RELEVANCE: Prior to our work, the function of the Fic domain in bacterial pathogenesis was unknown despite its presence in over 1400 proteins from a wide variety of bacteria. We have demonstrated that the Fic domain of IbpA, a large toxin found in Histophilus somni, can disrupt the actin cytoskeleton by using ATP as a substrate to catalyze the addition of AMP to several host GTPases. This covalent addition of AMP blocks signal transduction pathways in the host that are important for combating bacterial infections.

描述（由申请人提供）：somni组织是全球主要的感染剂。该细菌产生一种称为IBPA的大型纤维表面抗原（免疫球蛋白结合蛋白A）。 Multocida Pasturella Multocida是由于动物叮咬而引起的最常见细菌感染的致病药物，也会产生一种称为PFHB2的大型表面抗原。 PFHB2与IBPA具有广泛的氨基酸序列身份。这表明有一小群细菌可以容纳这4000个氨基酸毒素。我们将注意力集中在IBPA上，因为感染了H. somni的症状动物的康复血清识别IBPA，而无症状动物的血清则没有。因此，IBPA的存在直接与H. somni毒力直接相关。 IBPA的COOH末端与III型效应蛋白YOPT同源，Yopt是耶尔森氏菌用于损害宿主免疫系统的几种毒力因子之一。我们先前证明了Yersinia Yopt充当半胱氨酸蛋白酶，使Rho GTPase裂解并失活。我们的假设是，IBPA的丝状血凝素样域介导了宿主细胞的附着，而其含有YOPT同源性序列的COOH-末端则在宿主细胞中内化时作为细胞毒性效应子。与我们的期望相反，我们观察到，尽管保存了关键的催化C/H/D三合会，但IBPA的YOPT样结构域并未破坏肌动蛋白的细胞骨架。取而代之的是，我们确定了IBPA内的毒力决定因素，该决定因素位于被称为FIC（C-AMP诱导的丝）域的一部分。在大约1500种由细菌编码的蛋白质中发现了FIC结构域，并在许多真核基因组中作为单拷贝基因存在。这些FIC域的功能尚不清楚。我们证明了IBPA的FIC结构域通过靶向宿主GTPases，RhoA，RAC和CDC42诱导细胞毒性。这些GTPase的IBPA块信号传导的FIC结构域通过使用ATP催化在GTPase开关I区域中的酪氨酸（TYR）残基的共价添加到酪氨酸（TYR）残基中。这种共价AMP的添加导致GTPases下游信号传导的块，从而导致细胞毒性。将AMP添加到GTPase的能力取决于FIC域的核心基序HPFXXGNGR中保守的组氨酸（HIS）的存在。总而言之，我们已经确定了一类新的蛋白质，它们在细菌发病机理中起着重要的新作用。我们的结果还表明，在原核生物和真核生物中，将AMP添加到宿主蛋白质中可能是一种不足的翻译后修饰。该应用的具体目的是：（1）确定来自细菌和较高真核生物的FIC域是否具有腺苷转移酶活性。 （2a）研究FIC介导的腺苷酸化的动力学特性和催化机制。 （2B）阐明了含有酶的FIC结构域的细胞底物。 （3）确定IBPA如何进入哺乳动物细胞。 （4）确定IBPA FIC结构域的X射线结构以及与ATP的非水解类似物复合的FIC结构域。 （5）确定含有FIC结构域，非氢化ATP类似物和RhoA的蛋白质复合物的X射线结构。这些研究将详细了解FIC领域用于进行这种新型翻译后修饰的结构和机制。这将共同提高我们对含有蛋白质蛋白质的FIC结构域在细菌发病机理中的作用的理解。 公共卫生相关性：在我们工作之前，尽管在各种细菌中存在超过1400种蛋白质中，但FIC结构域在细菌发病机理中的功能尚不清楚。我们已经证明，IBPA的FIC结构域，一种在组织中发现的大型毒素，可以通过使用ATP用作底物来催化在几个宿主GTP酶中添加AMP。 AMP的共价添加阻断宿主中的信号转导途径，对于对抗细菌感染很重要。

项目成果

Comparative analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other fic domain-containing enzymes reveals differences in substrate and nucleotide specificities.

• DOI: 10.1074/jbc.m111.227603
• 发表时间: 2011-09-16
• 期刊: The Journal of biological chemistry
• 作者: Mattoo S;Durrant E;Chen MJ;Xiao J;Lazar CS;Manning G;Dixon JE;Worby CA
• 通讯作者: Worby CA

Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

睡眠嗜组织菌 IbpA DR2/Fic 对天然宿主肺泡上皮屏障的毒力和免疫保护作用。

• DOI: 10.1128/iai.01277-09
• 发表时间: 2010
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Zekarias,Bereket;Mattoo,Seema;Worby,Carolyn;Lehmann,Jason;Rosenbusch,RicardoF;Corbeil,LynetteB
• 通讯作者: Corbeil,LynetteB