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）：睡眠嗜组织菌是世界范围内的主要传染原。这种细菌产生一种称为 IbpA（免疫球蛋白结合蛋白 A）的大纤维状表面抗原。多杀性巴斯德氏菌是动物咬伤引起的最常见细菌感染的病原体，它也会产生一种称为 PfhB2 的大表面抗原。 PfhB2 与 IbpA 具有广泛的氨基酸序列同一性。这表明有一个细菌小家族含有这 4000 种氨基酸毒素。我们将注意力集中在 IbpA 上，因为感染睡眠嗜血菌的有症状动物的恢复期血清可识别 IbpA，而无症状动物的血清则不能。因此，IbpA 的存在与睡眠嗜血菌毒力直接相关。 IbpA 的 COOH 末端与耶尔森氏菌 III 型效应蛋白 YopT 同源，YopT 是耶尔森氏菌用来损害宿主免疫系统的几种毒力因子之一。我们之前证明耶尔森氏菌 YopT 作为半胱氨酸蛋白酶发挥作用，可裂解 Rho GTP 酶并使其失活。我们的假设是，IbpA 的丝状血凝素样结构域介导与宿主细胞的附着，而其含有 YopT 同源序列的 COOH 末端在内化到宿主细胞中时充当细胞毒性效应子。与我们的预期相反，我们观察到 IbpA 的 YopT 样结构域不会破坏肌动蛋白细胞骨架，尽管它保留了关键的催化 C/H/D 三联体。相反，我们在 IbpA 中发现了一个毒力决定簇，该决定簇位于蛋白质的 Fic（由 c-AMP 诱导的丝状结构）结构域部分。 Fic 结构域存在于细菌编码的大约 1500 种蛋白质中，并且在许多真核生物基因组中作为单拷贝基因存在。这些 Fic 结构域的功能尚不清楚。我们证明 IbpA 的 Fic 结构域通过靶向宿主 GTPases、RhoA、Rac 和 Cdc42 诱导细胞毒性。 IbpA 的 Fic 结构域通过使用 ATP 催化单磷酸腺苷 (AMP) 与 GTP 酶开关 I 区域中的酪氨酸 (Tyr) 残基共价添加来阻断这些 GTP 酶的信号传导。这种共价 AMP 添加会导致 GTP 酶下游信号传导受阻，进而导致细胞毒性。将 AMP 添加到 GTP 酶的能力取决于 Fic 结构域的核心基序 HPFxxGNGR 中保守组氨酸 (His) 的存在。总之，我们发现了一类新的蛋白质，它们在细菌发病机制中发挥着重要的新作用。我们的结果还表明，在原核生物和真核生物中，向宿主蛋白添加 AMP 可能是一种未被充分认识的翻译后修饰。该应用的具体目标是：（1）确定来自细菌和高等真核生物的Fic结构域是否都具有腺苷酸转移酶活性。 (2A) 研究Fic介导的腺苷酰化的动力学特性和催化机制。 (2B) 阐明含有 Fic 结构域的酶的细胞底物。 (3)确定IbpA如何进入哺乳动物细胞。 (4)确定IbpA的Fic结构域以及与ATP的非水解类似物复合的Fic结构域的X射线结构。 (5)确定含有Fic结构域、非水解ATP类似物和RhoA的蛋白质复合物的X射线结构。这些研究将提供对 Fic 结构域用于进行这种新颖的翻译后修饰的结构和机制的详细了解。这将共同推进我们对含有 Fic 结构域的蛋白质如何在细菌发病机制中发挥作用的理解。 公共健康相关性：在我们开展工作之前，尽管 Fic 结构域存在于来自多种细菌的 1400 多种蛋白质中，但它在细菌发病机制中的功能尚不清楚。我们已经证明，IbpA（一种在睡眠嗜组织菌中发现的大毒素）的 Fic 结构域可以通过使用 ATP 作为底物催化 AMP 添加到几种宿主 GTP 酶中来破坏肌动蛋白细胞骨架。 AMP 的这种共价添加会阻断宿主中对于对抗细菌感染非常重要的信号转导途径。

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