Role of Fic Proteins in Cellular Signaling

Fic 蛋白在细胞信号转导中的作用

• 批准号: 9277094
• 负责人: Seema Mattoo
• 金额: $ 15.15万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9277094
• 关键词: Active Sites; Adenosine; Alveolar; Antibodies; Antitoxins; Bacteria; Bacterial Proteins; Bacteriophage P1; Binding; Bioinformatics; Biological Models; Biological Process; Cell division; Cessation of life; Characteristics; Defect; Drug Design; Enzymes; Escherichia coli; Event; Family; Genes; Genetic; Glutamate-ammonia-ligase adenylyltransferase; Goals; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Histophilus; Human; Huntington gene; Immunoglobulin binding proteins; In Vitro; Legionella pneumophila; Mediating; Modification; Mono-S; Mutation; Nucleotides; Organism; Pathogenesis; Post-Translational Protein Processing; Protein S; Proteins; Reaction; Reporting; Role; SWI1; Signal Transduction; Specificity; Stress; Tertiary Protein Structure; Testing; Threonine; Toxin; Tyrosine; Variant; Vibrio; Vibrio parahaemolyticus; Yeasts; analog; innovation; inorganic phosphate; microbial; pathogen; pathogenic bacteria; prevent; protein E; protein protein interaction; respiratory; rho GTP-Binding Proteins; tool

DESCRIPTION (provided by applicant): In 2009, a new family of adenylyltransferases, defined by the presence of a 'Fic' domain, was discovered to catalyze the addition of adenosine mono-phosphate (AMP) to RhoGTPases. This adenylylation event inactivates RhoGTPases by preventing them from binding to their downstream effectors. The goal of this proposal is to investigate the functional repertoire of the Fic (filamentation induced by cyclic AMP) family of enzymes and understand the role of adenylylation as a post-translational modification in microbial pathogenesis and cellular signaling. Fic domains contain an HPFxxGNGR motif and are conserved from bacteria to humans. In E. coli, the Fic protein controls bacterial cell division, as a mutation in the fic gene results in aberrant septation3. Additionally, bacteriophage P1 encodes a Fic protein called Doc that is involved in stress survival4. We reported that the Fic domain(s) of the protein IbpA from the pathogenic bacterium Histophilus somni uses ATP to adenylylate and inhibit mammalian RhoGTPases, RhoA, Rac1 and Cdc42. This event induces host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and become septicemic. The adenylylation modification occurs on a tyrosine in the switch1 region of these GTPases, which is critical for function. We further demonstrated that the only human Fic domain-containing protein, HYPE/FicD, also has the ability to add AMP to RhoGTPases in vitro. Further, the secreted toxin VopS from Vibrio parahaemolyticus has been shown to catalyze the addition of AMP to a conserved switch1 threonine in the same family of GTPases. Another variation on the Fic domain was reported for the Legionella pneumophila type IV secreted protein AnkX, where the catalytic His of the Fic motif was required for breakdown of the golgi network. While it remains to be determined if AnkX, Fic and Doc also function as adenylyltransferases, these findings suggest that Fic-mediated adenylylation represents a new signaling paradigm that functions to alter the activity of the modified substrate and mediate protein-protein interactions. Over 3400 Fic domain-containing proteins are known, and our understanding of this large enzymatic family comes from the study of just two bacterial proteins, IbpA and VopS. Using innovative approaches, including the use of AMP-specific antibodies and ATP- analogs to capture adenylylation events, this study will take a 3-pronged approach to understanding the role of Fic-mediated adenylylation in regulating prokaryotic and eukaryotic signaling events. Specifically, we will 1) Determine the functional conservation of the Fic motif; 2) Elucidate the role of E. coli Fic in bacteria; and 3) Elucidate the role of HYPE in eukaryotic signaling. This study will establish parameters to define a Fic protein as a functional adenylyltransferase, and determine whether Fic proteins from evolutionarily distinct organisms use adenylylation as a conserved mechanism for regulating signal transduction networks.

描述（由申请人提供）：2009 年，发现一个新的腺苷酸转移酶家族（由“Fic”结构域的存在定义）可以催化将腺苷单磷酸 (AMP) 添加到 RhoGTP 酶中。该腺苷酸化事件通过阻止 RhoGTPase 与其下游效应子结合来使其失活。该提案的目标是研究 Fic（环 AMP 诱导的丝状形成）酶家族的功能，并了解腺苷酸化作为翻译后修饰在微生物发病机制和细胞信号传导中的作用。 Fic 结构域包含 HPFxxGNGR 基序，从细菌到人类都是保守的。在大肠杆菌中，Fic 蛋白控制细菌细胞分裂，因为 fic 基因的突变会导致异常分隔3。此外，噬菌体 P1 编码一种名为 Doc 的 Fic 蛋白，该蛋白参与应激生存4。我们报道了来自病原菌睡眠嗜组织菌的蛋白质 IbpA 的 Fic 结构域利用 ATP 腺苷酸化并抑制哺乳动物 RhoGTPase、RhoA、Rac1 和 Cdc42。这一事件会导致宿主细胞骨架崩溃，从而使睡眠嗜血菌突破肺泡屏障并导致败血症。腺苷酸化修饰发生在这些 GTPases 的 switch1 区域的酪氨酸上，这对于功能至关重要。我们进一步证明，唯一含有人类 Fic 结构域的蛋白质 HYPE/FicD 也具有在体外将 AMP 添加到 RhoGTPase 的能力。此外，副溶血弧菌分泌的毒素 VopS 已被证明可以催化 AMP 添加到同一 GTP 酶家族中的保守 switch1 苏氨酸上。据报道，Fic 结构域的另一种变异是嗜肺军团菌 IV 型分泌蛋白 AnkX，其中 Fic 基序的催化组氨酸是高尔基体网络分解所必需的。虽然 AnkX、Fic 和 Doc 是否也起到腺苷酰转移酶的作用仍有待确定，但这些发现表明 Fic 介导的腺苷酰化代表了一种新的信号传导范例，其功能是改变修饰底物的活性并介导蛋白质-蛋白质相互作用。已知超过 3400 种含有 Fic 结构域的蛋白质，我们对这一大型酶家族的了解仅来自对两种细菌蛋白质 IbpA 和 VopS 的研究。本研究将采用创新方法，包括使用 AMP 特异性抗体和 ATP 类似物来捕获腺苷酸化事件，从三管齐下的方法来了解 Fic 介导的腺苷酸化在调节原核和真核信号传导事件中的作用。具体来说，我们将1）确定Fic主题的功能守恒性； 2）阐明大肠杆菌Fic在细菌中的作用； 3) 阐明 HYPE 在真核信号传导中的作用。 这项研究将建立参数来将 Fic 蛋白定义为功能性腺苷酸转移酶，并确定来自进化上不同的生物体的 Fic 蛋白是否使用腺苷酸化作为调节信号转导网络的保守机制。