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; 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，发现了一个新的腺苷酸转移酶，该家族由“ FIC”域的存在定义，以催化腺苷单磷酸腺苷（AMP）与Rhogtpases添加。这个腺层化事件通过防止其与下游效应子结合而灭活Rhogtpase。该提案的目的是研究酶的FIC（由环状AMP引起的细丝）的功能库，并了解腺苷酸化作为微生物发病机理和细胞信号传导中翻译后修饰的作用。 FIC结构域包含HPFXXGNGR基序，并从细菌到人类保守。在大肠杆菌中，FIC蛋白控制细菌细胞分裂，作为FIC基因的突变导致异常分离3。此外，噬菌体P1编码了一种称为doc的FIC蛋白，该蛋白与应力存活有关。4。我们报道说，来自致病细菌的蛋白质IBPA的FIC结构域使用ATP使用ATP来腺苷酸化和抑制哺乳动物Rhogtpases，RhoA，Rac1和cdc42。该事件引起了宿主的细胞骨架塌陷，这使得Somni可以泄露肺泡屏障并成为败血症。腺层修饰发生在这些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也能够在体外向Rhogtpases添加AMP。此外，已显示出来自Vibrio parahayticus的分泌的毒素VOP可将AMP催化为在同一GTPase家族中的保守开关1苏氨酸中添加AMP。据报道，IV型军团菌的另一个变化是在IV型军团菌分泌的蛋白ANKX上，其中需要催化他的FIC基序才能分解Golgi网络。尽管ANKX，FIC和DOC是否也充当腺苷酸转移酶尚待确定，但这些发现表明FIC介导的腺苷酸化代表了一种新的信号范式，该范式可以改变修饰的底物的活性并介导蛋白质 - 蛋白质蛋白质相互作用。已知超过3400个含有FIC结构域的蛋白质，我们对这个大型酶促家族的理解仅来自对两种细菌蛋白IBPA和VOPS的研究。使用创新的方法，包括使用AMP特异性抗体和ATP-ailogs来捕获腺苷酸化事件，这项研究将采用一种三管齐下的方法来了解FIC介导的腺层层析在调节定原性和真核信号事件中的作用。具体而言，我们将1）确定FIC基序的功能保护； 2）阐明大肠杆菌在细菌中的作用； 3）阐明炒作在真核信号传导中的作用。 这项研究将建立参数以将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。