Mechanisms and Consequences of Mycobacterial N-terminal Protein Acetylation

项目概要： N-末端（Nt）乙酰化是细菌学研究不足的方面。N-乙酰化是将一个乙酰基 基团与蛋白质第一个氨基酸的α-碳上的氨基连接。的基本机制 促进和调节NT-乙酰化，这种修饰在细菌中的后果仍然不确定。 本更新申请的目的是确定NT-乙酰化的基本机制， 分枝杆菌核心假设是蛋白质N-乙酰化是一个动态的、受调控的过程， 影响分枝杆菌的毒力。中心假设将通过以下具体目标进行检验：1）定义 促进分枝杆菌中NT-乙酰化的酶。2)建立病毒的机制和后果- 分枝杆菌中的中性因子N-乙酰化。3)确定NT-乙酰化和分枝杆菌之间的联系 新陈代谢.在第一个目标下，申请人提出使用富集策略与定量富集策略相结合。 蛋白质组学来确定保守的分枝杆菌NAT的功能和底物特异性。根据证券交易委员会- 第二个目标是，体外生化分析将与靶向和定量蛋白质组学相结合，以确定 修饰必需的分枝杆菌毒力因子的NAT。遗传和分子方法将用于 定义分枝杆菌中预测NAT之间的功能关系。根据第三个目标，申请人将 联合收割机富集和蛋白质组学方法研究不同NT-乙酰化生长后， 分枝杆菌对宿主相关碳源的影响。申请人将使用邻近度相关标签来识别 NAT活性的潜在调节剂。这项提案的成功完成将有助于从根本上 了解促进NT-乙酰化的机制，并建立NAT，NT-乙酰化之间的联系 和基本的分枝杆菌毒力途径。这些贡献将是重要的，因为它们将- 万斯介绍了我们对一种未充分研究的蛋白质修饰的理解，这种修饰对分枝杆菌的毒力很重要， 可能适用于其他细菌物种。这个建议的主题在概念上是创新的，因为Nt 乙酰化在结核病研究和细菌学领域都是研究不足的蛋白质修饰。 此外，研究NT-乙酰化的代谢调控，以确定NT-乙酰化的关键事件 是一个创新的想法。该提案在技术上具有创新性，因为申请人 将生物化学筛选、富集方案与生物分析化学以及分子生物学方面的专业知识相结合， 和致病分枝杆菌的基因操作。申请人利用M.结核和M. marinum菌株以优化生产力。这些对细菌的研究将为集中和知情的 动物毒力模型的研究。通过严格研究Nt的机制和调节， 乙酰化，申请人可以建立新的治疗靶点，用于治疗分枝杆菌疾病， 放松。

PROJECT SUMMARY: N-terminal (Nt) acetylation is an understudied aspect of bacteriology. Nt-acetylation is the addition of an acetyl group to the amino group on the α-carbon of the first amino acid of a protein. The fundamental mechanisms promoting and regulating Nt-acetylation, and the consequences of this modification in bacteria remain undefined. The objective of this renewal application is to define the fundamental mechanisms underlying Nt-acetylation in mycobacteria. The central hypothesis is that protein Nt-acetylation is a dynamic, regulated process that directly impacts mycobacterial virulence. The central hypothesis will be tested by following these specific aims: 1) Define the enzymes promoting Nt-acetylation in mycobacteria. 2) Establish the mechanisms and consequences of vir- ulence factor Nt-acetylation in mycobacteria. 3) Determine the link between Nt-acetylation and mycobacterial metabolism. Under the first aim, the applicant proposes to use enrichment strategies combined with quantitative proteomics to determine the function and substrate specificity of conserved mycobacterial NATs. Under the sec- ond aim, in vitro biochemical assays will be combined with targeted and quantitative proteomics to identify the NATs that modify essential mycobacterial virulence factors. Genetic and molecular approaches will be used to define functional relationships between predicted NATs in mycobacteria. Under the third aim, the applicant will combine enrichment and proteomics approaches to investigate differential Nt-acetylation following growth of mycobacteria on host-relevant carbon sources. The applicant will use proximity-dependent labeling to identify potential regulators of NAT activity. The successful completion of this proposal will contribute a fundamental understanding of the mechanisms promoting Nt-acetylation and establish a link between NATs, Nt-acetylation and essential mycobacterial virulence pathways. These contributions will be significant because they will ad- vance our understanding of an understudied protein modification important for mycobacterial virulence, which may be applicable to other bacterial species. The topic of this proposal is conceptually innovative because Nt acetylation is an under-investigated protein modification in both areas of tuberculosis research and bacteriology. Furthermore, studying the regulation of Nt-acetylation by metabolism to identify Nt-acetylation events essential for mycobacterial virulence is an innovative idea. The proposal is technically innovative because the applicant combines biochemical screens, enrichment protocols with bioanalytical chemistry, and expertise in molecular and genetic manipulation of pathogenic mycobacteria. The applicant leverages both M. tuberculosis and M. marinum strains to optimize productivity. These studies in bacteria will lay a foundation for focused and informed studies in animal virulence models in the future. By rigorously studying the mechanisms and regulation of Nt- acetylation in mycobacteria, the applicant may establish new therapeutic targets for treating mycobacterial dis- ease.