Mechanisms and Consequences of Mycobacterial N-terminal Protein Acetylation

项目总结： N-末端(NT)乙酰化是细菌学研究较少的一个方面。NT-乙酰化是乙酰基的加成。 指蛋白质第一个氨基酸的α-Carbon上的氨基。基本机制 促进和调节NT-乙酰化，以及这种修饰在细菌中的后果仍未确定。 这一更新申请的目的是确定在基础上NT-乙酰化的基本机制。 分枝杆菌。中心假设是蛋白质NT-乙酰化是一个动态的、受调节的过程，直接 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 分枝杆菌中促进NT-乙酰化的酶。2)建立VIR的机制和后果-- 分枝杆菌的毒性因子NT-乙酰化。3)确定NT-乙酰化与分枝杆菌之间的联系 新陈代谢。在第一个目标下，申请人建议使用浓缩战略和量化相结合的战略。 蛋白质组学以确定保守的分枝杆菌NAT的功能和底物特异性。在证券交易委员会之下- 目的：体外生化分析将与靶向和定量蛋白质组学相结合，以鉴定 修饰基本的分枝杆菌毒力因子的NAT。将使用遗传和分子方法来 定义分支杆菌中预测的NAT之间的功能关系。在第三个目标下，申请人将 结合浓缩和蛋白质组学方法研究细胞生长后的差异NT-乙酰化 与宿主相关的碳源上的分枝杆菌。申请者将使用依赖于距离的标签来识别 NAT活动的潜在监管机构。这项提案的成功完成将为 了解促进NT-乙酰化的机制并在NAT、NT-乙酰化之间建立联系 和基本的分枝杆菌毒力途径。这些贡献将是重大的，因为它们将- Vance我们对一种未被研究的蛋白质修饰的理解对分枝杆菌的毒力很重要，它 可能适用于其他细菌物种。这项提议的主题在概念上是创新的，因为NT 乙酰化是一种在结核病研究和细菌学领域都未得到充分研究的蛋白质修饰。 此外，研究新陈代谢对NT-乙酰化的调节对识别NT-乙酰化事件至关重要 对分枝杆菌来说，毒力是一个创新的想法。该提案在技术上是创新的，因为申请人 将生化筛选、浓缩方案与生物分析化学和分子方面的专业知识相结合 以及致病分枝杆菌的基因操作。申请人利用结核分枝杆菌和结核分枝杆菌。 马丁尼菌株，以优化生产率。这些对细菌的研究将为聚焦和了解情况奠定基础 未来在动物毒性模型方面的研究。通过对NT-1的作用机制和调控机制的研究， 分枝杆菌的乙酰化，申请人可以建立治疗分枝杆菌疾病的新的治疗靶点。 放松点。

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.