Mechanisms and Consequences of Mycobacterial N-terminal Protein Acetylation

分枝杆菌 N 末端蛋白质乙酰化的机制和后果

• 批准号: 10680597
• 负责人: Patricia A Champion
• 金额: $ 46.95万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-10 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680597
• 关键词: Acetylation; Acetyltransferase; Affect; Amino Acids; Animals; Antibiotic Resistance; Antibiotics; Area; Bacteria; Bacterial Infections; Bacterial Proteins; Bacteriology; Binding; Biochemical; Biological Assay; Carbon; Charge; Chemistry; Cholesterol; Chronic; Data; Diagnosis; Disease; ELF3 gene; Enzymes; Epidemic; Event; Foundations; Funding; Future; Genetic; Genus Mycobacterium; Growth; Half-Life; Human; In Vitro; Individual; Infection; Knowledge; Label; Ligation; Link; Lipids; Lysine; Macrophage; Malignant Neoplasms; Metabolism; Methods; Mission; Modeling; Modification; Molecular; Mycobacterium marinum; Mycobacterium tuberculosis; N-terminal; Neurodegenerative Disorders; Organism; Parasitic infection; Pathogenesis; Pathogenicity; Pathway interactions; Peptides; Phase; Phenotype; Post-Translational Protein Processing; Prevention; Process; Productivity; Protein Acetylation; Proteins; Proteomics; Protocols documentation; Public Health; Publishing; Regulation; Research; Source; Substrate Specificity; System; Testing; Transfer RNA; Tuberculosis; United States National Institutes of Health; Virulence; Virulence Factors; amino group; chronic infection; genetic manipulation; human pathogen; innovation; mycobacterial; new therapeutic target; pathogen; protein function; small molecule; therapeutic development; therapeutic target; tool; tuberculosis treatment; vaccine development

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.

项目概要： N 末端 (Nt) 乙酰化是细菌学中尚未得到充分研究的一个方面。 Nt-乙酰化是添加一个乙酰基 基团连接到蛋白质第一个氨基酸的α-碳上的氨基。基本机制 促进和调节 Nt-乙酰化，并且这种修饰对细菌的影响仍不清楚。 本次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 Nt 乙酰化是一个动态的、受调节的过程，直接影响 影响分枝杆菌毒力。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vir-的机制和后果 分枝杆菌中的质量因子 Nt-乙酰化。 3) 确定Nt-乙酰化与分枝杆菌之间的联系 代谢。在第一个目标下，申请人建议采用富集策略与定量相结合的方法。 蛋白质组学以确定保守分枝杆菌 NAT 的功能和底物特异性。根据秒 另一个目标是，体外生化测定将与靶向和定量蛋白质组学相结合，以确定 改变分枝杆菌重要毒力因子的 NAT。遗传和分子方法将用于 定义分枝杆菌中预测的 NAT 之间的功能关系。根据第三个目标，申请人将 结合富集和蛋白质组学方法来研究生长后的差异 Nt 乙酰化 分枝杆菌对宿主相关碳源的依赖。申请人将使用邻近依赖标签来识别 NAT 活动的潜在监管者。该提案的顺利完成将为 了解促进 Nt-乙酰化的机制并建立 NAT、Nt-乙酰化之间的联系 和重要的分枝杆菌毒力途径。这些贡献将是重大的，因为它们将促进 促进我们对对分枝杆菌毒力至关重要的正在研究的蛋白质修饰的理解， 可能适用于其他细菌种类。该提案的主题在概念上具有创新性，因为 Nt 乙酰化是结核病研究和细菌学领域中一种尚未得到充分研究的蛋白质修饰。 此外，研究代谢对 Nt-乙酰化的调节以识别 Nt-乙酰化事件至关重要 对于分枝杆菌的毒力来说是一个创新的想法。该提案在技术上具有创新性，因为申请人 将生化筛选、富集方案与生物分析化学以及分子专业知识相结合 以及致病性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。 marinum 菌株可优化生产力。这些细菌研究将为重点和知情的研究奠定基础 未来动物毒力模型的研究。通过严格研究Nt-的机制和调控 分枝杆菌中的乙酰化，申请人可以建立新的治疗靶点来治疗分枝杆菌疾病 舒适。

项目成果

Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.

结核分枝杆菌毒力基因的合理工程促进了天然蛋白质 N 末端的蛋白质组学分析。

• DOI: 10.1038/srep33265
• 发表时间: 2016
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Reyna,Cristal;MbaMedie,Felix;Champion,MatthewM;Champion,PatriciaA
• 通讯作者: Champion,PatriciaA

An N-acetyltransferase required for ESAT-6 N-terminal acetylation and virulence in Mycobacterium marinum.

• DOI: 10.1128/mbio.00987-23
• 发表时间: 2023-10-31
• 期刊: mBio
• 影响因子: 6.4

Direct detection of bacterial protein secretion using whole colony proteomics.

• DOI: 10.1074/mcp.m112.017533
• 发表时间: 2012-09
• 期刊: Molecular & cellular proteomics : MCP
• 作者: Champion MM;Williams EA;Kennedy GM;Champion PA
• 通讯作者: Champion PA

EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System.

EspM 是一种保守转录因子，可调节基因表达以响应 ESX-1 系统。

• DOI: 10.1128/mbio.02807-19
• 发表时间: 2020
• 期刊: mBio
• 影响因子: 6.4
• 作者: Sanchez,KevinG;Ferrell,MicahJ;Chirakos,AlexandraE;Nicholson,KathleenR;Abramovitch,RobertB;Champion,MatthewM;Champion,PatriciaA
• 通讯作者: Champion,PatriciaA

Correlation of phenotypic profiles using targeted proteomics identifies mycobacterial esx-1 substrates.

• DOI: 10.1021/pr500484w
• 发表时间: 2014-11-07
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Champion, Matthew M.;Williams, Emily A.;Pinapati, Richard S.;Champion, Patricia A. DiGiuseppe
• 通讯作者: Champion, Patricia A. DiGiuseppe