The Role of Protein Acetylation in Stress Defense

蛋白质乙酰化在应激防御中的作用

• 批准号: 1656602
• 负责人: Jeffrey Lewis
• 金额: $ 57.13万
• 依托单位: University of Arkansas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1656602&HistoricalAwards=false
• 关键词: Role; Protein; Acetylation; Stress; Defense

All organisms experience stress and must rapidly adapt to changing conditions. Many cells have therefore evolved sophisticated rapid-response mechanisms that allow them to adapt to new environments. One such mechanism is post-translational modification of proteins, where the attachment or removal of small molecules to proteins after their initial formation allows for rapid and reversible regulation of protein activity. One type of protein modification, by the small 2-carbon acetyl unit (acetylation) has long been known to play a role in regulating which genes are expressed under different conditions. However, the surprising discovery of thousands of acetylated proteins in diverse organisms suggests a much broader role. The overall goal of this project is to understand how protein acetylation regulates stress defense in yeast. The project will identify which yeast proteins are modified by acetylation during the response to elevated temperatures, the enzymes responsible for those modifications, and how acetylation impacts protein function. In addition to addressing a fundamental knowledge gap, this project will provide broad scientific training for both graduate and undergraduate students. Undergraduates will include summer interns recruited through the George Washington Carver Research Program, which serves underrepresented students from Historically Black Colleges and Universities. Additionally, the project team will partner with a small local fermentation business to design a new Microbial Fermentation Laboratory course. The course will introduce students to the science behind fermentation, while lab exercises will provide hands-on experience in fermentation microbiology and biotechnology. The heat shock response is a universal mechanism for dealing with stress, and has been well studied in yeast and other model organisms. The proposed work will reveal novel and previously unexplored roles for protein acetylation in the heat shock response. Specifically, quantitative acetyl-proteomics will be used on yeast cells responding to heat stress over time to identify which proteins change in acetylation state, which sites are differentially acetylated, and the stoichiometry of acetylation. To implicate the enzymes responsible for protein acetylation changes during heat stress, acetyl-proteomics will be performed on acetyltransferase and deacetylase mutants. To gain fundamental insight into how acetylation affects protein function, the focus will be on the acetylation of stress-responsive transcription factors for detailed mechanistic characterization. Transcriptional profiling and network analysis will be used to connect the timing of changes in acetylation of transcription factors during heat stress with the timing of gene expression changes for those transcription factors? targets. Multiple genetic approaches will be used to probe the in vivo effects of transcription factor acetylation on protein function. To corroborate the in vivo findings, fully acetylated and fully unacetylated transcription factors will be purified to test the effects of acetylation in vitro. In addition to providing new insights into regulation of the yeast heat shock response, results from the proposed experiments will lay the groundwork for future studies exploring how acetylation impacts other stress responses, how acetylation changes are regulated under diverse conditions, and whether these regulatory phenomena are conserved in other diverse organisms.

所有生物都经历压力，必须迅速适应不断变化的条件。因此，许多细胞进化出复杂的快速反应机制，使它们能够适应新的环境。一种这样的机制是蛋白质的翻译后修饰，其中小分子在其初始形成后与蛋白质的连接或去除允许蛋白质活性的快速和可逆调节。一种类型的蛋白质修饰，通过小的2-碳乙酰基单元（乙酰化），长期以来一直被认为在调节哪些基因在不同条件下表达中发挥作用。然而，在不同生物体中发现的数千种乙酰化蛋白质的惊人发现表明了更广泛的作用。这个项目的总体目标是了解蛋白质乙酰化如何调节酵母的应激防御。该项目将确定哪些酵母蛋白在对高温的反应过程中被乙酰化修饰，负责这些修饰的酶，以及乙酰化如何影响蛋白质功能。除了解决基本的知识差距，该项目将为研究生和本科生提供广泛的科学培训。本科生将包括通过乔治华盛顿卡弗研究计划招募的暑期实习生，该计划为来自历史上黑人学院和大学的代表性不足的学生提供服务。此外，项目团队将与当地一家小型发酵企业合作，设计一门新的微生物发酵实验室课程。该课程将向学生介绍发酵背后的科学，而实验室练习将提供发酵微生物学和生物技术的实践经验。热休克反应是一种普遍的应对应激的机制，并且已经在酵母和其他模式生物中得到了很好的研究。这项工作将揭示蛋白质乙酰化在热休克反应中的新的和以前未探索的作用。具体而言，定量乙酰蛋白质组学将用于酵母细胞响应热应激随着时间的推移，以确定哪些蛋白质改变乙酰化状态，哪些网站是差异乙酰化，乙酰化的化学计量。为了揭示热应激过程中引起蛋白乙酰化变化的酶，将对乙酰转移酶和脱乙酰酶突变体进行乙酰蛋白质组学研究。为了深入了解乙酰化如何影响蛋白质功能，重点将放在应激反应转录因子的乙酰化上，以获得详细的机制表征。转录谱和网络分析将用于连接的时间在乙酰化的转录因子在热应激过程中的变化与这些转录因子的基因表达变化的时间？目标的多种遗传学方法将用于探测转录因子乙酰化对蛋白质功能的体内影响。为了证实体内发现，将纯化完全乙酰化和完全未乙酰化的转录因子以测试乙酰化的体外作用。除了为酵母热休克反应的调节提供新的见解外，拟议实验的结果将为未来的研究奠定基础，探索乙酰化如何影响其他应激反应，乙酰化变化如何在不同条件下受到调节，以及这些调节现象是否在其他不同的生物体中保守。

项目成果

Comparison of RNA isolation methods on RNA-Seq: implications for differential expression and meta-analyses

• DOI: 10.1186/s12864-020-6673-2
• 发表时间: 2020-03-20
• 期刊: BMC GENOMICS
• 影响因子: 4.4
• 作者: Scholes, Amanda N.;Lewis, Jeffrey A.
• 通讯作者: Lewis, Jeffrey A.

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags

使用串联质量标签对动态热休克蛋白质组进行准确、灵敏的定量

• DOI: 10.1021/acs.jproteome.9b00704
• 发表时间: 2020
• 期刊: Journal of Proteome Research
• 影响因子: 4.4
• 作者: Storey, Aaron J.;Hardman, Rebecca E.;Byrum, Stephanie D.;Mackintosh, Samuel G.;Edmondson, Rick D.;Wahls, Wayne P.;Tackett, Alan J.;Lewis, Jeffrey A.
• 通讯作者: Lewis, Jeffrey A.