The Role of Protein Acetylation in Stress Defense

点击翻译按钮获取中文摘要

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.