Understanding the reciprocal regulation between Hsp70 and the DNA damage response

了解 Hsp70 与 DNA 损伤反应之间的相互调节

基本信息

批准号：
10311502
负责人：
Andrew William Truman
金额：
$ 29.51万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-03 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10311502
关键词：
ATR gene Affect Alanine Alzheimer&apos s Disease Binding Binding Proteins Biological Process Cell Culture Techniques Cell Cycle Progression Cell Death Cell Survival Cells Client Code DNA Damage DNA Maintenance DNA Repair DNA biosynthesis Data Disease Exposure to Feedback Future Genetic Housekeeping Huntington Disease In Vitro Malignant Neoplasms Mammalian Cell Mediating Membrane Modification Molecular Biology Molecular Chaperones Mutation Neurodegenerative Disorders Nucleotides Nutrient Organism Pathway interactions Pattern Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Physiological Predisposition Process Proliferating Protein Dephosphorylation Protein Isoforms Proteins Proteomics Publishing Regulation Research Role Saccharomyces cerevisiae Signal Transduction Site Specificity System TEL1 Gene Techniques Transcriptional Regulation Work XRCC1 gene Yeasts anti-cancer therapeutic cancer type experimental study genome integrity in vivo inhibitor kinase inhibitor novel protein aggregation protein folding protein transport repaired response

项目摘要

Project Summary All organisms require maintenance of DNA integrity to grow and proliferate. Replication and repair of DNA damage requires the increased synthesis of DNA nucleotides, a process that is dependent on the activity of the kinases such as ATM and ATR. Misregulation of DNA replication can result in either cell death or cancer. Studies by our lab and others have shown that many DNA damage response (DDR) proteins (such as ATM, ATR, XRCC1 and RNR) are stabilized by the molecular chaperones Hsp70 and Hsp90. These proteins perform a variety of functions in the cell including protein folding of both newly synthesized and denatured proteins, protein transport across membranes and disaggregation of oligomerized proteins. Research has primarily focused on how chaperone function specificity arises through regulation of expression, isoform differences and the variety of co- chaperone proteins that bind to the Hsp70 and Hsp90 molecules. Despite the identification of several phosphorylation sites on both yeast and mammalian Hsp70 through global proteomic screens (known as the chaperone code), the biological function of these remains unclear. Our studies published in Cell determined that CDK-mediated phosphorylation of a single site on Hsp70 can regulate chaperone function by altering both co- chaperone and client protein interactions. In this proposal, we aim to understand how the activation of DDR can promote changes in the pattern of Hsp70 chaperone code. We predict that in line with several Hsp90-kinase interactions, Hsp70 phosphorylation during DNA damage creates a feedback system whereby chaperone phosphorylation increases stability of DDR proteins, amplifying the signal of the DNA damage response. We propose to use both molecular biology and state-of-the-art mass spectrometric techniques on both Saccharomyces cerevisiae and mammalian cell culture cells to achieve the aims of the objectives in our proposal. Identification and study of functional phosphorylation sites on Hsp70 in both yeast and mammalian cells will provide us with a completely novel way to target chaperone activity. Hsp70 activity may be suppressed using specific phosphatase/kinase inhibitors. It may be possible to target specific ‘client’ proteins though alteration of Hsp70 phosphorylation status and specific Hsp70 phospho-species may have a higher susceptibility to inhibitors. The scope of this work has broad implications for a variety of diseases associated with both the DNA damage response and molecular chaperone function, including many types of cancer and neurodegenerative illnesses caused by protein aggregation (Huntington’s disease, Alzheimer’s disease and Creutzfeld-Jakob disease).

项目摘要所有生物都需要维持DNA完整性以生长和增殖。 DNA的复制和修复损害需要增加DNA核动肽的合成，这一过程取决于激酶，例如ATM和ATR。 DNA复制的正调可能导致细胞死亡或癌症。研究由我们的实验室和其他实验室表明，许多DNA损伤反应（DDR）蛋白（例如ATM，ATR，XRCC1）和RNR）通过分子伴侣HSP70和HSP90稳定。这些蛋白质执行多种细胞中的功能，包括新合成和变性蛋白的蛋白质折叠，蛋白转运跨膜和寡聚蛋白的分解。研究主要关注伴侣酮功能特异性是通过调节表达，同工型差异和共同的种类而产生的结合HSP70和HSP90分子的伴侣蛋白。尽管有几个通过全球蛋白质组学筛选（称为酵母和哺乳动物HSP70）上的磷酸化位点（称为伴侣代码），它们的生物学功能尚不清楚。我们在细胞中发表的研究确定 HSP70上单个位点的CDK介导的磷酸化可以通过改变两个共同的链链功能伴侣和客户蛋白相互作用。在此提案中，我们旨在了解DDR的激活如何促进 HSP70伴侣代码。我们预测，根据几种HSP90-激酶相互作用，HSP70磷酸化在DNA损伤期间，会产生一个反馈系统，从而使伴侣磷酸化提高了DDR的稳定性蛋白质，扩增DNA损伤反应的信号。我们建议在两者上同时使用分子生物学和最先进的质谱技术酿酒酵母和哺乳动物细胞培养细胞在我们的提案中实现目标的目的。酵母和哺乳动物细胞中HSP70上功能磷酸化位点的鉴定和研究将为我们提供一种完全新颖的方法来靶向伴侣活动。 HSP70活动可以使用特异性磷酸酶/激酶抑制剂。通过改变，可以针对特定的“客户”蛋白 HSP70磷酸化状态和特定的HSP70磷酸物种可能对抑制剂具有更高的敏感性。这项工作的范围对与DNA损伤相关的多种疾病具有广泛的影响反应和分子链酮功能，包括许多类型的癌症和神经退行性疾病由蛋白质聚集（亨廷顿氏病，阿尔茨海默氏病和克鲁兹菲尔德 - 贾科布氏病）引起。