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：
10095512
负责人：
Andrew William Truman
金额：
$ 29.54万
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/10095512
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/antagonist 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).

项目概要所有生物体都需要维持 DNA 的完整性才能生长和增殖。需要增加 DNA 核苷酸的合成，这一过程依赖于 DNA 核苷酸的活性 ATM 和 ATR 等激酶 DNA 复制的失调可能导致细胞死亡或癌症。我们的实验室和其他人的研究表明，许多 DNA 损伤反应 (DDR) 蛋白（例如 ATM、ATR、XRCC1 和 RNR）由分子伴侣 Hsp70 和 Hsp90 稳定，这些蛋白质具有多种功能。细胞中的功能，包括新合成和变性蛋白质的蛋白质折叠、蛋白质运输研究主要集中在如何跨膜和寡聚蛋白的解聚上。伴侣功能特异性是通过表达调节、异构体差异和共轭体的多样性而产生的。尽管已鉴定出几种与 Hsp70 和 Hsp90 分子结合的伴侣蛋白。通过全局蛋白质组筛选（称为分子伴侣代码），但我们在《细胞》杂志上发表的研究表明，这些分子的生物学功能仍不清楚。 CDK 介导的 Hsp70 上单个位点的磷酸化可以通过改变两个共轭分子来调节伴侣功能。伴侣蛋白和客户蛋白相互作用。在这个提案中，我们旨在了解DDR的激活如何促进格局的改变。我们预测，Hsp70 磷酸化与几种 Hsp90-激酶相互作用一致。 DNA 损伤期间会创建一个反馈系统，伴侣磷酸化可提高 DDR 的稳定性蛋白质，放大 DNA 损伤反应的信号。我们建议在这两个方面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以达到我们建议中的目标的目的。酵母和哺乳动物细胞中 Hsp70 功能性磷酸化位点的鉴定和研究将为我们提供了一种全新的方法来靶向伴侣蛋白的活性，可以使用该方法来抑制 Hsp70 的活性。特定的磷酸酶/激酶抑制剂可以通过改变来靶向特定的“客户”蛋白质。 Hsp70 磷酸化状态和特定的 Hsp70 磷酸化种类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伴侣功能，包括多种癌症和神经退行性疾病由蛋白质聚集引起（亨廷顿病、阿尔茨海默病和克雅氏病）。