Understanding the reciprocal regulation between Hsp70 and the DNA damage response

项目摘要 所有生物体都需要维持DNA的完整性才能生长和增殖。DNA复制和修复 损伤需要增加DNA核苷酸的合成，这一过程依赖于DNA的活性。 激酶如ATM和ATR。DNA复制的失调可能导致细胞死亡或癌症。研究 我们的实验室和其他人已经表明，许多DNA损伤反应（DDR）蛋白（如ATM，ATR，XRCC 1 和RNR）由分子伴侣Hsp 70和Hsp 90稳定。这些蛋白质执行各种 在细胞中的功能包括新合成和变性蛋白质的蛋白质折叠，蛋白质转运 跨膜和解聚寡聚蛋白。研究主要集中在如何 伴侣蛋白功能特异性通过表达调节、同种型差异和多种共 与Hsp 70和Hsp 90分子结合的伴侣蛋白。尽管发现了几个 酵母和哺乳动物Hsp 70上的磷酸化位点，通过全球蛋白质组学筛选（称为 伴侣蛋白编码），这些的生物学功能仍然不清楚。我们在Cell上发表的研究表明， CDK介导的Hsp 70上单个位点的磷酸化可以通过改变Hsp 70上的两个共-磷酸化位点来调节分子伴侣的功能。 伴侣蛋白和客户蛋白相互作用。 在本提案中，我们的目的是了解复员方案的启动如何能够促进 Hsp 70分子伴侣编码。我们预测，与几种Hsp 90-激酶相互作用一致，Hsp 70磷酸化 在DNA损伤过程中产生反馈系统，由此伴侣磷酸化增加DDR的稳定性， 蛋白质，放大DNA损伤反应的信号。 我们建议同时使用分子生物学和最先进的质谱技术， 酿酒酵母细胞和哺乳动物细胞培养的目的，以达到我们建议的目标。 在酵母和哺乳动物细胞中鉴定和研究Hsp 70上的功能性磷酸化位点将有助于 为我们提供了一种全新的方法来靶向分子伴侣的活性。Hsp 70的活性可能会受到抑制， 特异性磷酸酶/激酶抑制剂。通过改变蛋白质的结构， Hsp 70磷酸化状态和特定的Hsp 70磷酸化物质可能对抑制剂具有更高的敏感性。 这项工作的范围对与DNA损伤和DNA损伤相关的各种疾病都有广泛的影响。 反应和分子伴侣功能，包括许多类型的癌症和神经退行性疾病 由蛋白质聚集引起的疾病（亨廷顿病、阿尔茨海默病和克雅氏病）。

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).