Understanding the reciprocal regulation between Hsp70 and the DNA damage response

项目摘要 所有的生物都需要保持DNA的完整性才能生长和繁殖。DNA的复制和修复 损伤需要增加DNA核苷酸的合成，这一过程取决于 诸如自动柜员机和自动柜员机之类的激活酶。DNA复制的错误调控可能会导致细胞死亡或癌症。研究 由我们的实验室和其他人已经证明，许多DNA损伤反应(DDR)蛋白(如ATM、ATR、XRCC1 和RNR)由分子伴侣Hsp70和Hsp90稳定。这些蛋白质执行各种不同的 在细胞中的功能，包括新合成和变性蛋白质的蛋白质折叠，蛋白质运输 跨膜和解聚寡聚蛋白质。研究主要集中在如何 伴侣功能的特异性是通过调节其表达、异构体差异和辅酶-2的变化而产生的。 与Hsp70和Hsp90分子结合的伴侣蛋白。尽管确认了几个 通过全球蛋白质组筛选在酵母和哺乳动物Hsp70上的磷酸化位点(称为 伴侣密码)，这些基因的生物学功能尚不清楚。我们发表在《细胞》杂志上的研究确定 CDK介导的Hsp70上单个位点的磷酸化可以通过改变两个共同的受体来调节伴侣功能。 伴侣蛋白和客户蛋白相互作用。 在这项提案中，我们旨在了解DDR的激活如何促进 HSP70伴侣代码。我们预测，在几个Hsp90-激酶的相互作用中，Hsp70的磷酸化 在DNA损伤过程中创建了一个反馈系统，伴侣蛋白的磷酸化增加了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学技术来研究这两种情况 实现酿酒酵母和哺乳动物细胞培养的目标，在我们的提案中。 酵母和哺乳动物细胞中Hsp70功能磷酸化位点的鉴定与研究 为我们提供了一种针对伴侣活动的全新方式。热休克蛋白70活性可被抑制 特定的磷酸酶/激酶抑制剂。有可能通过改变靶向特定的‘客户’蛋白 HSP70的磷酸化状态和特定的HSP70磷酸化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损伤相关的各种疾病具有广泛的影响 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(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).