Regulation of Hsp70-Mediated Cyclin D1 Destruction in Breast Cancer

项目摘要 HSP70是一种普遍保守的分子伴侣，在细胞中执行多种功能，包括 新合成的和变性的蛋白质的折叠，蛋白质的跨膜运输和 寡聚蛋白质的解聚。研究主要集中在Hsp70是如何发挥作用的 通过调节a)Hsp70的表达，b)Hsp70蛋白家族中的异构体差异和c) 与Hsp70分子结合的各种辅助伴侣蛋白。尽管确认了几个 通过全球蛋白质组筛选，酵母和哺乳动物HSP70上的磷酸化位点，生物 这些基因的功能尚不清楚。 所有的生物体都需要细胞周期的正确协调才能生长和增殖。细胞调节失调 周期进展可能导致细胞死亡或癌症。细胞从G1到G2的过程需要 细胞周期蛋白依赖性激酶(CDK)CDK4的活性。CDK4的临界活性是通过结合到 细胞周期蛋白，如细胞周期蛋白D1，在细胞周期中周期性地上升和下降。 由于Cyclin D1在细胞周期控制中的重要作用，Cyclin D1活性的错误调节(通过 转录增加或稳定性)通常在癌细胞中观察到。任何降低Cyclin D1活性的策略 可能形成新的抗癌疗法的基础。 我们最近的研究已经确定，Hsp70上单个位点的磷酸化调节伴侣蛋白 通过改变辅伴蛋白和客户蛋白的相互作用来发挥作用。特别是，我们已经证明了 Hsp70磷酸化增强促进细胞周期蛋白D1的破坏。 在这项提案中，我们期望获得进一步的机械性洞察Hsp70磷酸化如何改变 通过下调Cyclin D1和其他重要的细胞周期蛋白的水平来促进细胞周期的进展。 我们建议在乳房中使用分子生物学和最先进的质谱学技术。 来实现我们提案中目标的目标。理解热休克蛋白70的调控 哺乳动物细胞中的磷酸化将为我们提供一种全新的靶向伴侣活性的方法。 HSP70的活性可以用特定的磷酸酶/激酶抑制剂来抑制。有可能将目标对准 通过改变Hsp70的磷酸化状态和特定的Hsp70磷酸化物种来表达特定的‘客户’蛋白 可能对抑制剂有更高的敏感性。这项工作的范围对各种 与细胞周期和分子伴侣功能相关的疾病，包括许多类型的癌症 和蛋白质聚集引起的神经退行性疾病(亨廷顿病、阿尔茨海默病 和克雅氏病)。

Project Summary Hsp70 is a universally conserved molecular chaperone that performs 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 Hsp70 function specificity arises through regulation of a) expression of Hsp70, b) isoform differences in the Hsp70 protein family and c) the variety of co-chaperone proteins that bind to the Hsp70 molecule. Despite the identification of several phosphorylation sites on both yeast and mammalian Hsp70 through global proteomic screens, the biological function of these remains unclear. All organisms require correct coordination of the cell cycle to grow and proliferate. Misregulation of cell cycle progression can result in either cell inviability or cancer. Progression of the cell from G1 to G2 requires the activity of cyclin-dependent kinase (CDK) CDK4. The critical activity of CDK4 is regulated by binding to cyclin proteins such as Cyclin D1, which rises and falls in abundance periodically through the cell cycle. Because of the important role of Cyclin D1 in cell cycle control, misregulation of Cyclin D1 activity (through increased transcription or stability) is often observed in cancer cells. Any strategy that lowers Cyclin D1 activity in cells may form the basis of novel anticancer therapies. Our recent studies have determined that phosphorylation of a single site on Hsp70 regulates chaperone function by altering both co-chaperone and client protein interactions. In particular, we have shown that increased Hsp70 phosphorylation promotes Cyclin D1 destruction. In this proposal, we expect to gain further mechanistic insight into how Hsp70 phosphorylation can alter cell cycle progression by downregulating the levels of Cyclin D1 and other important cell cycle proteins. We propose to use both molecular biology and state-of-the-art mass spectrometric techniques in breast cancer cells to achieve the aims of the objectives in our proposal. Understanding the regulation of Hsp70 phosphorylation in 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 cell cycle 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).