Mechanisms of SIRT2 Function in the Replication Stress Response

描述（由申请人提供）：基因组的精确复制和对其完整性的持续监测对于细胞存活和避免癌症和早衰等疾病至关重要。复制应激反应（RSR）识别来自电离辐射等损伤的DNA复制挑战，并协调维持基因组完整性所必需的多种DNA修复和细胞周期检查点途径。然而，介导许多RSR活动的机制以及它们的失调如何导致致癌尚不清楚。因此，本提案的总体目标是确定细胞对复制应激反应的关键过程，并将这些知识应用于癌症的预防和治疗。初步数据表明SIRT2 （sirtuin家族去乙酰化酶）是一种新的RSR调节因子。先前的研究表明sirtuins在衰老和肿瘤抑制中都有作用。具体来说，Sirt2缺失的小鼠会发生乳腺癌和肝癌，而Sirt2在人类乳腺癌和肝癌中的表达降低，这表明Sirt2是一种合法的肿瘤抑制因子。最近来自实验室的数据表明，SIRT2在一个保守的赖氨酸残基上使CDK9去乙酰化，以促进从复制停滞中恢复。质谱分析方法还发现了一些与SIRT2相互作用并被SIRT2去乙酰化的额外RSR蛋白。这些发现表明SIRT2在RSR中的关键作用可能提供了衰老和致癌相关的潜在机制；然而，SIRT2在RSR中的确切功能尚不清楚。因此，本提案的总体目标是确定SIRT2在RSR中发挥作用以维持基因组完整性的机制。正在测试的假设是，SIRT2通过CDK9和其他关键底物的去乙酰化来指导RSR，至少在一定程度上维持基因组的完整性。提出了以下具体目标：确定并从机制上剖析SIRT2在指导RSR中所起的特定作用，2。确定sirt2介导的CDK9去乙酰化在RSR中的功能意义，2。鉴定在RSR中起作用的SIRT2底物。该建议在概念上具有创新性，因为它提出了一种新的RSR调控机制，其中SIRT2通过关键RSR蛋白的去乙酰化作为RSR的协调者，建立了一种新的范式，通过去乙酰化调节RSR蛋白，并为SIRT2以及更普遍的sirtuins在RSR调控中的作用建立了新的作用。这些目标的完成将为SIRT2如何维持基因组完整性和预防癌症提供新的见解，阐明SIRT2乙酰基在调节RSR中的意义，并提高我们对衰老与致癌之间联系的理解。最近，NCI提出了一个新的研究重点，即设计严谨和创新的研究策略，以解决癌症研究中的具体问题和悖论，即NCI的“挑衅性问题”。这项研究计划解决了这样一个问题：“除了突变的积累之外，衰老的什么机制能促进或防止癌症的发展？”

DESCRIPTION (provided by applicant): Accurate replication of the genome and continuous surveillance of its integrity are essential for cell survival and avoidance of diseases such as cancer and premature aging. The replication stress response (RSR) recognizes challenges to DNA replication from insults such as ionizing radiation and coordinates diverse DNA repair and cell cycle checkpoint pathways necessary to maintain genome integrity. However, the mechanisms mediating many RSR activities and how their dysregulation leads to carcinogenesis are poorly understood. Thus, the overarching goal of this proposal is to determine the critical processes by which cells respond to replication stress and apply this knowledge for cancer prevention and treatment. Preliminary data identify SIRT2, a sirtuin family deacetylase, as a novel regulator of the RSR. Prior work has implicated sirtuins in both aging and tumor suppression. Specifically, mice deficient in Sirt2 develop breast and liver cancers, and SIRT2 expression is decreased in human breast and liver cancers, suggesting that SIRT2 is a legitimate tumor suppressor. Recent data from the lab show that SIRT2 deacetylates CDK9 at a conserved lysine residue to promote recovery from replication arrest. Mass spectrometry approaches also identified a number of additional RSR proteins that interact with and are deacetylated by SIRT2. These findings imply a critical role for SIRT2 in the RSR that may provide an underlying mechanism by which aging and carcinogenesis are connected; however, the precise functions of SIRT2 in the RSR are not clear. As such, the overall objective of this proposal is to determine the mechanisms by which SIRT2 functions in the RSR to maintain genome integrity. The hypothesis being tested is that SIRT2 maintains genome integrity, at least in part, by directing the RSR through deacetylation of CDK9 and other key substrates. The following specific aims are proposed: 1. Determine and mechanistically dissect the specific role that SIRT2 plays in directing the RSR, 2. Determine the functional significance of SIRT2-mediated deacetylation of CDK9 in the RSR, 3. Identify SIRT2 substrates that function in the RSR. This proposal is conceptually innovative in that it proposes a novel mechanism for regulation of the RSR that involves SIRT2 as an orchestrator of the RSR through deacetylation of critical RSR proteins, establishing a novel paradigm by which RSR proteins are regulated by deacetylation and establishing a novel role for SIRT2, and more generally for sirtuins, in the regulation of the RSR. Completion of these aims will provide new insights into how SIRT2 maintains genome integrity and prevents cancer, elucidate the significance of the SIRT2 acetylome in regulating the RSR, and improve our understanding of the link between aging and carcinogenesis. Recently, the NCI has proposed a new research emphasis to design rigorous and innovative research strategies to solve specific problems and paradoxes in cancer research identified as the NCI's "Provocative Questions." This research proposal addresses one such question "What mechanisms of aging, beyond the accumulation of mutations, promote or protect against cancer development?"