Mechanisms of SIRT2 Function in the Replication Stress Response

描述(申请人提供)：基因组的准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过早衰老等疾病至关重要。复制应激反应(RSR)识别来自电离辐射等侮辱对DNA复制的挑战，并协调维持基因组完整性所必需的各种DNA修复和细胞周期检查点途径。然而，调节许多RSR活性的机制以及它们的失调如何导致癌症发生的机制还知之甚少。因此，这项建议的首要目标是确定细胞对复制压力做出反应的关键过程，并将这一知识应用于癌症预防和治疗。初步数据表明，SIRT2是Sirtuin家族的脱乙酰酶，是RSR的一个新的调节因子。先前的工作已经证明sirtuins在衰老和肿瘤抑制中都有作用。具体地说，缺乏SIRT2的小鼠会患上乳腺癌和肝癌，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。实验室的最新数据显示，SIRT2在保守的赖氨酸残基上去乙酰化CDK9，以促进从复制停滞中恢复。质谱学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。这些发现暗示了SIRT2在RSR中的关键作用，这可能为衰老和癌症的发生提供了一个潜在的机制；然而，SIRT2在RSR中的确切功能尚不清楚。因此，这项建议的总体目标是确定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至少在一定程度上通过CDK9和其他关键底物的脱乙酰化来指导RSR，从而维持基因组的完整性。提出了以下具体目标：1.确定并机械地剖析SIRT2在RSR中所起的具体作用；2.确定SIRT2介导的CDK9脱乙酰基在RSR中的功能意义；3.确定SIRT2底物在RSR中的作用。这一建议在概念上是创新的，因为它提出了一种新的RSR调控机制，涉及SIRT2作为RSR的协调者，通过关键RSR蛋白的脱乙酰化来调节RSR，建立了一种新的范式，通过脱乙酰化来调节RSR蛋白，并建立了SIRT2，更广泛地说，SIRT2在RSR调节中的新角色。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乙酰组在调节RSR中的意义，并提高我们对衰老和癌症发生之间的联系的理解。最近，NCI提出了一个新的研究重点，以设计严格和创新的研究策略，以解决癌症研究中被确定为NCI的“挑衅性问题”的具体问题和悖论。这项研究提案解决了这样一个问题：除了突变的积累，还有什么衰老机制可以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?"