Dissecting functional interplay of deubiquitinases and the cell cycle machinery

描述(由申请人提供)：准确的DNA复制对于正常发育和预防癌症等疾病至关重要。E3泛素连接酶在协调无数过程以确保一轮精确的复制中的重要性是众所周知的，即使在不利的条件(复制压力)下也是如此。然而，调节这些连接酶有序活动的机制还不是很清楚。解剖这些机制不仅对于理解这一基本过程的病理生理变化至关重要， 也是为了利用这些变化来开发新的治疗策略。最近，我们发现去泛素酶(DUB)USP37与E3连接酶APCCDh1结合并拮抗，以促进及时的复制启动。此外，蛋白质组学分析在USP37-相互作用组中鉴定了多个APCCDh1底物和增殖细胞核抗原相关因子。USP37与肿瘤的发生和发展有关。然而，它的生物学功能(S)几乎是未知的。到目前为止，我们的数据表明USP37在复制过程中是核心的。继续这一新的研究路线，我们发现，首先，USP37对底物的顺序靶向控制复制检查点活动。在G1末/S早期，USP37促进了检查点的激活。在S/G2晚期，USP37促进了检查点恢复和细胞周期恢复。我们的数据支持这样一个模型，即USP37在S期的磷酸化(可能由ATR)改变其底物特异性，允许关键检查点蛋白的积累和检查点活性的增加。其次，在S期，我们发现USP37被募集到染色质中，并与分子支架增殖细胞核抗原相互作用。我们的数据表明，USP37通过调节增殖细胞核抗原的相互作用来防止复制因子的虚假招募，从而促进基因组的稳定性。与复制中的中心角色一致，USP37耗尽的细胞在相对惰性复制压力下的存活率降低。在本申请中，我们建议阐明控制复制检查点活性和调控增殖细胞核抗原相互作用的依赖于USP37的机制；然后这些机制将作为USP37与底物相互作用的工作模型，用于识别和表征其他USP37底物和功能。我们的中心假设是，在细胞周期中对USP37底物选择的调节促进了忠实的基因组复制和对复制压力的耐受。为了验证这一假设，我们将：1.确定受调控的USP37底物选择控制复制检查点。2.研究USP37与增殖细胞核抗原相互作用对复制应激耐受性的影响。3.研究USP37调控的蛋白质组。这些研究的结果将为细胞如何维持基因组完整性提供重要的新见解。最终，该项目为剖析USP37在胚胎发育中的作用以及USP37在癌症中表达增加的功能奠定了基础，这可能最终为将抑制USP37作为一种有效的手段使细胞对复制应激敏感提供理论基础。

DESCRIPTION (provided by applicant): Accurate DNA replication is essential for normal development and preventing diseases such as cancer. The importance of the E3 ubiquitin ligases in coordinating myriad processes to ensure a single, precise round of replication, even under unfavorable conditions (replication stress), is well known. However, the mechanisms that regulate the ordered activities of these ligases are less clear. Dissecting these mechanisms is critical not only for understanding the pathophysiological alterations of this fundamental process, but also for exploiting these changes to develop novel therapeutic strategies. Recently, we showed that the deubiquitinating enzyme (DUB) USP37 binds and antagonizes the E3 ligase APCCdh1 to facilitate timely replication initiation. Further, proteomic analysis identified multipl APCCdh1 substrates and PCNA-associated factors in the USP37- interactome. USP37 is implicated in development and oncogenesis. However, its biological function(s) are nearly unknown. Our data thus far signify that USP37 is central to the replication process. Continuing this novel line of research, we have found that, first, sequential targeting of substrates by USP37 controls replication checkpoint activity. In late G1/early S, USP37 promotes the activation of the checkpoint. In late S/G2, USP37 facilitates checkpoint recovery and cell cycle resumption. Our data support a model whereby phosphorylation of USP37 in S-phase (likely by ATR) alters its substrate specificity, allowing the accumulation of key checkpoint proteins and increased checkpoint activity. Second, during S-phase we found that USP37 is recruited to chromatin and interacts with the molecular scaffold PCNA. Our data suggest that USP37 promotes genomic stability by regulating PCNA interactions to prevent spurious recruitment of replication factors. Consistent with a central role in replication, USP37-depleted cells had diminished survival on exposure to relatively inert replication stress. In this application we propose to elucidate the USP37-dependent mechanisms underlying control of replication checkpoint activity and regulation of PCNA interactions; these mechanisms will then serve as working models of USP37-substrate interactions for identifying and characterizing additional USP37 substrates and functions. Our central hypothesis is that regulation of USP37 substrate selection during the cell cycle promotes faithful genome replication and tolerance of replication stress. To test this hypothesis we will: 1. Determine that regulated USP37 substrate selection controls the replication checkpoint. 2. Characterize the impact of the USP37-PCNA interaction on the tolerance of replication stress 3. Investigate the USP37-regulated proteome. The results of these studies will provide significant new insight into how cells maintain genomic integrity. Ultimately, the project lays the foundation for dissecting the role of USP37 in embryonic development and the function of increased USP37 expression in cancer, which may ultimately provide the rationale for pursuing USP37 inhibition as a potent means of sensitizing cells to replication stress.