Proteolytic control of DNA interstrand cross-link repair and genome integrity

项目摘要 由不正确的DNA修复系统引起的基因组不稳定性是肿瘤发生的主要驱动力。我们长久以来- 本学期的目标是了解细胞蛋白水解如何控制负责修复DNA的途径 从而保护基因组的完整性。由于DNA修复因子的稳态对于 DNA修复的活性，阐明了DNA中泛素蛋白水解途径的潜在机制 当癌症发生时，修复对于理解癌症的病因是必不可少的。我们感兴趣的是 将蛋白水解与范可尼贫血（FA）DNA修复途径的信号传导联系起来的机制， 与DNA复制过程中遇到的DNA链间交联（ICL）。它的缺陷会导致高风险的 由于基因组不稳定性升高而导致的多种癌症，并且已知其异常活性会影响治疗效果。 对利用DNA交联剂（包括铂）的细胞毒性化疗的反应。因此，在本发明中， 关于控制FA途径的分子和遗传因素的知识有望帮助我们利用它们。 放松管制以开发改进的癌症治疗剂。一个基本的监管 蛋白质降解的机制是蛋白质靶点的可逆磷酸化，这标志着蛋白质 被泛素-蛋白酶体系统破坏。我们最近发现， FAAP 20，FA途径所必需的FA核心泛素E3连接酶复合物的关键组分 激活，并表明FAAP 20的失调导致FA核心复合物的功能破坏， 损害细胞修复DNA ICL损伤的能力。具体来说，我们将SCFFBW 7定义为泛素E3 连接酶复合物负责磷酸化依赖性FAAP 20降解，并证明了其如何 去调节影响FA途径。我们的初步研究还表明， 顺反异构酶PIN 1调节FAAP 20构象变化调节泛素信号转导 FAAP 20降解，从而决定FA核心复合物的命运并影响FA的效率 DNA ICL修复。在此，我们建议解释PIN 1-SCFFBW 7蛋白水解信号在控制FA 途径及其对基因组不稳定性的影响。具体来说，我们将（1）剖析FAAP 20的信号通路 （2）阐明了PIN 1驱动的SCFFBW 7的结构变化的机制， FAAP 20作为调节开关起作用以控制FAAP 20稳定性，以及（3）确定PIN 1在以下中的作用： 通过FA信号通路调节DNA ICL修复和乳腺癌对铂的治疗反应 使用癌细胞系和小鼠模型。我们的研究有望揭示 人类癌症中高度失调的PIN 1-SCFFBW 7轴与DNA ICL修复之间的关系。这项工作将 通过提供独特的机会来设计治疗干预措施， FA相关恶性肿瘤和一般癌症中的异常DNA修复相关蛋白水解信号传导。

PROJECT SUMMARY Genome instability caused by incorrect DNA repair system is a major driver for tumorigenesis. Our long- term goal is to understand how cellular proteolysis controls the pathway responsible for repairing DNA damage, thereby preserving the integrity of the genome. Since homeostasis of DNA repair factors is critical for the activity of DNA repair, elucidating underlying mechanisms for the ubiquitin-proteolytic pathway in DNA repair is essential for understanding the etiology of cancer when it is derailed. We are interested in the mechanisms that link proteolysis to signaling of the Fanconi anemia (FA) DNA repair pathway, which deals with DNA interstrand cross-links (ICL) encountered during DNA replication. Its defects lead to a high risk of multiple cancers due to elevated genome instability, and its aberrant activity is known to influence therapeutic response to cytotoxic chemotherapy that utilizes DNA cross-linking agents including platinum. Thus, knowledge on molecular and genetic factors that control the FA pathway is expected to help us exploit their deregulation for the development of improved cancer therapeutics. One of the fundamental regulatory mechanisms for protein degradation is reversible phosphorylation of protein targets, which marks a protein to be destroyed by ubiquitin-proteasome system. We recently discovered the proteolytic signaling pathway of FAAP20, a key component of the FA core ubiquitin E3 ligase complex necessary for the FA pathway activation, and showed that deregulation of FAAP20 leads to a functional disruption of the FA core complex, impairing the ability of cells to repair DNA ICL lesions. Specifically, we defined SCFFBW7 as an ubiquitin E3 ligase complex responsible for phosphorylation-dependent FAAP20 degradation and demonstrated how its deregulation affects the FA pathway. Our preliminary studies also indicate that phosphorylation-dependent conformational change of FAAP20 regulated by cis-trans isomerase PIN1 modulates ubiquitin signaling of FAAP20 degradation, thereby determining the fate of the FA core complex and influencing the efficiency of DNA ICL repair. Herein, we propose to explicate PIN1-SCFFBW7 proteolytic signaling in controlling the FA pathway and its impact to genome instability. Specifically, we will (1) dissect the signaling pathway of FAAP20 degradation regulated by SCFFBW7, (2) elucidate the mechanisms by which PIN1-driven structural change of FAAP20 functions as a regulatory switch to control FAAP20 stability, and (3) determine the role of PIN1 in regulating DNA ICL repair and the therapeutic response of breast cancer to platinum via FA pathway signaling using cancer cell lines and a mouse model. Together, our studies are expected to reveal the first direct link between a highly deregulated PIN1-SCFFBW7 axis in human cancer and DNA ICL repair. This work will ultimately benefit human health by offering a unique opportunity to design therapeutic interventions that exploit aberrant DNA repair-associated proteolytic signaling in FA-related malignancy and cancer in general.