CTDP1 Regulates FANCI and the Response to DNA Interstrand Crosslinks

项目摘要/摘要：CTDP 1调节FANCI和DNA链间反应 交联 RNA聚合酶II亚基A C-末端结构域磷酸酶CTDP 1是RNA聚合酶II亚基A C-末端结构域磷酸酶中唯一的磷酸酶。 一种含有BRCA 1 C-末端（BRCT）结构域的人类蛋白质组。这个模块域只存在于 介导DNA损伤反应（DDR）的一组小而特化的蛋白质，但 该过程中的CTDP 1尚未描述。对BRCT相互作用组的分析已经鉴定出蛋白质 CTDP 1和范可尼贫血（FA）蛋白FANCI之间发生的相互作用，FANCI是 DNA链间交联（ICL）损伤解决和对ICL诱导剂丝裂霉素的抗性 c和顺铂。初步结果表明，CTDP 1表达促进FANCI激活， 抑制细胞对ICL诱导化合物丝裂霉素C和美法仑的敏感性。镇压 CTDP 1表达还与细胞周期S期细胞积累增加有关 在正常生长条件下和对ICL的响应下。CTDP 1表达是促进 ICL诱导的FANCI与DNA修复蛋白相互作用，包括DNA损伤灶蛋白γ H2 AX。 这些结果代表了首次报道的FANCI和磷酸酶之间的蛋白质相互作用， 规范其功能。因此，这项研究的中心假设是CTDP 1是必需的， 通过调节FANCI激活对ICL损伤的细胞反应，以及靶向抑制 CTDP 1可以使细胞对引起ICL的化疗药物敏感。该项目的具体目标是：1） 表征CTDP 1介导的FANCI磷酸化对FA途径对ICL反应的影响; 2） 表征CTDP 1抑制对乳腺癌原位模型中ICL敏感性的影响; （3）采用高通量筛选方法筛选CTDP 1的小分子抑制剂。这些 实验将利用磷酸化蛋白质组学来表征磷酸化的时间调节 DNA损伤后由CTDP 1调节的FANCI上的位点。乳腺癌的异种移植模型将是 用于确定CTDP 1抑制对癌症生长、转移和对顺铂的反应的影响 使用体内成像。潜在CTDP 1 BRCT-FANCI的虚拟和物理高通量筛选 将完成蛋白质相互作用破坏化合物，并选择先导化合物， CTDP 1调节的FANCI和RPB 1上的磷酸化事件的经验证的蛋白质印迹分析。 这项研究的成功完成将导致对CTDP 1介导的 调节FANCI和ICL DNA损伤反应，其可用于敏化交联剂 对DNA损伤疗法有抵抗力的癌症。

Project Summary/Abstract: CTDP1 Regulates FANCI and the Response to DNA Interstrand Crosslinks RNA polymerase II subunit A C-terminal domain phosphatase, CTDP1, is the only phosphatase in the human proteome that contains a BRCA1 C-Terminal (BRCT) domain. This modular domain only exists in a small and specialized group of proteins that mediate the DNA damage response (DDR), but the role of CTDP1 in this process has not been delineated. Analysis of the BRCT interactome has identified protein interactions that occur between CTDP1 and the Fanconi anemia (FA) protein FANCI, which is required for the resolution of DNA interstrand crosslink (ICL) lesions and resistance to ICL-inducing agents mitomycin c and cisplatin. Preliminary results indicate that CTDP1 expression promotes FANCI activation and represses cellular sensitivity to the ICL-inducing compounds mitomycin c and melphalan. Repression of CTDP1 expression is also associated with an increased accumulation of cells in S-phase of the cell cycle under both normal growth conditions and in response to ICL. CTDP1 expression is required to facilitate ICL-induced FANCI interactions with DNA repair proteins, including the DNA damage foci protein γH2AX. These results represent the first reported protein interaction between FANCI and a phosphatase that can regulate its function. Thus, the central hypothesis of this research is that CTDP1 is required for the cellular response to ICL damage through the regulation of FANCI activation, and that targeted inhibition of CTDP1 can sensitize cells to ICL-causing chemotherapeutics. The Specific Aims of this project are: 1) To characterize the impact of CTDP1-mediated FANCI phosphorylations on FA pathway response to ICL; 2) To characterize the impact of CTDP1 inhibition on ICL sensitivity in an orthotopic model of breast cancer; and 3) To identify small molecule inhibitors of CTDP1 using high-throughput screening. These experiments will utilize phosphoproteomics to characterize the temporal regulation of phosphorylation sites on FANCI regulated by CTDP1 following DNA damage. Xenograft models of breast cancer will be used to determine the effects of CTDP1 inhibition on cancer growth, metastasis, and response to cisplatin using in vivo imaging. Virtual and physical high-throughput screening of potential CTDP1 BRCT-FANCI protein interaction disrupting compounds will be completed, and selected lead compounds will be validated western blot analysis of CTDP1 regulated phosphorylation events on FANCI and RPB1. Successful completion of this research will lead to a detailed understanding of the CTDP1-mediated regulation of FANCI and the ICL DNA damage response, which could be used to sensitize crosslinker resistant cancers to DNA damaging therapies.