Cell cycle regulation by C. elegans CUL-2 E3 complexes

描述（由申请人提供）：本研究的长期目标是了解动物发育背景下的细胞周期调控。受调节的蛋白水解是控制细胞周期的核心，而亚奎素介导的蛋白水解缺陷与癌症有关。细胞周期依赖性蛋白降解主要通过泛素蛋白水解途径发生。人CUL2泛素连接酶作为肿瘤抑制复合体的一部分起预防癌症的作用，但其在调节细胞周期中的作用尚未被直接研究。在秀丽隐杆线虫中，CUL-2参与细胞周期调控的多个方面，包括：G1期到S期的转变；染色体缩合;减数分裂II中期向后期转变；有丝分裂进程;有丝分裂周期蛋白B. CUL-2的降解在极性和性别决定的调节中具有额外的作用。CUL-2作为多亚基泛素连接酶的核心成分，使用不同的底物识别亚基（SRSs）。识别几种cul -2依赖性细胞周期途径中底物的SRSs的身份尚不清楚，其泛素化允许细胞周期事件发生的关键底物也是如此。本课题的实验将从三方面阐明CUL-2泛素连接酶的功能。首先，将通过免疫亲和纯化和双杂交筛选鉴定与CUL-2复合体物理相关的SRSs和蛋白质。相互作用蛋白将被表征为细胞功能和表达模式。其次，将通过改进的亲和纯化方案和双杂交筛选鉴定被CUL-2复合物泛素化以调节细胞周期的底物。这些底物将被表征以确定它们在其中起作用的cul -2依赖过程及其在该过程中的作用。最后，对CUL-2复合体SRS基因的抑制基因进行遗传筛选，将鉴定出调节CUL-2复合体活性、允许绕过CUL-2功能或作为CUL-2关键底物的相互作用基因。了解CUL-2调控细胞周期的分子途径将为基本细胞周期事件的控制提供重要的见解。癌症起源于不受调控的细胞增殖，对细胞周期控制的全面了解将为理解癌症生物学和开发抗癌疗法提供基础。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand cell cycle regulation in the context of animal development. Regulated proteolysis is central to the control of the cell cycle, and defects in ubquitin- mediated proteolysis have been linked to cancer. Cell cycle-dependent protein degradation occurs principally through the ubiquitin proteolytic pathway. The human CUL2 ubiquitin ligase functions as part of a tumor suppressor complex to prevent cancer, but its role in regulating the cell cycle has not been directly studied. In C. elegans, CUL-2 is required for multiple aspects of cell cycle regulation, including: the G1 to S phase transition; chromosome condensation; the meiosis II metaphase to anaphase transition; mitotic progression; and the degradation of mitotic cyclin B. CUL-2 has additional roles in the regulation of polarity and sex determination. CUL-2 functions as a central component of a multisubunit ubiquitin ligase that employs distinct substrate recognition subunits (SRSs). The identity of the SRSs that recognize substrates in several of the CUL-2-dependent cell cycle pathways are unknown, as are the critical substrates, whose ubiquitination allows the cell cycle events to occur. The experiments in this proposal will clarify the functions of the CUL-2 ubiquitin ligase in three major ways. First, SRSs and proteins that physically associate with the CUL-2 complex will be identified by immunoaffinity purification and two-hybrid screens. Interacting proteins will be characterized for cellular function and expression pattern. Second, the substrates that are ubiquitinated by CUL-2 complexes to regulate the cell cycle will be identified by a modified affinity purification protocol and two-hybrid screen. These substrates will be characterized to determine the CUL-2-dependent process in which they function and their role in that process. Finally, genetic screens for suppressors of CUL-2 complex SRS genes will identify interacting genes that either modulate CUL-2 complex activity, allow a bypass of CUL-2 function, or are critical substrates of CUL-2. Understanding the molecular pathways through which CUL-2 regulates the cell cycle will provide important insights into the control of fundamental cell cycle events. Cancer derives from unregulated cell proliferation, and a complete understanding of cell cycle control will provide a foundation for understanding cancer biology and developing anti-cancer therapeutics.