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中心，泛素介导的蛋白水解的缺陷与癌症有关。细胞周期依赖性蛋白质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物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。in C.在线虫中，CUL-2是细胞周期调控的多个方面所必需的，包括：G1到S期转变;染色体浓缩;减数分裂II中期到后期转变;有丝分裂进程;以及有丝分裂细胞周期蛋白B的降解。CUL-2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中心组分起作用，所述多亚基泛素连接酶采用不同的底物识别亚基（SRS）。识别几种CUL-2依赖性细胞周期途径中底物的SRS的身份是未知的，关键底物也是未知的，其泛素化允许细胞周期事件发生。本实验将从三个方面阐明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复合物物理缔合的SRS和蛋白质。相互作用蛋白质将表征细胞功能和表达模式。其次，将通过改良的亲和纯化方案和双杂交筛选来鉴定被CUL-2复合物泛素化以调节细胞周期的底物。将对这些底物进行表征，以确定它们发挥功能的CUL-2依赖性过程及其在该过程中的作用。最后，对CUL-2复合物SRS基因的抑制因子进行基因筛选将识别相互作用的基因，这些基因要么调节CUL-2复合物活性，允许绕过CUL-2功能，要么是CUL-2的关键底物。了解CUL-2调节细胞周期的分子途径将为控制基本细胞周期事件提供重要的见解。癌症起源于不受调节的细胞增殖，对细胞周期控制的完整理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.