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

描述(申请人提供)：这项研究的长期目标是在动物发育的背景下了解细胞周期调节。调节的蛋白分解是控制细胞周期的中心，泛素介导的蛋白分解缺陷与癌症有关。依赖细胞周期的蛋白质降解主要通过泛素蛋白分解途径发生。人类CUL2泛素连接酶作为肿瘤抑制复合体的一部分，用于预防癌症，但其在调节细胞周期中的作用尚未被直接研究。在线虫中，细胞周期调控的多个方面都需要Cul-2，包括：G1期到S期的转变；染色体凝集；减数分裂II中期到后期的转变；有丝分裂的进程；以及有丝分裂周期蛋白B的降解。Cul-2在极性和性别决定中还发挥着额外的作用。CuL-2是使用不同底物识别亚单位(SRSS)的多亚基泛素连接酶的中心成分。在一些依赖CuL-2的细胞周期通路中识别底物的SRSS的身份尚不清楚，关键底物也是如此，其泛素化允许细胞周期事件的发生。这项提议中的实验将从三个主要方面阐明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来鉴定物理上与CuL-2复合体相关的SRSS和蛋白质。相互作用的蛋白质将以细胞功能和表达模式为特征。其次，将通过改良的亲和纯化方法和双杂交筛选来鉴定被CuL-2络合物泛素化以调节细胞周期的底物。这些底物将被表征，以确定它们在其中发挥作用的CuL-2依赖的过程及其在该过程中的作用。最后，对Cul-2复合体SRS基因抑制子的遗传筛选将确定相互作用的基因，这些基因要么调节Cul-2复合体的活性，要么允许Cul-2功能的旁路，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的细胞周期事件提供重要的见解。癌症起源于不受调控的细胞增殖，对细胞周期调控的全面了解将为理解癌症生物学和开发抗癌治疗药物提供基础。

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.