STRUCTURE OF THE UBIQUITIN LIGASE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 泛素介导的蛋白分解调控一系列的生物过程，包括生长控制和癌症、信号转导和应激反应。靶蛋白的泛素化通过三重酶的级联反应进行，包括泛素激活(E1)、结合(E2)和连接(E3)活性。尽管对这种泛素化反应进行了深入的结构和功能分析，但目前我们对泛素如何从E2转移到E3结合底物的特定赖氨酸(S)的了解非常有限。多亚单位SCF(Skp1-cul1-Rbx1-F box蛋白)E2-和底物募集复合体构成了最大的E3超家族。SCFFbx4(上标表示F盒蛋白)负责调节细胞周期激活因子细胞周期蛋白D的周转。在许多癌症中，细胞周期蛋白D水平高与肿瘤分级高、预后差和转移增加有关。我们正试图获得SCFFbx4单独的以及与其底物细胞周期蛋白D和E2酶形成的复合体的晶体结构。这项工作的长期目标是通过表征泛素化位点的选择、SCF二聚化的要求以及细胞周期蛋白D泛素化反应的E2-SCF相互作用来阐明泛素转移的结构和机制基础。我们的研究将解决SCF E3泛素化反应的一般方面，以及关于细胞周期蛋白D降解的具体问题。所获得的知识将为开发旨在操纵癌症中细胞周期蛋白D水平的治疗方法提供一条新的途径。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ubiquitin-mediated proteolysis regulates a vast array of biological processes, including growth control and cancer, signal transduction, and the stress response. Ubiquitination of a target protein proceeds through a triple-enzyme cascade, entailing ubiquitin-activating (E1), conjugating (E2), and ligating (E3) activities. Despite intensive structural and functional analysis of this ubiquitination reaction, our current knowledge of how ubiquitin is transferred from E2 to particular lysine(s) of the bound substrate in E3 is extremely limited. The multisubunit SCF (Skp1-Cul1-Rbx1-F box protein) E2- and substrate-recruiting complexes comprise the largest superfamily of E3s. SCFFbx4 (the superscript denotes the F box protein) are responsible for regulated turnover of the cell-cycle activator cyclin D. High cyclin D levels are associated with high tumor grade, poor prognosis, and increased metastasis in many cancers. We are trying to get the crystal structures of SCFFbx4 alone and in complex with its substrate cyclin D and the E2 enzyme. The long-term objective of this work is to elucidate the structural and mechanistic basis for ubiquitin transfer by characterizing ubiquitination site selection, requirement for SCF dimerization, and E2-SCF interactions of the cyclin D ubiquitination reaction. Our studies will address both general aspects of the ubiquitination reaction by the SCF E3s and also specific questions about cyclin D degradations. The knowledge gained will provide a novel avenue for the development of therapeutics designed to manipulate cyclin D levels in cancer.