Structural biology of SCFs and related ubiquitin Ligases

SCF 和相关泛素连接酶的结构生物学

• 批准号: 6838748
• 负责人: NIKOLA P PAVLETICH
• 金额: $ 33.29万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6838748
• 关键词: Baculoviridae; Escherichia coli; X ray crystallography; acid aminoacid ligase; computer simulation; crystallization; enzyme complex; enzyme mechanism; enzyme model; enzyme substrate complex; growth factor receptors; intermolecular interaction; isozymes; molecular dynamics; molecular site; phosphoproteins; physical model; protein degradation; protein engineering; protein purification; protein structure function; site directed mutagenesis; structural biology; transcription factor; tumor suppressor proteins; ubiquitin

DESCRIPTION (provided by applicant): Ubiquitin-dependent proteolysis regulates diverse biological processes such as the cell cycle, signaling pathways, tumor suppressor and oncogene networks and the immune response. The ubiquitination of a target protein proceeds through a multi-enzyme pathway involving ubiquitin-activating (El), ubiquitin-conjugating (E2) and ubiquitin-protein ligase (E3) activities. The E3 is responsible for target specificity, and represents the primary regulated step in the process. E3s comprise a large superfamily of proteins and protein complexes. RING E3s are the biggest and most diverse class of E3s. They promote ubiquitination in a reaction that is poorly understood. This proposal focuses on the crystallographic and biochemical characterization of four families of RING E3s, of their complexes with substrates and E2s, and of ubiquitination reaction intermediates. These are the 4-protein SGF, the 5-protein VHL/SOCS, the 10-protein APC and the single chain c-CbI proto-oncogene. The SCF E3 consists of an F-box protein, Skp1, Cull and Rbxl, and is by far the largest E3 family known to date, with hundreds of SCF subunits in the databases. Structures of two human SCFs, SCFSKp2 and SCFbTRCP, which ubiquitinate the p27Kipl tumor suppressor and the beta-catenin oncoprotein, respectively, will be determined. A divergent SCF-like E3 is exemplified by the VHL tumor suppressor, which binds ElonginC, ElonginB, Cul2 and Rbx1, and induces the degradation of the hypoxia-inducible transcription factor 1alpha (HIF1), negatively regulating angiogenesis. The structure of the entire VHL complex bound to an HIF1 substrate peptide will be determined. The APC (anaphase promoting complex) E3 has a central role in coordinating mitosis. Two APC subunits, Apc2 and Apc1 1, share limited homology to the Cul1-Rbx1 components of the SCF. The function of the other subunits is not yet known. In the first step, the structure of the Apc2-Apc1 1 catalytic core will be determined. Several additional subunits that interact with the core will then be assembled and their structures determined. Cbl ubiquitinates the activated forms of several receptor tyrosine kinases (RTKs) and terminates signaling. The minimal portion of PDGFR that binds to CbI and gets ubiquitinated will be determined. Building on the Cbl-E2 structure, the structure of the CbI-E2-substrate complex will be determined. These studies will address questions about substrate recognition, mechanism(s) of ubiquitination, specificity in ubiquitination sites and between different E3 subunits, and the architectures of E3s, with implications for the function and regulation of these large and diverse complexes. They will also help address whether E3s, many of which are implicated in diseases such as cancer, are likely targets for drug discovery.

描述（由申请人提供）：泛素依赖性蛋白水解调节多种生物过程，例如细胞周期、信号传导途径、肿瘤 抑制基因和癌基因网络以及免疫反应。的泛素化 靶蛋白通过多酶途径进行，涉及 泛素激活 (E1)、泛素缀合 (E2) 和泛素蛋白 连接酶 (E3) 活性。 E3 负责目标特异性，并且 代表该过程中的主要监管步骤。 E3 包括一个大 蛋白质和蛋白质复合物的超家族。 RING E3 是最大且 最多样化的 E3 类别。它们促进泛素化的反应是 不太了解。该提案重点关注晶体学和 RING E3 四个家族及其复合物的生化特征 与底物和 E2，以及泛素化反应中间体。这些 是 4 蛋白 SGF、5 蛋白 VHL/SOCS、10 蛋白 APC 和 单链 c-CbI 原癌基因。 SCF E3 由 F-box 蛋白组成， Skp1、Cull 和 Rbxl，是迄今为止已知的最大的 E3 家族，其中 数据库中有数百个 SCF 亚基。两种人类 SCF 的结构， SCFSKp2 和 SCFbTRCP，泛素化 p27Kipl 肿瘤抑制因子和 将分别测定β-连环蛋白癌蛋白。一个发散的 SCF 样 E3 的例子是 VHL 肿瘤抑制因子，它与 ElonginC 结合， ElonginB、Cul2 和 Rbx1，并诱导缺氧诱导蛋白的降解 转录因子 1α (HIF1)，负调节血管生成。这 与 HIF1 底物肽结合的整个 VHL 复合物的结构将是 决定。 APC（后期促进复合物）E3 在 协调有丝分裂。两个 APC 亚基 Apc2 和 Apc1 1 具有有限的同源性 到 SCF 的 Cul1-Rbx1 组件。其他亚基的功能是 尚不清楚。第一步，Apc2-Apc1 1催化的结构 核心将被确定。几个额外的亚基与相互作用 然后将组装核心并确定其结构。 Cbl 泛素化 几种受体酪氨酸激酶 (RTK) 的激活形式并终止 发信号。 PDGFR 与 CbI 结合并获得的最小部分 泛素化将被确定。在 Cbl-E2 结构的基础上， CbI-E2-底物复合物的结构将被确定。这些研究 将解决有关底物识别、机制的问题 泛素化、泛素化位点和不同 E3 之间的特异性 子单元和 E3 的架构，以及对功能和功能的影响 对这些大型且多样化的综合体的监管。他们还将帮助解决 E3s（其中许多与癌症等疾病有关）是否是 药物发现的可能目标。