Structure and Activation Mechanism of Ubiquitin Conjugating Enzymes

泛素结合酶的结构和激活机制

• 批准号: 8116551
• 负责人: VINCENT CHAU
• 金额: $ 30万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8116551
• 关键词: Address; Antineoplastic Agents; Area; Binding; Biochemical; Biological Assay; Chimeric Proteins; Complex; Data Collection; Development; Docking; Enzymes; Goals; Growth; Hand; Human; Individual; Kinetics; Knowledge; Laboratories; Lead; Link; Lymphoma; Maps; Measurement; Mediating; Methods; Modeling; Multiple Myeloma; Nature; Pathway interactions; Polyubiquitin; Process; Protein Engineering; Protein-Protein Interaction Map; Proteins; Reaction; Research; Resolution; Solutions; Staging; Structure; Structure-Activity Relationship; Surface; Testing; Ubiquitin; Ubiquitin-Conjugating Enzymes; Velcade; Yeasts; base; data structure; drug development; genome wide association study; improved; insight; multicatalytic endopeptidase complex; mutant; novel; protein protein interaction; protein structure; public health relevance; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The assembly of polyUb chains on substrate proteins requires the concerted action of a ubiquitin-conjugating enzyme E2 and a partner ubiquitin-protein ligase E3. The RING-E3 gp78 protein is known to function with Ubc7 in ER-mediated degradation. We have shown with unanchored K48-specific polyUb chain synthesis that the RING domain from gp78 is a potent activator of this activity in Ubc7. We have defined the RING:E2 binding interface of this interacting pair by solution NMR measurements and by determination of a 2.2 A resolution crystal structure of an engineered protein construct in which the gp78 RING is fused to the N-terminus of Ubc7. These studies reveal details of interaction at the interface. We have on hand a panel of RING domains that are activators of either Ubc7, E2-25K, or both. In addition to activation function, these RING domains can be segregated into four major structural subtypes. With these preliminary results, we proposed to achieve a detailed understanding of RING-mediated E2 activation with the following specific aims: I. To establish the nature of the E2:RING domain interface formed with structural subtypes of the RING domain fold. II. To determine how individual pair-wise interactions in a RING:E2 interface contribute to selective recognition and E2 activation. III. To map protein:protein interactions in E2~Ub complexes. Public Health Relevance: The proposed research will lead to novel insights on how proteins in the ubiquitin pathway interact with each other. This pathway has been successfully exploited in the development of Velcade, an anti-cancer drug to treat myeloma and lymphoma. We expect knowledge to be gained will further improve the accessibility of this pathway for drug development.

描述（由申请人提供）：多聚泛素链在底物蛋白上的组装需要泛素缀合酶 E2 和伴侣泛素蛋白连接酶 E3 的协同作用。已知 RING-E3 gp78 蛋白与 Ubc7 一起参与 ER 介导的降解。我们通过非锚定的 K48 特异性多聚泛素链合成表明，来自 gp78 的 RING 结构域是 Ubc7 中这种活性的有效激活剂。我们通过溶液 NMR 测量和确定工程蛋白构建体的 2.2 A 分辨率晶体结构，定义了该相互作用对的 RING:E2 结合界面，其中 gp78 RING 融合到 Ubc7 的 N 末端。这些研究揭示了界面交互的细节。我们手头有一组 RING 域，它们是 Ubc7、E2-25K 或两者的激活剂。除了激活功能之外，这些 RING 结构域还可分为四种主要结构亚型。有了这些初步结果，我们建议详细了解 RING 介导的 E2 激活，具体目标如下： I. 确定由 RING 结构域折叠的结构亚型形成的 E2:RING 结构域界面的性质。二.确定 RING:E2 界面中的各个成对相互作用如何有助于选择性识别和 E2 激活。三．绘制 E2~Ub 复合物中蛋白质：蛋白质相互作用的图谱。公共健康相关性：拟议的研究将对泛素通路中的蛋白质如何相互作用产生新的见解。该途径已成功用于开发治疗骨髓瘤和淋巴瘤的抗癌药物 Velcade。我们期望获得的知识将进一步提高药物开发途径的可及性。