Di-ubiquitin modification of ubiquitin ligase adaptors in membrane protein downregulation

泛素连接酶接头的双泛素修饰在膜蛋白下调中的作用

• 批准号: 10521677
• 负责人: SCOTT D EMR
• 金额: $ 41.07万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10521677
• 关键词: Adaptor Signaling Protein; Amino Acids; Architecture; Arrestins; Autophagocytosis; Binding; Binding Sites; Biological Assay; C-terminal; C2 Domain; Cell Cycle; Cell membrane; Cell physiology; Cells; Collaborations; Complex; Cryoelectron Microscopy; Crystallography; DNA Repair; Data; Defect; Distal; Down-Regulation; Endocytosis; Enzymes; Eukaryota; Evolution; Family; Family member; Fostering; Genetic Transcription; Goals; Immune response; In Vitro; Ligase; Light; Link; Lysine; Maintenance; Malignant Neoplasms; Mammals; Mediating; Membrane; Membrane Proteins; Methionine; Modeling; Modification; Molecular; N-terminal; Nature; Neurodegenerative Disorders; PHEMX gene; Physiological; Play; Post-Translational Protein Processing; Process; Proteins; Quality Control; Research; Role; Signal Transduction; Site; Specificity; System; Testing; Ubiquitin; Ubiquitin family; Ubiquitin-Activating Enzymes; Ubiquitination; Yeasts; base; human disease; in vivo; interest; member; protein degradation; protein function; proteostasis; recruit; therapeutic development; therapeutic target; trafficking; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY/ABSTRACT Post-translational modification by ubiquitin is an essential mechanism to alter protein function in eukaryotes. Ubiquitin, a 76 amino acid protein, is attached to specific proteins via a cascade of ubiquitin activating enzyme E1, conjugating enzyme E2, and ubiquitin ligase E3. Ubiquitination plays an essential role in a broad aspect of cellular processes, including transcription, DNA repair, signal transduction, autophagy, cell cycle, immune response, and membrane trafficking. Aberration in the ubiquitination system leads to a number of human diseases, such as neurodegenerative diseases and cancers. Within the ubiquitination cascade, E3s primarily dictate the specificity of the ubiquitination system and thus are often the focal points of research and attractive therapeutic targets. The Nedd4 family E3s are an essential family of HECT-type E3s, members of which contain an N-terminal C2 domain followed by 2-4 WW domains and the C-terminal HECT domain. Nedd4 E3s recognize substrates carrying a “PPxY” motif through their WW domains. However, most substrates lack such a motif but engage with the ligase through “PPxY” motif-containing adaptors. We recently discovered that in yeast, the ubiquitin E3 ligase adaptor protein Art1 is primed with di-ubiquitination and the attachment of the di-Ub chain to a specific lysine residue in Art1 is warranted for its full activity. In this proposal, we plan to investigate the physiological function of adaptor di-ubiquitination and to elucidate the molecular mechanisms of the modular ubiquitination platform form with Nedd4 E3 ligase and di-ubiquitinated adaptors. Specifically, we will pursue the following aims: Aim 1: To investigate the mechanism of Nedd4 E3 adaptor di-ubiquitination. Aim 2: To determine the role of di-ubiquitinated adaptor-E3 complexes in substrate ubiquitination. Aim 3: To elucidate the molecular architecture of di-ubiquitinated adaptors with the Nedd4 E3 ligases. Uncovering the physiological role of Need4 E3 adaptors di-ubiquitination will be of critical importance to understand the molecular basis of how E3 adaptors specifically recognize substrate proteins and efficiently present the substrates for ubiquitination by HECT E3 ligases. We expect the successful implementation of this proposal will not only make significant contributions to the understanding of the molecular mechanisms underlying the Nedd4 E3 ligase/adaptor mediated ubiquitination, but also shed light on the mechanism of protein quality control governed by targeted ubiquitination.

项目概要/摘要 泛素翻译后修饰是改变蛋白质功能的重要机制 泛素是一种由 76 个氨基酸组成的蛋白质，通过泛素级联连接到特定的蛋白质上。 激活酶 E1、结合酶 E2 和泛素连接酶 E3 起着至关重要的作用。 在细胞过程的广泛方面发挥作用，包括转录、DNA 修复、信号转导、 自噬、细胞周期、免疫反应和泛素化中的膜运输畸变。 系统导致许多人类疾病，例如神经退行性疾病和癌症。 在泛素化级联中，E3 主要决定泛素化系统的特异性，因此 Nedd4 家族 E3 通常是研究的焦点和有吸引力的治疗靶点。 HECT 型 E3 家族，其成员包含 N 端 C2 结构域，后跟 2-4 个 WW 结构域和 C 端 HECT 结构域识别携带“PPxY”基序的底物。 然而，大多数底​​物缺乏这样的基序，但通过其与连接酶结合。 我们最近在酵母中发现了含有“PPxY”基序的接头。 蛋白质 Art1 经双泛素化处理并将二 Ub 链附着到特定的赖氨酸上 Art1 中的残留物保证了其全部活性。在本提案中，我们计划研究其生理学。 接头双泛素化的功能并阐明模块化的分子机制 具体来说，我们将使用 Nedd4 E3 连接酶和双泛素化适配器形成泛素化平台。 追求以下目标： 目标1：研究Nedd4 E3接头双泛素化机制。 目标 2：确定双泛素化接头-E3 复合物在底物泛素化中的作用。 使用 Nedd4 E3 连接酶阐明双泛素化接头的分子结构。 Need4 E3 接头双泛素化的生理作用对于理解至关重要 E3 接头如何特异性识别底物蛋白并有效呈现的分子基础 HECT E3 连接酶泛素化的底物我们预计该项目能够成功实施。 该提案不仅将对分子机制的理解做出重大贡献 Nedd4 E3 连接酶/接头介导的泛素化的基础，同时也阐明了 由靶向泛素化控制的蛋白质质量控​​制。