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

项目总结/摘要 泛素的翻译后修饰是改变蛋白质功能的重要机制， 真核生物泛蛋白是一种由76个氨基酸组成的蛋白质，通过泛蛋白级联反应附着在特定蛋白质上 活化酶E1、缀合酶E2和泛素连接酶E3。泛在化是一个至关重要的 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、免疫应答和膜运输。泛素化的畸变 系统导致许多人类疾病，如神经退行性疾病和癌症。内 在遍在蛋白化级联中，E3主要决定遍在蛋白化系统的特异性，因此是 通常是研究的焦点和有吸引力的治疗靶点。Nedd 4系列E3是 HECT型E3家族，其成员含有一个N-末端C2结构域，后接2-4个WW 结构域和C-末端HECT结构域。Nedd 4 E3识别携带“PPxY”基序的底物 通过他们的WW域。然而，大多数底物缺乏这样的基序，但通过连接酶与连接酶接合。 含有“PPxY”基序的衔接子。我们最近发现，在酵母中，泛素E3连接酶接头 蛋白质Art 1被双泛素化和双泛素链与特定赖氨酸的连接引发 第1条中的残留物保证其充分活性。在这项提案中，我们计划研究生理学 衔接子双泛素化的功能，并阐明模块化的分子机制， 泛素化平台与Nedd 4 E3连接酶和双泛素化衔接子形成。具体来说，我们将 目的1：探讨Nedd 4 E3衔接子双泛素化的机制。 目的2：研究双泛素化衔接子-E3复合物在底物泛素化中的作用。目标3： 阐明具有Nedd 4 E3连接酶的双泛素化衔接子的分子结构。揭开 Need 4 E3衔接子双泛素化的生理作用对于理解Need 4 E3衔接子双泛素化的生理作用将是至关重要的 E3衔接子如何特异性识别底物蛋白并有效呈递的分子基础 HECT E3连接酶泛素化的底物。我们期待着成功地实施这一计划。 这一建议不仅将对理解分子机制做出重大贡献， 潜在的Nedd 4 E3连接酶/接头介导的泛素化，但也阐明了机制， 蛋白质质量控制由靶向泛素化控制。

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.