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

项目摘要/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。泛素是一种由76个氨基酸组成的蛋白质，它通过一系列泛素与特定的蛋白质结合 激活酶E1、结合酶E2和泛素连接酶E3。泛素化起着至关重要的作用 在广泛的细胞过程中发挥作用，包括转录，DNA修复，信号转导， 自噬、细胞周期、免疫反应和膜运输。泛素化过程中的像差 系统会导致许多人类疾病，如神经退行性疾病和癌症。在 泛素化级联，E3主要决定泛素化系统的特异性，因此 往往是研究的焦点和吸引人的治疗靶点。Nedd4系列E3是必不可少的 Hect型E3家族，其成员包含N-末端C2结构域，后跟2-4个WW 结构域和C-末端的Hect结构域。Nedd4 E3识别带有“PPxY”基序的底物 通过他们的WW域名。然而，大多数底物缺乏这样的基序，但通过与连接酶结合 含有“PPxY”基序的适配器。我们最近发现，在酵母中，泛素E3连接酶适配器 蛋白质Art1被二泛素化和二-Ub链连接到特定的赖氨酸所启动 Art1中的残留物是其充分活性的保证。在这项提案中，我们计划研究生理上的 接头二泛素化的功能及模块作用的分子机制 具有Nedd4 E3连接酶和二泛素化接头的泛素化平台形式。具体来说，我们将 追求以下目标：目的1：研究Nedd4 E3适配器二泛素化的机制。 目的2：确定二泛素化适配器-E3复合体在底物泛素化中的作用。目标3： 目的：阐明Nedd4 E3连接酶二泛素化接头的分子结构。揭开面纱 了解Need4 E3适配器二泛素化的生理作用将是至关重要的 E3接头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。我们期待着这一目标的成功实施。 这一提议不仅将对理解分子机制做出重大贡献 Nedd4 E3连接酶/接头介导的泛素化，但也揭示了 靶向泛素化管理的蛋白质质量控制。

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.