Mechanisms of Gap Junction Phosphodegradation

间隙连接磷酸降解机制

• 批准号: RGPIN-2018-04261
• 负责人: Chen, Vincent
• 金额: $ 2.7万
• 依托单位: Brandon University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=747820
• 关键词: Mechanisms; Gap; Junction; Phosphodegradation

Gap junctions (GJs) are intercellular channels that allow for the exchange of low molecular weight (1kDa) ions, molecules and secondary messengers between adjacent cells. Intercellular communication permitted by GJs is critical for tissue homeostasis as it provides the most direct means for metabolic and electrical synchronization. Short half-life and demonstrated trafficking patterns within the secretory pathway consistent with complex network to maintain connexin levels, including synthesis, transport, assembly, gating and internalization from the plasma membrane. Gap junctions comprised of connexin43 (Cx43) undergo high rates of production and degradation, such that gap junctions within the human body are replaced daily (half-life 1-5.5 hr). Intercellular communication is know to regulate a diverse set of cellular and physiological processes. The structures making up gap junctions appear to be incredibly complex, with a large number of proteins supporting the assembly and turnover. Despite significant efforts, very little is known about the basic mechanisms that governing gap junction degradation. We have previously characterize the proteome of Cx43 of gap junction and provided evidence for phosphorylation-induced degradation (phosphodegron) associated with the E3 ubiquitin ligase TRIM21. The research proposed in this application will reveal the precise molecular mechanisms the turnover of these structures in responses to epidermal growth factor, a know regulator of gap junctional communication loss. The proteomic characterization of Cx43 post-translational modification, protein-protein interactions will form the core focus of this program of study.We propose Cx43/TRIM21 is involved with gap junction internalization and loss of intercellular communication. Our plan is to: 1) identify the functional domains of TRIM21 that are responsible for interaction with Cx43; 2) conduct the in depth analysis phosphoproteomic analysis Cx43/TRIM21 complexes. The research proposed will reveal the signalling pathways and the temporal events responsible for gap junction loss. We expect that our work will reveal new concepts governing the loss of gap junction intercellular communication that will provide critical insights into how communicating gap junctions are lost in disease. At molecular resolution, the research proposed will reveal the temporal events underlying the loss of intercellular communication. Students supported by this NSERC grant will obtain expert skills in cellular proteomics, chromatography and mass spectrometry. The training of highly qualified personnel with such skills after currently in demand within multiple areas of diagnostic testing and life science research.

间隙连接（GJ）是允许相邻细胞之间交换低分子量（1 kDa）离子、分子和第二信使的细胞间通道。GJ所允许的细胞间通信对于组织稳态是至关重要的，因为它为代谢和电同步提供了最直接的手段。半衰期短，分泌途径内的运输模式与维持连接蛋白水平的复杂网络一致，包括合成、运输、组装、门控和质膜内化。 由连接蛋白43（Cx43）组成的间隙连接经历高速率的产生和降解，使得人体内的间隙连接每天被替换（半衰期1-5.5小时）。细胞间通讯被认为是调节一系列不同的细胞和生理过程。构成缝隙连接的结构看起来非常复杂，有大量的蛋白质支持组装和周转。尽管付出了巨大的努力，但人们对间隙连接退化的基本机制知之甚少。我们以前的特点缝隙连接的蛋白质组，并提供证据磷酸化诱导的降解（磷酸降解决定子）与E3泛素连接酶TRIM 21。本申请中提出的研究将揭示这些结构响应表皮生长因子（一种已知的间隙连接通讯丢失调节剂）的周转的精确分子机制。Cx43的翻译后修饰、蛋白质间相互作用的蛋白质组学研究将成为本研究的核心内容，我们提出Cx43/TRIM 21可能参与缝隙连接的内化和细胞间通讯的丧失。我们的计划是：1）鉴定TRIM 21的负责与Cx43相互作用的功能结构域; 2）进行深入分析磷酸蛋白质组学分析Cx43/TRIM 21复合物。这项研究将揭示信号通路和负责间隙连接损失的时间事件。我们希望我们的工作将揭示新的概念，管理的间隙连接细胞间通讯的损失，将提供关键的见解，如何沟通间隙连接在疾病中丢失。 在分子分辨率下，这项研究将揭示细胞间通讯丢失的时间事件。由NSERC资助的学生将获得细胞蛋白质组学，色谱和质谱方面的专业技能。在诊断测试和生命科学研究的多个领域中，目前需要培训具有此类技能的高素质人员。