Mechanisms of Gap Junction Phosphodegradation

缝隙连接(GJS)是一种细胞间通道，允许相邻细胞之间交换低分子(1 KDa)离子、分子和二级信使。GJS允许的细胞间通讯对组织内稳态至关重要，因为它为代谢和电同步提供了最直接的手段。半衰期短，并显示出与维持连接蛋白水平的复杂网络一致的分泌途径内的运输模式，包括合成、运输、组装、门控和质膜内化。由连接蛋白43(Cx43)组成的缝隙连接经历了高速率的产生和降解，以至于人体内的缝隙连接每天(半衰期1-5.5小时)被替换。众所周知，细胞间通讯可以调节一系列不同的细胞和生理过程。构成缝隙连接的结构似乎非常复杂，有大量蛋白质支持组装和周转。尽管进行了大量的努力，但人们对控制缝隙结退化的基本机制知之甚少。我们以前已经鉴定了缝隙连接的Cx43的蛋白质组，并为与E3泛素连接酶TRIM21相关的磷酸化诱导的降解(磷酸化降解)提供了证据。这项应用中提出的研究将揭示这些结构在响应表皮生长因子时翻转的精确分子机制，表皮生长因子是缝隙连接通讯损失的已知调节因子。Cx43翻译后修饰、蛋白质-蛋白质相互作用的蛋白质组学特征将成为本研究的核心内容。*我们认为Cx43/TRIM21参与了缝隙连接内化和细胞间通讯的丧失。我们的计划是：1)确定TRIM21与Cx43相互作用的功能结构域；2)对Cx43/TRIM21复合体进行深入的磷酸蛋白质组分析。这项研究将揭示导致缝隙连接损耗的信号通路和时间事件。我们希望我们的工作将揭示管理缝隙连接细胞间通讯丢失的新概念，这将为疾病中通讯缝隙连接如何丢失提供关键的见解。在分子分辨率上，这项研究将揭示细胞间通讯丧失背后的时间事件。由NSERC资助的学生将获得细胞蛋白质组学、层析和质谱学方面的专家技能。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。*

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.*****