Mechanisms of Gap Junction Phosphodegradation

间隙连接（GJs）是细胞间通道，允许相邻细胞之间交换低分子量（1kDa）离子、分子和次级信使。GJs允许的细胞间通讯对组织稳态至关重要，因为它为代谢和电同步提供了最直接的手段。半衰期短，分泌通路内的运输模式与维持连接蛋白水平的复杂网络一致，包括质膜的合成、运输、组装、门控和内化。由connexin43 （Cx43）组成的间隙连接经历高速率的产生和降解，因此人体内的间隙连接每天都会被替换（半衰期1-5.5小时）。众所周知，细胞间通讯调节着一系列不同的细胞和生理过程。构成间隙连接的结构似乎非常复杂，有大量的蛋白质支持其组装和周转。尽管做出了巨大的努力，但人们对控制间隙结退化的基本机制知之甚少。我们之前已经表征了间隙连接Cx43的蛋白质组，并提供了与E3泛素连接酶TRIM21相关的磷酸化诱导降解（phosphodegron）的证据。本应用程序的研究将揭示这些结构在响应表皮生长因子时的精确分子机制，表皮生长因子是间隙连接通信丢失的已知调节剂。Cx43翻译后修饰、蛋白-蛋白相互作用的蛋白质组学表征将成为本项目研究的核心重点。

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.