Pannexin Interactome: from Discovery to Functional Screening

Pannexin Interactome：从发现到功能筛选

• 批准号: RGPIN-2021-03801
• 负责人: Cowan, Kyle
• 金额: $ 2.33万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742721
• 关键词: Pannexin; Interactome; Discovery; Functional; Screening

Pannexin 1 (Panx1) forms single membrane channels that are widely expressed throughout the body. As such, Panx1 channels have been implicated in a variety of cellular processes through its ability to release ATP or other nucleotides, highlighting the importance of its localization at the cell surface for proper function. However, while progress has been made in identifying and understanding Panx1 channel functions, little is known with regards to the regulation of Panx1 channel trafficking and the signaling pathways that link Panx1 channels to their downstream functions. As protein-protein interactions regulate numerous processes that occur within a cell, identifying the proteins that interact with Panx1 is essential to understand Panx1 regulation and function.      My goal is to develop an unbiased platform to identify pannexin (family of 3 members: Panx1, Panx2, and Panx3) interactomes in cell-specific contexts and determine the functional relevance of these interactions. In order to develop our `interactome-to-function' high-throughput platform, we will be using keratinocytes as a cell model based on my previous work and experience with these cells. Moreover, the importance of Panx1 in regulating the cellular properties of keratinocytes has been demonstrated using Panx1 knockout mice. The Panx1 interactome will be discovered through the combination of two complementary approaches: BioID and co-immunoprecipitation performed using Panx1-enriched membranes. The Panx1 interactors will be identified by mass spectrometry and the interactions validated using skin from female and male mice. Genome-wide high-throughput functional screens using RNA interference technology will then reveal, in an unbiased manner, which interactions regulate Panx1 localization at the cell surface and its inhibitory effect on keratinocyte proliferation. Functional relevance of these interactions will be further validated using a CRISPR-Cas9 approach and off-target effects will be ruled-out by genetic rescue experiments.      In addition to bringing important insights into the molecular mechanisms that regulate Panx1 and its functions in keratinocytes, this work will constitute the proof-of-principle for the use of an unbiased genome-wide `interactome-to-function' high-throughput platform to identify the functional relevance of pannexin interactors. My long-term vision is to expand this research program into a powerful, comprehensive, and versatile platform to discover novel components of the signaling pathways that regulate Panx1, Panx2, and Panx3 expression, localization, channel activity, and function in relevant cellular contexts.

Pannexin 1 (Panx1) 形成单膜通道，在全身广泛表达。因此，Panx1 通道通过其释放 ATP 或其他核苷酸的能力参与多种细胞过程，凸显了其定位在细胞表面对于正常功能的重要性。然而，虽然在识别和理解 Panx1 通道功能方面取得了进展，但人们对 Panx1 通道运输的调节以及将 Panx1 通道与其下游功能连接的信号通路知之甚少。由于蛋白质-蛋白质相互作用调节细胞内发生的许多过程，因此识别与 Panx1 相互作用的蛋白质对于了解 Panx1 调节和功能至关重要。      我的目标是开发一个公正的平台来识别细胞特异性环境中的 pannexin（由 3 个成员组成的家族：Panx1、Panx2 和 Panx3）相互作用组，并确定这些相互作用的功能相关性。为了开发我们的“相互作用组到功能”高通量平台，我们将根据我之前的工作和这些细胞的经验，使用角质形成细胞作为细胞模型。此外，Panx1 敲除小鼠已证明 Panx1 在调节角质形成细胞的细胞特性中的重要性。 Panx1 相互作用组将通过两种互补方法的结合来发现：BioID 和使用富含 Panx1 的膜进行的免疫共沉淀。 Panx1 相互作用子将通过质谱法进行鉴定，并使用雌性和雄性小鼠的皮肤验证相互作用。使用 RNA 干扰技术的全基因组高通量功能筛选将以公正的方式揭示哪些相互作用调节 Panx1 在细胞表面的定位及其对角质形成细胞增殖的抑制作用。这些相互作用的功能相关性将使用 CRISPR-Cas9 方法进一步验证，并且脱靶效应将通过基因拯救实验排除。      除了对调节 Panx1 及其在角质形成细胞中的功能的分子机制带来重要见解外，这项工作还将构成使用无偏见的全基因组“相互作用组到功能”高通量平台来识别 pannexin 相互作用子的功能相关性的原理验证。我的长期愿景是将这一研究项目扩展为一个强大、全面和多功能的平台，以发现在相关细胞环境中调节 Panx1、Panx2 和 Panx3 表达、定位、通道活性和功能的信号通路的新组件。