Pannexin Interactome: from Discovery to Functional Screening

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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.