Saturday, September 7, 2024
9/7/2024
PROJECT ABSTRACT Cell death pathways play a central role in several biological processes—from programmed cell death during the early stages of development to destroying infected cells that serve as niches for pathogens, such as bacteria. Thus, proper cell death is critical to multiple biological processes and diseases and must be precisely regulated and coordinated. The processes that regulate cell death pathways are not well defined, and this gap in knowledge limits the ability to design targeted treatment and prevention strategies for diseases that result when cell death is dysregulated. A critical regulatory point for several cell death pathways is receptor-interacting serine/threonine-protein kinase 1 (RIPK1), which serves as a pivotal decision point for several cell death and inflammatory pathways. Our lab has performed foundational studies on RIPK1, identifying this protein as a regulator of caspase-8-dependent gasdermin D activation and inflammasome-mediated responses following exposure to the bacteria Yersinia. Using a genome-wide CRISPR/Cas9 screen in Yersinia-infected primary macrophages, we identified putative regulators of RIPK1/caspase-8-induced cytotoxicity, including the splicing factors Raver1 and Ptbp1, which were among the top five hits. These identified splicing factors are potential regulators of RIPK1, and elucidating their mechanism of action will be an important advance in understanding cell death and inflammation. We generated Raver1-/- mice and cell lines, and our studies indicate that Raver1 and Ptbp1 promote canonical RNA splicing of the RIPK1 kinase domain. We hypothesize that Raver1 and Ptbp1 control the splicing of RIPK1 and determine the potency of cell death and inflammation signals. In Aim 1, we will determine the roles of Raver1 and Ptbp1 on RIPK1/caspase-8 mediated cytotoxicity and inflammation by examining macrophages and other cells deficient in Raver1 or Ptbp1. In Aim 2, we will define how Raver1, Ptbp1, and other factors influence RIPK1 by examining how these factors control the splicing, expression and nonsense-mediated decay of RIPK1. In Aim 3, we will assess the impact of Raver1 splicing on cytotoxicity and inflammation in vivo to test the hypothesis that the absence of Raver1 profoundly alters host resistance to infection and inflammation using our Raver1-/- mouse line. This proposal will address an essential gap in knowledge by elucidating how a central node in multiple cell death pathways is regulated. The completion of these aims will establish novel roles of Raver1 and Ptbp1 in regulating key inflammatory and cytotoxic responses and enable novel targeted strategies to prevent and treat diseases that result from dysregulation in cell death pathways.
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