Friday, April 26, 2024
4/26/2024
Project summary Necroptosis is a recently discovered immunogenic necrotic cell death pathway. Distinct from apoptosis, it is characterized by cellular swelling, membrane rupture and release of damaged-associated molecular patterns (DAMPs). It is implicated in an array of human diseases, including infection, inflammation, tissue injury, cancer and neurodegeneration. Upstream signals, including cytokines such as TNF, viral infection or bacterial infection converge on receptor-interacting kinase 3 (RIPK3) and its substrate mixed lineage kinase-like protein MLKL. Phosphorylation of MLKL by RIPK3 drives MLKL polymerization and membrane translocation, leading to plasma membrane disruption. However, how MLKL membrane translocation is regulated and how membrane rupture is achieved are still under debate. The overall goal of this proposal is to define the role of a novel membrane protein MADMAN in necroptosis during development and disease state. Using a whole-genome CRISPR-Cas9 knockout screen, we identified a novel protein that is required for necroptosis induced by dimerization of N-terminal domain (NTD) of MLKL. We named this protein MADMAN for MLKL-associated membrane activator of necroptosis. MADMAN contains two putative transmembrane domains and forms disulfide bond-linked oligomers on the lysosomal membrane. Our preliminary results demonstrate that MADMAN interacts with MLKL upon necroptosis induction and recruits MLKL to the lysosomal membrane, leading to lysosomal membrane permeabilization (LMP). Furthermore, overexpression of MADMAN is sufficient to induce MLKL-dependent necroptosis. Importantly, Madman-/- mice are resistant to TNF-induced systemic inflammatory response syndrome (SIRS), confirming its essential function in necroptosis in vivo. In this proposal, we want to define the mechanism by which MADMAN regulates MLKL activation to activate necroptosis. Specifically, we will characterize membrane localization of MADMAN and define how MADMAN and MLKL interaction regulates MLKL localization. We will also investigate how MADMAN promotes MLKL polymerization which leads to LMP. Furthermore, we will examine how Madman-/- mice respond to TNF-induced systemic inflammatory response syndrome and if loss of Madman rescues embryonic lethality caused by Caspase 8 deficiency. Lastly, we will investigate the role of LMP and lysosomal proteases in necroptosis execution. Answering these questions will lead us to a better understanding of how necroptosis is executed at the molecular level and provide new therapeutic strategies for diseases associated with hyperactivation of necroptosis. Our proposal uses a combination of forward genetics, reverse genetics, biochemical and cell biology approaches to decipher the role of a novel membrane protein MADMAN in necroptosis. It will answer very important questions regarding MLKL membrane translocation, polymerization and membrane disruption. These studies will provide novel molecular insights into the necroptosis pathway, which may lead to new therapeutic strategies for treating necroptosis-associated inflammatory and infectious disease.
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