Molecular and Chemical Regulation of Cell Death in Disease

Abstract My research program aims to understand the molecular mechanism of cell death and develop small molecular inhibitors to treat diseases associated with dysregulated cell death, including systemic inflammatory response syndrome (SIRS), infection, and heart failure. Apoptosis and necroptosis are essential pathways of cell death with distinct biochemical mechanisms, pivotal in development and disease. Apoptosis involves caspase activation, while necroptosis triggers the assembly of the necrosome, containing receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) and the mixed lineage kinase-like protein MLKL. Despite advancements, questions persist regarding RIPK1 activation, necroptosis execution, and the contribution of necroptosis to heart failure. Through a sensitized CRISPR whole-genome knockout screen, we have discovered the essential role of protein phosphatase 1 regulatory subunit 3G (PPP1R3G) in cell death. PPP1R3G partners with PP1γ to dephosphorylate RIPK1, activating both apoptosis and necroptosis. Ppp1r3g-/- mice exhibit protection against TNF-induced SIRS, affirming its in vivo importance. However, little is known about PPP1R3G’s involvement in cardiac cell death and injury. Project 1 aims to elucidate the role of PPP1R3G in myocardial infarction (MI) and doxorubicin (DOX)-induced cardiac toxicity, enhancing our understanding of cardiomyocyte death and its therapeutic implications. Furthermore, we have developed a novel peptidomimetic that disrupts the interaction between PPP1R3G and PP1γ, blocking cell death in vitro and protecting against TNF-induced SIRS. In Project 2, we will further investigate its mechanism of inhibition and explore its efficacy in mouse models of MI and DOX-induced cardiotoxicity. Project 3 focuses on unraveling necroptosis execution mechanisms. While MLKL is traditionally believed to oligomerize on the plasma membrane, our recent findings suggest that significant portions of MLKL polymerizes on the lysosomal membrane, inducing lysosomal membrane permeabilization (LMP) and cell death. Through a CRISPR screen, we identified a novel membrane protein, that we named MADMAN for MLKL-associated membrane activator of necroptosis, as a crucial protein recruiting MLKL to the lysosomal membrane. We'll investigate MADMAN's role in necroptosis and its in vivo function in TNF-induced SIRS and MI. Our proposal employs forward and reverse genetics, biochemistry, and structure-based drug design to examine the mechanisms of cell death systematically and rigorously. The outcomes of our studies will provide innovative molecular insights into inflammation and cell death pathways and uncover potential drug candidates, paving the way for the development of novel therapeutic strategies for human diseases linked to cell death dysregulation.