The Mitochondrial Protein Card19 Regulates Membrane Integrity And Terminal Lysis Downstream Of Caspase Activation And Gasdermin Cleavage

During pyroptosis, inflammatory caspases cleave gasdermin D to release an N-terminal fragment that generates plasma membrane pores, which in turn mediate cell lysis and IL-1 release. However, certain stimuli or cell types release IL-1 cytokines in a gasdermin D-dependent manner while remaining viable, a process termed hyperactivation. How these distinct cell fate choices are regulated downstream of caspase activation is unknown. Here, we demonstrate that a mitochondrial CARD-containing protein, CARD19, promotes the progression to terminal lysis following initial pore formation downstream of caspase activation and gasdermin cleavage. CARD19-deficient macrophages were protected from cell death, but had no discernable defects in caspase activation, IL-1 secretion, or gasdermin cleavage. Differential expression of CARD19 was linked to differential cell death in populations of peritoneal macrophages. Mechanistically, Card19-/- macrophages exhibited a subtle reduction in cleaved gasdermins present in light membrane fractions. Our data also indiate that full-length gasdermin D and E partially localize in close proximity to the cortical actin network and cell periphery in transfected cells. These preliminary findings provide a potential alternative mechanism by which gasdermins are regulated, in which full-length gasdermin D and E might be associated with the plasma membrane and poised for pore formation. These results implicate CARD19 as a cellular factor that positively regulates cell death by potentially promoting association of cleaved gasdermin proteins with cellular membranes and provide insight into regulation of terminal steps of cell death following caspase activation.