PROJECT SUMMARY Several human pattern-recognition receptors detect intracellular danger-associated signals, oligomerize into multiprotein complexes called inflammasomes, and trigger a lytic form of cell death called pyroptosis. Inflammasomes are involved in mounting immune responses to pathogens and in maintaining organismal homeostasis, but their hyperactivation can cause cancer, autoimmune disorders, and metabolic dysfunction. As such, it is critically important to characterize the molecular mechanisms that regulate inflammasome activation. NLRP1 and CARD8 are related pattern-recognition receptors that form inflammasomes, but the danger signals that they sense have not been fully established. Notably, ligands that bind to the serine dipeptidyl peptidases 8 and 9 (DPP8/9), including endogenous peptides with Xaa-Pro (where Xaa is any amino acid) N-termini, have been reported to activate these inflammasomes. However, why the innate immune system monitors Xaa-Pro peptide levels is unknown and constitutes a major knowledge gap. Recently, reductive stress, or a profound lack of reactive oxygen species (ROS), was also reported to activate the NLPR1 and CARD8 inflammasomes. The central hypothesis of this application is that reductive stress and Xaa-Pro peptide accumulation are intimately related danger signals that together comprise an overall “danger state” that causes rapid and full NLRP1 and CARD8 inflammasome activation. Specifically, it is proposed that the disordered regions of many cytosolic proteins, including the autoinhibitory N-terminal region of CARD8, are stabilized by intramolecular disulfide bonds; reductive stress abolishes these bonds, destabilizing these sequences and triggering their degradation into peptides by the proteasome. Proline is the most abundant amino acid in disordered protein regions, and therefore reductive stress likely generates many Xaa-Pro peptides. In this way, Xaa-Pro peptide accumulation can serve to confirm that reductive stress is occurring. This central hypothesis has been formulated based on preliminary data produced in the applicant’s laboratory and described in this application. The objective of this project is to determine the relationship between reductive stress, disordered protein degradation, and Xaa-Pro peptide accumulation. This project consists of three Specific Aims: 1) to determine how reductive stress induces the proteasome-mediated degradation of CARD8, 2) to characterize the relationship between reductive stress and Xaa-Pro peptide accumulation, and 3) to determine the relationship between cell metabolism and inflammasome activation. The successful completion of this work will not only clarify the primordial function of these enigmatic inflammasomes, but will also reveal a previously unknown a connection between intracellular redox state and protein stability. Moreover, this work will provide the foundation for future efforts to therapeutically control these inflammasomes for the treatment of h...