Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9–CARD8 interaction. We show that the DPP9–CARD8 interaction, unlike the DPP9–NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9’s catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.

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Weak polyampholytes are charged polymers, where the charge asymmetry can be tuned by varying the solution pH. We determine the size of a randomly charged weak polyampholyte in dilute solution as a function of charge asymmetry, Bjerrum length, salt concentration, pH, and degree of polymerization, using a self‐consistent method. It is known that in the limiting cases of low and high charge asymmetries, polyampholytes behave as neutral polyampholytes and polyelectrolytes, respectively. We explore in detail the regime of intermediate charge asymmetry where a polyampholyte show non‐monotonic change in the chain size as a function of Bjerrum length. A hierarchy of structures exists at different length scales, ranging from ideal coils at low Bjerrum length, extended rod‐like state at intermediate Bjerrum length to globular states at high Bjerrum length. The transition between ideal coil and rod‐like states is continuous, while that between rod‐like and globular states is discontinuous. The addition of salt changes the nature of the rod‐to‐globule transition from discontinuous to continuous. The effective free energy shows a double minimum at intermediate charge asymmetry, indicating the coexistence of globules and extended states. The size as a function of the solution pH shows a minimum at the isoelectric point. The size of neutral polyampholytes at the isoelectric pH increases with the increase in the salt concentration. The size of charge excess polyampholytes far away from the isoelectric pH decreases with the increase in the salt concentration. magnified image

Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS.