Radoslaw M. Sobota

Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca(2+) flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock.

The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1β. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases.

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.

Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases.