MEX3B as a Potent Regulator of Inflammasome Signaling 2267250

Abstract Introduction Inflammasomes are crucial for innate immune defense to many microbial infections but are also subjected to stringent cellular regulation to avoid persistent, aberrant activation that could lead to lethal sepsis and autoinflammatory conditions. Ubiquitination is one of the most important posttranslational modifications involved in many immune signaling pathways, including the nucleotide-binding and oligomerization domain (NOD)-like receptor 3 (NLRP3) inflammasome. However, its role in the non-canonical caspase-4 inflammasomes is poorly understood. Methods To address this significant gap, we constructed 375 individual ubiquitin E3 ligase knockout lines by CRISPR-Cas9 and performed an unbiased screening. This library represents almost all the currently known definite E3 ligases (total ∼377). Results Our screen identified 15 positive regulators of the caspase-4 inflammasome; one of the top hits was Muscle Excess 3B (MEX3B), an RNA-binding protein with a ubiquitin ligase domain. We found that deletion of MEX3B inhibited caspase-4 and gasdermin D (GSDMD) activation, pyroptosis, and secretion of inflammasome-dependent inflammatory cytokines in human cell lines and murine primary macrophages upon priming with interferon gamma (IFN-γ) and transfection with lipopolysaccharide (LPS). Notably, MEX3B was also crucial for canonical inflammasome signaling such as NLRP3 and NLRC4. Mechanistically, MEX3B was required for caspase-1 activation but not NLRP3 oligomerization. The role of MEX3B in inflammasome signaling was reliant on its RNA-binding, but not E3 ligase activity. However, the expression of caspase-4 and GSDMD, IFN-γ and Toll-like receptor (TLR4) signaling, and apoptosis remained intact in MEX3B-/- cells. Conclusion Our results suggest that MEX3B is a pan-inflammasome regulator targeting inflammatory caspases. Ongoing work is to investigate both the NLRP3 and caspase-4 inflammasome signaling in Mex3b-/- mice and elucidate the molecular mechanism of action of MEX3B. Funding Source National Institute of Allergy and Infectious Diseases, USA Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)