Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

The non-canonical caspase-4 inflammasome is a crucial anti-bacterial immune mechanism, yet, when dysregulated, may contribute to sepsis pathogenesis. Its regulation is heavily reliant on transcriptional control of caspase-4 expression. However, posttranslational regulation of the caspase-4 inflammasome remains poorly understood. Here, we report that UBX domain-containing protein 1 (UBXN1) facilitates the non-canonical inflammasome via unanchored lysine 48- or 63-linked polyUb (K48/63-Ub) chains. UBXN1 deficiency impairs the LPS-induced caspase-4 inflammasome and pyroptosis, renders mice resistant to LPS and polymicrobial sepsis. Depleting cellular unanchored polyUb with ubiquitin-specific proteinase 5 (USP5) reduces, while inhibiting USP5 enhances, caspase-4 activation in a UBXN1-dependent manner. In vitro , unanchored K48/63-Ub chains enhance LPS-induced caspase-4 enzymatic activity in a chain length- and UBXN1-dependent manner. Mechanistically, UBXN1 directly interfaces with and bridges unanchored K48/63-Ub and caspase-4, forming a tripartite complex to facilitate caspase-4 activation. Our findings uncover a previously unrecognized UBXN1- and unanchored K48/63-Ub-dependent regulatory layer in the caspase-4 inflammasome.