The role of unanchored ubiquitin chains in non-canonical inflammasome signaling - Inflammasomes are large cytosolic multiprotein complexes formed in response to infections and cellular stresses that drive auto-activation of inflammatory caspases, production of inflammatory mediators, and pyroptosis. Inflammasomes are important for controlling bacterial infections; however, if hyperactive and persistent, are damaging to tissues due to their nature of inflammation. Notably, inflammasomes are involved in the pathogenesis of sepsis, a life-threatening illness due to the human body’s extreme inflammatory response to infections, usually bacterial infections. There are >1.5 million cases of sepsis and >250,000 sepsis-related deaths each year in the United States. However, there are no effective therapies. Most inflammasomes are assembled by a pathogen- or danger-associated pattern recognition receptor (PRR) and an adaptor (optional), which serve as a platform for the recruitment and auto-processing caspase-1. However, human caspase-4/5 and murine caspase-11 can be directly bound and activated by a bacterial endotoxin, lipopolysaccharide (LPS); therefore, they are recognized as the non-canonical inflammasomes. Although much has been learned about the regulatory mechanisms for the canonical inflammasomes, little is known for the non-canonical inflammasomes. To address this gap, we employed an unbiased manner to identify caspase-4 interactors and found that ubiquitin regulatory X (UBX) domain-containing protein 1 (UBXN1) was a top hit. Preliminary studies showed that UBXN1 was positive regulator of the non-canonical inflammasome signaling and facilitated the pathogenesis of LPS and polymicrobial sepsis. Mechanistically, our initial results suggest that UBXN1 enriches and tethers lysine (K) 63- and K48-linked unanchored ubiquitin chains to caspase-4/11, thus promoting the assembly and activation of caspase-4/11. In contrast to the conventional ubiquitination that adds a ubiquitin molecule (Ub) via the covalent bond to a substrate protein and additional Ub molecules in tandem (polyubiquitination), unanchored (free) ubiquitin chains are derived from deubiquitylation or de novo synthesis and interact with a target protein non- covalently. However, the research on free polyUb is still in its infancy. We further demonstrated that tetrameric free K63-Ub4 and K48-Ub4 enhanced caspase-4 activation. Herein, we hypothesize that unanchored K48/63-Ub chains bind and facilitate the non-canonical inflammasome activation in a UBXN1-dependent manner. We will employ multiple approaches including cell-free, biochemical, microscopic, genetic, pharmacological, in vivo tools and murine models. We hope to advance the fundamental knowledge about unanchored polyubiquitin chains and comprehensively understand the in vivo physiological functions of UBXN1 in the pathogenesis of polymicrobial and single bacterial sepsis and its molecular mechanism of action. These pieces of knowledge will lay a solid foundation for the future development of small molecular inhibitors of UBXN1 to treat sepsis and other autoinflammatory diseases.
