Sepsis is the 10th leading cause of death in the US. An unresolved systemic cytokine storm caused by bacterial infection is a hallmark of sepsis. The robust acute inflammatory response, through Toll Like Receptors (TLRs) and interleukin-1R like receptors (ILRs), trigger detrimental effects including multi-organ failure. Despite extensive research, therapies for sepsis focus on the use of antimicrobials that lead to multi-drug resistance. Hence, an unmet scientific need is to understand the molecular regulation of anti-inflammatory responses that diminish the severity of tissue injury. Single immunoglobulin interleukin-1-related receptor (SIGIRR), which is also known as Toll/IL-1 receptor 8, exhibits an anti-inflammatory effect against TLRs and ILRs signaling. Recently, IL-37, which is a suppressor of innate immunity, has been identified as the SIGIRR ligand. Both IL-37 and SIGIRR have been recognized as major therapeutic targets to lessen cytokine storm, however, very little is known regarding the molecular regulation of SIGIRR stability. Receptor degradation, a negative feedback regulation of receptor function, is a highly regulated process by post-translational modification, such as phosphorylation and ubiquitination. Ubiquitination is a molecular signal for protein degradation in either the proteasome or lysosome. De-ubiquitination, which is mediated by deubiquitinating enzymes (DUBs), tightly controls protein stability by removal of ubiquitin chains from target proteins. In our preliminary data, we discovered that (i) SIGIRR is poly-ubiquitinated and degraded in the proteasome in response to its ligand binding; (ii) Ubiquitin-specific protease (USP13), a member of DUBs, targets and stabilizes SIGIRR by hydrolyzing the ubiquitin chains from SIGIRR; (iii) glycogen synthase kinase 3? (GSK3?) phosphorylates SIGIRR and interrupts the association between SIGIRR and USP13, thereby reducing SIGIRR stability; (iv) USP13 increases survival rate in experimental sepsis. These observations have led to the following hypothesis: USP13 ameliorates cytokine storm and septic shock through deubiquitination and stabilization of the anti-inflammatory receptor, SIGIRR. To evaluate this hypothesis we will determine 1) molecular signature of USP13-promoted SIGIRR stability; 2) if GSK3?-induced disruption of USP13/SIGIRR interaction lessens anti-inflammatory effects of SIGIRR; 3) if stabilization of SIGIRR by USP13 mitigates endotoxin-induced pro-inflammatory responses. In summary, this application provides molecular mechanisms by which SIGIRR is degraded via phosphorylation-driven ubiquitination. Our preliminary data has uncovered two previously unrecognized post-translational modifications of SIGIRR: phosphorylation and ubiquitination. Two mediators were revealed: GSK3?, which phosphorylates SIGIRR; and USP13, which de- ubiquitinates SIGIRR. These studies will be the first to elucidate that phosphorylation of SIGIRR promotes its ubiquitination by disassociating USP13 from SIGIRR. These studies will lay the foundation for a significant mechanistic advance regarding the molecular regulation of the inflammatory response through modulation of anti-inflammatory receptor stability.
Gram-negative bacterial infection-induced sepsis and lung injury is a highly virulent pathogen etiologically linked to high morbidity and mortality during fulminant infection. These studies will be the first to elucidate the role of deubiquitinating enzyme, protein kinase, and anti- inflammatory cytokine receptor stability in the pathogenesis of systemic inflammation. These studies will be critical in developing novel therapeutics by enhancing SIGIRR stability by USP13 to lessen the severity of septic shock and lung injury.
