Molecular regulation of anti-inflammatory cytokine receptor in sepsis

摘要 败血症是美国第十大死亡原因。未解决的系统性细胞因子风暴 是败血症的标志强烈的急性炎症反应， 通过Toll样受体（TLR）和白细胞介素-1R样受体（ILR）， 包括多器官功能衰竭。尽管有广泛的研究，脓毒症的治疗方法主要集中在 使用导致多重耐药性的抗菌药物。因此，一个尚未满足的科学需求是， 了解抗炎反应的分子调控，减少严重性 组织损伤单免疫球蛋白白细胞介素-1相关受体（SIGIRR），也是 称为Toll/IL-1受体8，对TLR和ILR表现出抗炎作用 信号最近，作为先天免疫抑制剂的IL-37已被鉴定为免疫抑制剂。 SIGIRR配体。IL-37和SIGIRR都已被认为是主要的治疗靶点， 然而，关于细胞因子风暴的分子调控知之甚少， SIGIRR稳定性。受体降解是一种负反馈调节受体功能的过程， 通过翻译后修饰，如磷酸化， 泛素化泛素化是蛋白质降解的分子信号， 蛋白酶体或溶酶体。去泛素化，由去泛素化酶介导 （DUBs）通过从靶蛋白去除泛素链来严格控制蛋白质稳定性。在 我们的初步数据，我们发现（i）SIGIRR是多聚泛素化和降解的， 蛋白酶体响应其配体结合;（ii）泛素特异性蛋白酶（USP 13）， DUBs的成员，通过水解来自 （iii）糖原合成酶激酶3 β（GSK 3 β）磷酸化SIGIRR并中断SIGIRR的表达。 SIGIRR和USP 13之间的关联，从而降低SIGIRR稳定性;（iv）USP 13 增加实验性败血症的存活率。这些意见导致了以下情况 假设：USP13通过去泛素化改善细胞因子风暴和脓毒性休克， 抗炎受体SIGIRR的稳定。为了验证这个假设，我们将 确定1）USP 13促进的SIGIRR稳定性的分子特征; 2）如果GSK 3 β诱导 USP13/SIGIRR相互作用的破坏降低了SIGIRR的抗炎作用; 3）如果 通过USP 13稳定SIGIRR减轻了内毒素诱导的促炎反应。在 总之，本申请提供了SIGIRR通过以下途径降解的分子机制： 磷酸化驱动的泛素化。我们的初步数据显示， SIGIRR的未识别的翻译后修饰：磷酸化和泛素化。 揭示了两种介质：GSK3 β，磷酸化SIGIRR;和USP 13， 泛素化SIGIRR。这些研究将首次阐明SIGIRR的磷酸化 通过使USP13与SIGIRR分离来促进其泛素化。这些研究将奠定 为一个重要的机制进展的基础，关于分子调控的， 通过调节抗炎受体稳定性来调节炎症反应。

Abstract 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 3β (GSK3β) 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 GSK3β-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: GSK3β, 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.