HECT-domain E3 Ligase and Acute Lung Injury

急性呼吸窘迫综合征(ARDS)每年影响近25万患者 死亡率超过40%。其主要原因是肺炎和败血症。中心到 这种肺损伤的病理生理学是一种持续的免疫反应。带着夸张的口吻 免疫应答，NF-κB介导的细胞因子释放导致 肺水肿、多器官衰竭和休克。最近，我们发现了一条新的途径 通过蛋白质泛素化的炎症，泛素E3的普遍机制 连接酶是降解的目标蛋白质。我们发现一种支持炎症的孤儿- 结构域泛素(Ub)E3连接酶被称为KIAA0317，在微生物感染后被激活。这款E3 连接酶泛素化一种名为SOCS2(细胞因子抑制因子)的有效抗炎蛋白 信号传递2)，从而将其标记为在蛋白酶体中降解。SOCS2不仅抑制了 信号转导和转录激活子(STATs)的激活，但也防止 核因子-κB依赖的基因激活。有选择地调节丰度的策略 SOCS2可能成为一种新的治疗干预策略。然而，到目前为止，非常 关于SOCS2在蛋白质稳定性水平上的分子调控，人们知之甚少。我们的 初步数据表明：(I)细菌激活KIAA0317，它足以泛化 并介导炎症抑制因子SOCS2的降解，(Ii)KIAA0317是一种促进 体内和体外的炎性蛋白，(Iii)蛋白激酶Cα使SOCS2磷酸化，从而 为KIAA0317靶向创造独特的分子信号，以及(Iv)一种新的小分子 KIAA0317抑制剂BC-1365减弱内毒素和铜绿假单胞菌诱导的细胞因子分泌 在活体内。这些数据导致了我们的新假设，转录上调 KIAA0317专门针对SOCS2进行泛素化和降解，从而诱导 炎症和组织损伤。我们将确定KIAA0317是如何转录调控的 通过细菌病原体和SOCS2如何被KIAA0317靶向的分子基础， 从而上调NF-κB介导的炎症反应。我们还将确定KIAA0317是否 其基因变异可作为炎症性疾病的新生物标志物(目标1)。我们 将在各种实验性肺损伤模型中测试KIAA0317基因敲除小鼠以证实 KIAA0317是一个可下药的靶点。此外，我们将设计一个最佳的KIAA0317拮抗剂，使用一个 基于定量构效关系(QSAR)的设计和毒性测试 以及体外和体内的抗炎活性(目标2)。最后，这项建议 揭示了肺损伤的一个新的分子模型，因为它与细胞因子信号有关。我们的 初步数据发现了一种新的蛋白质KIAA0317，它与细胞因子有关 通过SOCS2蛋白信号进行应答。这些研究将是第一个阐明 KIAA0317的酶行为，似乎激活了NF-κB-细胞因子轴。行刑 将为一种根本性的、改变范式的治疗方法奠定基础 调节先天免疫和治疗炎症性疾病的进展 为新的翻译计划做好准备。

Acute Respiratory Distress Syndrome (ARDS) affects almost a quarter million patients annually and has a mortality rate of over 40%. Its primary causes are pneumonia and sepsis. Central to the pathophysiology of this lung injury is a sustained immune response. With an exaggerated immune response, NF-κB mediated cytokine release leads to the devastating effects of pulmonary edema, multi-organ failure, and shock. Recently, we discovered a novel pathway for inflammation through protein ubiquitination, a universal mechanism whereby ubiquitin E3 ligases target proteins for degradation. We discovered that a pro-inflammatory, orphan HECT- domain ubiquitin (Ub) E3 ligase, termed KIAA0317, is activated after microbial infection. This E3 ligase ubiquitinates a potent anti-inflammatory protein termed SOCS2 (Suppressor of Cytokine Signaling 2), thereby marking it for degradation in the proteasome. SOCS2 not only suppresses the activation of signal transducers and activators of transcription (STATs), but also prevents NF-κB-dependent gene activation. Maneuvers designed to selectively modulate the abundance of SOCS2 might serve as a novel strategy for therapeutic intervention. However, to date, very little is known regarding the molecular regulation of SOCS2 at the level of protein stability. Our preliminary data suggest that (i) bacteria activates KIAA0317, which is sufficient to ubiquitinate and mediate the degradation of the inflammatory repressor SOCS2, (ii) KIAA0317 is a pro- inflammatory protein in vivo and in vitro, (iii) the kinase PKCα phosphorylates SOCS2, thereby creating a unique molecular signal for KIAA0317 targeting, and (iv) a novel small molecule inhibitor of KIAA0317, BC-1365, attenuates LPS and P. aeruginosa-induced cytokine secretion in vivo. These data led to our novel hypothesis that transcriptionally upregulated KIAA0317 specifically targets SOCS2 for ubiquitination and degradation, thus inducing inflammation and tissue injury. We will determine how KIAA0317 is regulated transcriptionally by bacterial pathogens and the molecular basis of how SOCS2 is targeted by KIAA0317, thereby upregulating NF-κB mediated inflammation. We will also determine whether KIAA0317 and its genetic variants can be used as novel biomarkers for inflammatory diseases (Aim 1). We will test KIAA0317 knockout mice in various experimental lung injury models to confirm that KIAA0317 is a druggable target. Further, we will design an optimal KIAA0317 antagonist using a quantitative structure-activity relationship (qSAR) based design and test its toxicity, target engagement, and anti-inflammatory activity both in vitro and in vivo (Aim 2). Last, this proposal unveils a new molecular model of lung injury as it relates to cytokine signaling. Our preliminary data have uncovered a novel protein, KIAA0317, which is linked to cytokine response through SOCS2 protein signaling. These studies will be the first to elucidate the enzymatic behavior of KIAA0317, which appears to activate the NF-κB-cytokine axis. Execution of these studies will lay the groundwork for a fundamental, paradigm-changing therapeutic advance for regulating innate immunity and treating inflammatory diseases that will ultimately set the stage for a new translational initiative.