Systematic Investigation of Protein Ubiquitination in ARDS

急性呼吸窘迫综合征(ARDS)每年影响近25万患者， 并负责360万天的住院日。ARDS的死亡率接近40%， 其主要原因是肺炎和败血症。在肺的病理生理学中起中心作用 损伤是一种持续的炎症反应，导致线粒体损伤，肺泡 上皮细胞损伤，并导致水肿和气体交换障碍的形成 穿过牙槽。ARDS的临床治疗在很大程度上是支持性的，并有许多干预措施 (例如T细胞受体阻滞剂、血管紧张素II受体拮抗剂、细胞因子阻断抗体 或皮质类固醇)并不能改善ARDS的预后。因此，有一种未得到满足的需求 减少与ARDS相关的高发病率和死亡率的新疗法。蛋白 泛素化是细胞内主要的蛋白质加工途径，泛素(Ub)通过它来标记一个 通过26s蛋白酶体或溶酶体降解的靶向蛋白质。它扮演了一个如此关键的角色 在生物过程中的作用及其失调会导致许多疾病。不幸的是， 细菌感染通常会扰乱蛋白质的泛素化过程。我们和其他许多人 研究人员已经证明，感染或其他炎症刺激会改变信使核糖核酸和 Ub E3连接酶的蛋白水平，从而影响其靶蛋白的水平和功能。 因此，发现了新的基于Ub E3连接酶的与肺有关的分子通路 伤害提供了独特的机会，潜在地设计出新的减重策略 阿兹。我们提出了对ARDS中蛋白质泛素化网络的系统分析，它已经 从未被处死过。我们已经确定了几个高价值的蛋白质靶标并放置了 拿出实验计划。这些研究将增加对这一令人兴奋和 肺损伤和炎症的关键区域。具体地说，我们将重点确定 调控炎症级联反应的新分子途径--线粒体 功能/有丝分裂，抑制肺内炎症小体的激活，并开发新的 ARDS的治疗学。在所有这三个领域，我们将系统地调查如何以及哪些 在肺损伤过程中，蛋白质泛素化过程处于失调状态。我们将实施国情咨文。 高通量筛选(HTS)以确定相关E3连接酶/底物通路 在肺损伤的过程中。我们将研究蛋白质泛素化的过程 先进的生化工具，将提供详细的机械数据，如底物 泛素化位点和E3连接酶结合基序。分子和遗传方法(例如 CRISPR/Cas9基因编辑和体内基因转移)将用于研究E3的功能 连接酶/底物与肺损伤我们还将利用我们的药物发现专业知识开发小型 用于临床前肺损伤模型的分子。这项提议将为以下工作奠定基础 涉及发现靶向蛋白质的小分子的未来研究 ARDS中的泛素化途径。

Acute Respiratory Distress Syndrome (ARDS) affects almost a quarter million patients annually, and is responsible for 3.6 million hospital days. ARDS has a mortality rate approaching 40%, and its primary causes are pneumonia and sepsis. Central to the pathophysiology of this lung injury is a sustained inflammatory response, which leads to mitochondrial damage, alveolar epithelia injury, and contributes to the formation of edema and impairment of gas exchange across the alveolus. Clinical treatment for ARDS is largely supportive, and many interventions (e.g. T-cell receptor blockades, angiotensin II receptor antagonists, cytokine blocking antibodies or corticosteroids) have not improved outcomes in ARDS. Thus, there is an unmet need for new therapies to reduce the high morbidity and mortality associated with ARDS. Protein ubiquitination is the major protein processing pathways in cells by which ubiquitin (Ub) flags a targeted protein for degradation through the 26s proteasome or lysosome. It plays such a critical role in biological processes and its dysregulation leads to many diseases. Unfortunately, bacterial infection often disrupts the protein ubiquitination process. We and many other investigators have shown that infection or other inflammatory stimuli will alter the mRNA and protein levels of Ub E3 ligases, thus affecting the levels and functions of their target proteins. Thus, uncovering new Ub E3 ligase-based molecular pathways that contribute to lung injury provides unique opportunities to potentially devise new strategies to attenuate ARDS. We propose a systematic analysis of protein ubiquitination networks in ARDS, which has not been executed before. We have already identified several high value protein targets and laid out the experiment plans. These studies will add to the investigation into this exciting and critically important area of lung injury and inflammation. Specifically, we will focus on identifying novel molecular pathways that modulate the inflammatory cascade, mitochondria function/mitophagy, and DAMPs/inflammasome activation in the lung, and develop novel therapeutics for ARDS. In all three areas, we will systematically investigate how and which protein ubiquitination processes are dysregulated during lung injury. We will carry out state-of- the-art High-Throughput Screening (HTS) to identify the relevant E3 ligase/substrate pathways in the lung injury process. We will examine the process of protein ubiquitination using sophisticated biochemical tools, which will provide detailed mechanistic data such as substrate ubiquitination site and E3 ligase binding motif. Molecular and genetic approaches (such as Crispr/Cas9 gene editing and in vivo gene transfer) will be used to study the functions of E3 ligase/substrate in lung injury. We will also use our drug discovery expertise to develop small molecules for use in preclinical lung injury models. This proposal will lay the groundwork for futures studies involving the discovery of small molecules targeting protein ubiquitination pathways in ARDS.