Systematic Investigation of Protein Ubiquitination in ARDS

ARDS 中蛋白质泛素化的系统研究

• 批准号: 10320731
• 负责人: Beibei Chen
• 金额: $ 93.9万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-26 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320731
• 关键词: 26S proteasome; Acute Respiratory Distress Syndrome; Adrenal Cortex Hormones; Affect; Alveolus; Angiotensin II Receptor; Area; Attenuated; Bacterial Infections; Binding; Biological Process; Blocking Antibodies; CRISPR/Cas technology; Cells; Clinical Treatment; Data; Disease; Edema; Functional disorder; Future; Gases; Gene Transfer; Genes; Impairment; Infection; Inflammasome; Inflammatory; Inflammatory Response; Intervention; Investigation; Length of Stay; Lung; Lysosomes; Messenger RNA; Mitochondria; Modeling; Molecular; Molecular Genetics; Morbidity - disease rate; Pathway interactions; Patients; Play; Pneumonia; Process; Protein Analysis; Proteins; Pulmonary Inflammation; Research Personnel; Role; Sepsis; Site; Stimulus; T-Cell Receptor; Ubiquitin; Ubiquitination; alveolar epithelium; antagonist; base; biochemical tools; cytokine; drug discovery; epithelial injury; experimental study; genetic approach; high throughput screening; in vivo; lung injury; mortality; novel; novel therapeutics; pre-clinical; protein degradation; small molecule; ubiquitin-protein ligase

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.

急性呼吸窘迫综合征（ARDS）每年影响近25万患者， 并负责360万医院的日子。 ARDS的死亡率接近40％， 它的主要原因是肺炎和败血症。该肺病理生理学的中心 受伤是一种持续的炎症反应，导致线粒体损伤，肺泡 上皮损伤，并有助于形成水肿和气体交换的损害 跨过肺泡。 ARDS的临床治疗在很大程度上是支持的，许多干预措施 （例如T细胞受体阻滞，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）没有改善ARDS的预后。因此，有未满足的需求 降低与ARDS相关的高发病率和死亡率的新疗法。蛋白质 泛素化是泛素（UB）标志A的细胞中的主要蛋白质加工途径 通过26S蛋白酶体或溶酶体降解的靶向蛋白质。它发挥了如此关键 在生物过程中的作用及其失调导致许多疾病。很遗憾， 细菌感染通常会破坏蛋白质泛素化过程。我们和许多其他 研究人员表明，感染或其他炎症刺激会改变mRNA，并 UB E3连接酶的蛋白质水平，因此影响其靶蛋白的水平和功能。 因此，发现新的UB E3连接酶的分子途径，有助于肺 伤害提供了独特的机会，可以设计新的策略来减弱 ARDS。我们建议对ARDS中蛋白质泛素化网络进行系统分析，该网络具有 以前没有执行。我们已经确定了几个高价值蛋白质靶标和奠定 消除实验计划。这些研究将增加对这一令人兴奋和的调查 肺损伤和炎症至关重要的区域。具体来说，我们将专注于确定 调节炎症级联反应，线粒体的新型分子途径 肺中的功能/线粒体和潮湿/炎症体激活，并发展出新颖的 ARDS的治疗学。在这三个领域，我们将系统地研究如何以及哪些 肺部损伤期间蛋白质泛素化过程失调。我们将执行最先进 ART高通量筛选（HTS）以识别相关的E3连接酶/底物途径 在肺部损伤过程中。我们将使用 复杂的生化工具，该工具将提供详细的机械数据，例如基板 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 肺损伤中的连接酶/底物。我们还将使用我们的药物发现专业知识来发展小型 用于临床前肺损伤模型的分子。该提议将为 期货研究涉及发现针对蛋白质的小分子 ARDS中的泛素化途径。