Regulation of Cardiolin Byosynthesis in Epithelial Injury

上皮损伤中心磷脂合成的调节

基本信息

批准号：
8643329
负责人：
Rama K Mallampalli
金额：
$ 39.08万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-03 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8643329
关键词：
Acute Acute Lung Injury Adult Respiratory Distress Syndrome Adverse effects Alveolar Anabolism Apoptosis Binding Biogenesis Cardiolipins Cell Death Cell Survival Cells Chemicals Clinical Cullin Proteins Cytochromes Data Defect Elements Environment Enzymes Epithelial Epithelial Cells Epithelium Extracellular Space F Box Domain F-Box Proteins Family Generations Homeostasis Human Human Biology Infection Injury Knockout Mice Lead Link Lipids Lung Mediating Mediator of activation protein Membrane Mitochondria Molecular Molecular Profiling Nature Orphan Oxygen PINK1 gene Pathway interactions Patients Pattern Peptide Hydrolases Phosphorylation Phosphotransferases Pneumonia Post-Translational Protein Processing Process Proteins Recruitment Activity Regulation Resistance Respiratory physiology Role Secondary to Sepsis Severities Signal Transduction Staphylococcus aureus System Testing Translating Ubiquitin base cardiolipin synthase design efficacy testing extracellular in vivo inhibitor/antagonist injured mitochondrial dysfunction mutant novel pathogen small molecule ubiquitin-protein ligase

项目摘要

A hallmark of patients with ARDS is the inability to utilize oxygen secondary to mitochondrial damage that profoundly limits generation of chemical energy needed in critically injured patients. The mechanistic basis for mitochondrial injury in ARDS patients is unknown. Cardiolipin is a critical mitochondrial structural lipid that when deficient, leads to loss of mitochondria and cell death. But because of its resemblance to bacterial membranes, we discovered that cardiolipin is a highly toxic damage signal that profoundly disrupts lung homeostasis when released from dying cells (Nature Med 2010). Thus, factors that impair cardiolipin availability might reduce mitochondrial integrity and trigger cell death, releasing preformed cardiolipin externally to elicit adverse effects. In this Project, we have preliminary data indicating that S. aureus linked to sepsis-induced ARDS degrades the key enzyme, cardiolipin synthase 1 (CLS1) required for cardiolipin biosynthesis leading to mitochondrial dysfunction and apoptosis, thereby releasing preformed cardiolipin extracellularly. Further, S. aureus activates an orphan ubiquitin E3 ligase F box protein, termed FBX015, that when recruited to CLS1 is sufficient to ubiquitinate and mediate degradation of CLS1 in epithelia. CLS1 phosphorylation by a kinase, termed Pink1, is a critical recognition signal for FBX015. These observations have led to the overall hypothesis that bacterial-induced mitochondrial dysfunction involves post-translational modification of CLS1 that severely limits intracellular availability of this lipid thereby triggering cell death and extracellular release of preformed cardiolipin. To test this hypothesis, we will determine if acute infection with S. aureus inhibits cardiolipin biosynthesis via Pink1 kinase-induced phosphorylation of CLS1 (Aim 1). We will test how the F-box protein, FBX015, triggers ubiquitin-dependent degradation of CLS1 in a phosphorylation¿ dependent manner after S. aureus infection to impair mitochondrial homeostasis (Aim 2). These studies will translate our basic observations to an in vivo system by testing efficacy of CLS1 phosphorylation and protease-resistant enzyme mutants, adoptive cell based transfer strategies, and initial design of Pink1/F box inhibitors to lessen the severity of alveolar injury. Execution of these studies will serve as a basis for generation of new small molecule CLS1 activators or novel ubiquitin-kinase antagonists. Completion of these studies will lay the groundwork for a potentially significant conceptual advance with regard to the control of mitochondrial integrity and epithelial cell viability during alveolar injury.

ARDS 患者的一个特点是无法利用继发于线粒体损伤的氧气严重限制了严重受伤患者所需的化学能量的产生。心磷脂是一种关键的线粒体结构脂质，缺乏时会导致线粒体损失和细胞死亡。我们发现心磷脂是一种剧毒损伤信号，从垂死细胞中释放时会严重破坏肺稳态（Nature Med 2010），因此，损害心磷脂可用性的因素可能会减少。线粒体完整性并触发细胞死亡，向外部释放预先形成的心磷脂以引起不良反应。在该项目中，我们的初步数据表明，与脓毒症诱导的 ARDS 相关的金黄色葡萄球菌会降解心磷脂生物合成所需的关键酶心磷脂合成酶 1 (CLS1)。导致线粒体功能障碍和细胞凋亡，将预先形成的心磷脂释放到细胞外。此外，金黄色葡萄球菌还会激活孤儿泛素 E3 连接酶 F 盒蛋白， FBX015 被招募到 CLS1 后，足以泛素化并介导上皮细胞中 CLS1 的磷酸化，称为 Pink1，这是 FBX015 的关键识别信号。这些观察结果得出了细菌诱导线粒体的总体假设。功能障碍涉及 CLS1 的翻译后修饰，这严重限制了其在细胞内的可用性，从而导致脂质触发细胞死亡和预先形成的心磷脂的细胞外释放。为了检验这一假设，我们将确定金黄色葡萄球菌的急性感染是否通过 Pink1 激酶诱导的 CLS1 磷酸化抑制心磷脂生物合成（目标 1），我们将测试 F-box 蛋白 FBX015 如何触发 CLS1 的泛素依赖性降解。在磷酸化中¿金黄色葡萄球菌感染后以依赖的方式损害线粒体稳态（目标 2），这些研究将通过测试 CLS1 磷酸化和蛋白酶抗性酶突变体的功效、基于细胞的过继转移策略和初步设计，将我们的基本观察结果转化为体内系统。 Pink1/F 盒抑制剂可减轻肺泡损伤的严重性，这些研究的执行将作为产生新的小分子 CLS1 激活剂或新型的基础。这些研究的完成将为肺泡损伤期间线粒体完整性和上皮细胞活力的控制方面的潜在重大概念进步奠定基础。