Regulation of Cardiolin Byosynthesis in Epithelial Injury

上皮损伤中心磷脂合成的调节

• 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
• 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

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患者的一个标志是继发于线粒体损伤的不能利用氧， 严重限制了重伤患者所需的化学能的产生。ARDS患者线粒体损伤的机制基础尚不清楚。心磷脂是一种重要的线粒体结构脂质，当缺乏时，会导致线粒体丢失和细胞死亡。但由于其与细菌膜的相似性，我们发现心磷脂是一种高度毒性的损伤信号，当从垂死的细胞中释放出来时，会严重破坏肺的体内平衡（Nature Med 2010）。因此，损害心磷脂可用性的因素可能会降低线粒体完整性并触发细胞死亡，在外部释放预先形成的心磷脂以引起不良反应。在本项目中，我们有初步的数据表明，S。与脓毒症诱导的ARDS相关的金黄色葡萄球菌降解心磷脂生物合成所需的关键酶心磷脂合酶1（CLS 1），导致线粒体功能障碍和细胞凋亡，从而在细胞外释放预先形成的心磷脂。此外，S。金黄色葡萄球菌激活孤儿泛素E3连接酶F盒蛋白，称为FBX 015，当招募到CLS 1时，足以泛素化并介导上皮细胞中CLS 1的降解。被称为Pink 1的激酶磷酸化的CLS 1是FBX 015的关键识别信号。这些观察结果导致了细菌诱导的线粒体功能障碍涉及CLS 1的翻译后修饰的总体假设，这严重限制了这种脂质的细胞内可用性，从而触发细胞死亡和预先形成的心磷脂的细胞外释放。为了验证这一假设，我们将确定急性感染S。金黄色葡萄球菌通过Pink 1激酶诱导的CLS 1（Aim 1）磷酸化抑制心磷脂生物合成。我们将测试F-box蛋白FBX 015如何在S.金黄色葡萄球菌感染损害线粒体稳态（目的2）。这些研究将通过测试CLS 1磷酸化和蛋白酶抗性酶突变体的功效、基于过继细胞的转移策略以及Pink 1/F盒抑制剂的初始设计来将我们的基本观察转化为体内系统，以减轻肺泡损伤的严重程度。这些研究的执行将作为产生新的小分子CLS 1激活剂或新型泛素激酶拮抗剂的基础。这些研究的完成将奠定一个潜在的重大概念方面的进步，在肺泡损伤的线粒体完整性和上皮细胞活力的控制基础。