Regulation of Cardiolin Byosynthesis in Epithelial Injury

ARDS患者的一个特征是无法利用继发于线粒体损伤的氧气， 极大地限制了重伤患者所需化学能的产生。ARDS患者线粒体损伤的机制尚不清楚。心磷脂是一种重要的线粒体结构脂类，当它缺乏时，会导致线粒体丢失和细胞死亡。但由于它与细菌膜相似，我们发现心磷脂是一种剧毒的损害信号，当从死亡细胞中释放出来时，它会深刻地破坏肺的动态平衡(自然医学2010)。因此，削弱心磷脂可获得性的因素可能会降低线粒体的完整性并引发细胞死亡，从而从外部释放预制的心磷脂来引发不良反应。在本项目中，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降解了心磷脂生物合成所需的关键酶-心磷脂合成酶1(CLS1)，从而导致线粒体功能障碍和细胞外释放预先形成的心磷脂。此外，金黄色葡萄球菌激活一种孤儿泛素E3连接酶F盒蛋白，称为FBX015，当被招募到CLS1时，该蛋白足以泛素化并介导上皮细胞中CLS1的降解。CLS1被一种被称为PINK1的激酶磷酸化，是FBX015的关键识别信号。这些观察结果导致了一个总体假设，即细菌诱导的线粒体功能障碍涉及CLS1的翻译后修饰，严重限制了这种脂类的细胞内可获得性，从而引发细胞死亡和预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金黄色葡萄球菌是否通过PINK1激酶诱导CLS1的磷酸化来抑制心磷脂的生物合成(目标1)。我们将测试F-box蛋白FBX015如何在金黄色葡萄球菌感染后以磷酸化依赖的方式触发CLS1泛素依赖的降解，从而破坏线粒体动态平衡(目标2)。这些研究将把我们的基本观察转化为体内系统，通过测试CLS1磷酸化和蛋白酶抗性酶突变体的有效性，基于过继细胞的转移策略，以及初步设计PINK1/F盒抑制剂来减轻肺泡损伤的严重程度。这些研究的实施将作为产生新的小分子CLS1激活剂或新型泛素-激酶拮抗剂的基础。这些研究的完成将为在控制肺泡损伤期间线粒体完整性和上皮细胞活性方面取得潜在的重大概念性进展奠定基础。

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.