Mechanisms of Lung Phospholipid Homeostasis

肺磷脂稳态机制

基本信息

批准号：
7925693
负责人：
Rama K Mallampalli
金额：
$ 40.17万
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：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7925693
关键词：
Acute Acute Lung Injury Address Adult Respiratory Distress Syndrome Alveolar Animal Model Animals Bacteria Bacterial Infections Binding Biochemical Biological Assay Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Calmodulin Calpain Cartoons Cell Nucleus Cell membrane Cells Choline-Phosphate Cytidylyltransferase Clinical Trials Complex Cytidylyltransferase Cytoplasm Data Degradation Pathway Disease Effectiveness Enzymes Epithelium Equilibrium Event Exhibits Foundations Generations Goals Homeostasis In Vitro Infection Inflammatory Laboratories Lead Lecithin Left Lung Lysine Maps Mediating Membrane Membrane Lipids Metabolism Modeling Modification Molecular Molecular Chaperones Molecular Profiling Monoubiquitination Mus Newborn Respiratory Distress Syndrome Nuclear Nuclear Envelope Nuclear Import Nuclear Localization Signal Pathway interactions Peptide Hydrolases Pharmaceutical Preparations Phase Phospholipids Phosphorylation Phosphotransferases Play Process Production Proteins Proteolysis Pseudomonas aeruginosa Recruitment Activity Regulation Replacement Therapy Resistance Respiratory physiology Role Sepsis Testing Ubiquitin Virulent alveolar epithelium alveolar homeostasis enzyme activity exhaust genetic regulatory protein in vitro activity in vivo inhibitor/antagonist lipid biosynthesis lung injury molecular site mutant novel pathogen protein transport public health relevance respiratory distress syndrome response sensor septic small molecule surfactant surfactant deficiency synthetic enzyme tool ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Sepsis due to infection with highly virulent bacteria, such as P. aeruginosa, is the most common cause of the acute respiratory distress syndrome (ARDS). P. aeruginosa causes lung injury by greatly decreasing synthesis and availability of phosphatidylcholine (PC), the major surfactant phospholipid. This proposal will investigate how P. aeruginosa modulates surfactant production by altering function of the key enzyme, cytidylyltransferase (CCT). The PI has uncovered several novel preliminary observations: (i) P. aeruginosa rapidly triggers CCT nuclear import in lung epithelia, a process involving CCT interaction with Ca??? kinase I, and 14-3-3?, the latter which regulates protein trafficking, (ii) P. aeruginosa potently degrades CCT protein via a Caactivated E3-ligase mediated monoubiquitin-dependent degradation mechanism, and (iii) that CaM protects CCT from E3-ligase ubiquitin degradation. Thus, alveolar epithelia harbor two intrinsic homeostatic control mechanisms to maintain surfactant PC synthesis after bacterial infection. The first mechanism involves CCT nuclear entry as an acute salvage response; the second mechanism is protection of CCT proteolysis by CaM. When these pathways are overwhelmed by bacterial infection, CCT is degraded by ubiquitin-dependent processing. These results led to the overall hypothesis that calcium-CaM interactions play a central regulatory role in salvage and degradative pathways for the surfactant enzyme, CCT, in response to bacterial infection in lung epithelia. The PI will determine if 14-3-3? is a molecular chaperone that escorts CCT for nuclear import in vitro and in vivo in a Ca??? kinase 1-regulated manner. This mechanism may serve as an initial salvage pathway after P. aeruginosa infection to preserve surfactant synthesis (Aim 1). Second, the PI will determine if the E3 ligase, FBL2, triggers ubiquitin-dependent degradation of CCT in a Ca?regulated manner after long-term P. aeruginosa infection, an effect antagonized by CaM (Aim 2). The PI will use molecular and biochemical approaches to identify the molecular signatures that direct interactions between these CCT regulatory proteins and will determine their functional significance in murine models of bacterial infection. Studies will entail expression of novel E3 ligase resistant CCT enzyme mutants that exhibit robust catalytic activity and yet are less sensitive to proteolytic modification after P. aeruginosa-induced acute lung injury. These studies lay the foundation for generating small molecule (i.e. drug) CCT or 14-3-3 activators or specific E3 ligase inhibitors for use in surfactant-deficient states. Execution of these studies will lay the groundwork for a significant mechanistic advance with regard to the pathobiology of surfactant metabolism and mechanisms for alveolar homeostasis during inflammatory lung injury. PUBLIC HEALTH RELEVANCE: Sepsis-induced acute lung injury results in decreased production of surfactant, an essential material that stabilizes lung function. We have discovered that in animal models of septic lung injury, there are two mechanisms that protect a key surfactant synthetic enzyme, CCT: i) CCT shifts to the nucleus of lung epithelia by binding to a nuclear trafficking protein, termed 14-3-3, and ii) CCT is stabilized from its breakdown by calmodulin. After severe infection, these mechanisms are exhausted and CCT is rapidly degraded. In this application we will use several tools to confirm that 14-3-3 and calmodulin are indispensable for CCT to maintain sufficient surfactant production and uncover how CCT is degraded.

描述（由申请人提供）：由于高度毒细菌感染而引起的败血症，例如铜绿假单胞菌，是急性呼吸窘迫综合征（ARDS）的最常见原因。铜绿假单胞菌通过大大降低磷脂酰胆碱（PC）的可用性（主要表面活性剂磷脂）而导致肺损伤。该提案将通过改变关键酶，细胞质转移酶（CCT）的功能来调节铜绿假单胞菌如何调节表面活性剂的产生。 PI发现了几个新型的初步观察：（i）铜绿假单胞菌迅速触发肺上皮中的CCT核进口，这是一个涉及与CA相互作用的过程？激酶I和14-3-3？，后者调节蛋白质运输的后者，（ii）铜绿假单胞菌通过烧生激活的E3-岩合酶介导的单纤维蛋白依赖性降解机制有效降解CCT蛋白，并且（iii）（iii）CAM可保护CCT免受e3-烯丙基酶Ubiquitin ubiquitin degradeation的cct。因此，肺泡上皮具有两种固有的稳态控制机制，可在细菌感染后维持表面活性剂PC合成。第一种机制涉及CCT核进入作为急性打捞反应。第二种机制是CAM对CCT蛋白水解的保护。当这些途径被细菌感染淹没时，CCT会因泛素依赖性加工而降解。这些结果导致了一个总体假设，即钙-CAM相互作用在肺上皮细菌感染的表面活性剂酶CCT的挽救和降解途径中起着核心调节作用。 PI将确定是否14-3-3？是否在CAC中陪同CCT进行CCT的分子伴侣在CA中的体外进口？？？激酶1调节的方式。该机制可以用作铜绿假单胞菌感染后的初始打捞途径，以保存表面活性剂合成（AIM 1）。其次，PI将确定E3连接酶FBL2是否会在长期铜绿假单胞菌感染后以CA受调节的方式触发CCT的泛素依赖性降解，这是由CAM拮抗的效果（AIM 2）。 PI将使用分子和生化方法来识别这些CCT调节蛋白之间直接相互作用的分子特征，并在细菌感染的鼠模型中确定它们的功能意义。研究将需要表达新型的E3连接酶抗性CCT酶突变体，该酶突变体表现出强大的催化活性，但对铜绿假单胞菌诱导的急性肺损伤后对蛋白水解的修饰不太敏感。这些研究为产生小分子（即药物）CCT或14-3-3激活剂或特定的E3连接酶抑制剂奠定了基础，以用于表面活性剂缺乏状态。这些研究的执行将为炎症性肺损伤期间的表面活性剂代谢的病理学和肺泡稳态机制的病理学奠定基础。公共卫生相关性：败血症引起的急性肺损伤导致表面活性剂的产生降低，表面活性剂的产生是稳定肺功能的重要材料。我们发现，在败血性肺损伤的动物模型中，有两种机制可以保护关键的表面活性剂合成酶，CCT：i）CCT通过与核交通蛋白结合，称为14-3-3和II）CCT通过降解稳定稳定CCT，将CCT转移到肺上皮核的细胞核中。严重感染后，这些机制耗尽，CCT迅速降解。在此应用中，我们将使用几种工具来确认14-3-3和钙调蛋白是必不可少的，即可维持足够的表面活性剂生产并发现CCT如何降解。