Membrane Phospholipid Distribution/Mediator Translocatio

说明（改编自申请人的摘要）在激活炎性细胞之后，乱序酶的质膜磷脂（触发器）的增强的整体跨双层运动导致深刻的生物学后果。磷脂酰丝氨酸在质膜外叶中的出现导致凝血的显著增强，无论是在止血中适当，还是在急性肺损伤中不适当。类似地，磷脂酰丝氨酸的出现显著增强补体激活，并作为吞噬细胞识别和摄取凋亡细胞的信号。此外，乱序酶激活将导致介质磷脂（血小板活化因子（PAF）、溶血磷脂和氧化磷脂）或其前体（花生四烯酸酯递送至分泌的PLA2）的释放（从合成细胞）或摄取（进入靶细胞）。根据细胞类型、刺激和乱序蛋白酶的同种型，假设至少有四种机制参与调节乱序蛋白酶活性：a）通过PKC磷酸化乱序蛋白酶（例如fMLP刺激的嗜中性粒细胞或Jurkat细胞凋亡），B）在磷酸化不存在的情况下细胞内钙的升高（例如凝血酶刺激的血小板），c）向膜和从膜的易位（fMLP刺激的嗜中性粒细胞）和d）干扰素介导的扰乱酶表达的上调（干扰素α处理的Raji细胞）。本项目的目的是通过磷酸化来确定scramblase的调节，并确定scramblase增强膜磷脂跨双层运动的机制。描述了整合的生物化学、结构和遗传方法，其中确定了乱序蛋白酶磷酸化活性，并定义了磷酸化对乱序蛋白酶活性的影响，特别是关于钙需求的影响。类似地，这些相同的整合方法将被用来定义的机制，通过该机制，scramblase介导的磷脂的transbilayer运动，假设需要自我关联或与其他蛋白质的关联。

Description (Adapted from Applicant's Abstract) Following activation of inflammatory cells, enhanced bulk transbilayer movement of plasma membrane phospholipids (flip-flop) of scramblase results in profound biological consequences. The appearance of phosphatidylserine in the plasma membrane outer leaflet results in the marked enhancement of coagulation, either appropriate as in hemostasis, or inappropriate as in acute lung injury. Similarly, the appearance of phosphatidylserine markedly enhances complement activation and serves as a signal for the recognition and uptake of apoptotic cells by phagocytes. Furthermore, scramblase activation will result in either release (from the synthesizing cell) or uptake (into target cells) of mediator phospholipids (platelet activating factor (PAF), lysophospholipids, and oxidized phospholipids) or their precursors (delivery of arachidonate to secreted PLA2). Depending on cell type, stimulus, and isoform of the scramblase, it is hypothesized that at least four mechanisms are involved in regulating scramblase activity: a) phosphorylation of scramblase by PKC (e.g. fMLP-stimulated neutrophils or Jurkat cell apoptosis), b) elevation of intracellular calcium in the absence of phosphorylation (e.g. thrombin-stimulated platelets), c) translocation to and from the membrane (fMLP-stimulated neutrophil) and d) cytokine-mediated up-regulation of scramblase expression (interferon alpha treated Raji cells). The aims of this project are to define the regulation of scramblase by phosphorylation and determine the mechanism(s) by which scramblase enhances transbilayer movement of membrane phospholipids. Integrated biochemical, structural and genetic approaches are described in which the scramblase phosphorylation activity, be identified and the effect of phosphorylation on scramblase activity, particularly with regard to calcium requirement, defined. Similarly, these same integrated approaches will be used to define the mechanism by which scramblase mediates the transbilayer movement of phospholipids, hypothesized to require either self-association or association with other proteins.