Membrane Phospholipid Distribution/Mediator Translocatio

描述（改编自申请人摘要）炎症细胞激活后，质膜磷脂的大体积跨双分子层运动增强（触发器）导致深远的生物学后果。磷脂酰丝氨酸在质膜外小叶的出现导致凝血明显增强，这在止血时是适当的，在急性肺损伤时则是不适当的。同样，磷脂酰丝氨酸的出现显著增强了补体活化，并作为吞噬细胞识别和摄取凋亡细胞的信号。此外，超燃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）或它们的前体（将花生四烯酸酯递送到分泌的PLA2）的释放（从合成细胞）或摄取（进入靶细胞）。根据细胞类型、刺激和超燃酶的异构体，假设至少有四种机制参与调节超燃酶的活性：a)由PKC磷酸化（如fmlp刺激的中性粒细胞或Jurkat细胞凋亡），b)在没有磷酸化的情况下细胞内钙的升高（如凝血酶刺激的血小板），c)向膜转运（fmlp刺激的中性粒细胞）和d)细胞因子介导的促裂酶表达上调（干扰素α处理的Raji细胞）。这个项目的目的是通过磷酸化来定义对超燃酶的调节，并确定超燃酶增强膜磷脂跨双分子层运动的机制。综合生化，结构和遗传方法，其中鉴定了超燃酶磷酸化活性，并定义了磷酸化对超燃酶活性的影响，特别是对钙需求的影响。同样，这些相同的综合方法将用于定义炒合酶介导磷脂跨双分子层运动的机制，假设需要自结合或与其他蛋白质结合。

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.