ACTIVATION OF PI-SPECIFIC PHOSPHOLIPASE C ENZYMES

PI 特异性磷脂酶 C 酶的激活

• 批准号: 6363328
• 负责人: Mary Fedarko Roberts
• 金额: $ 19.65万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363328
• 关键词: G protein; conformation; enzyme activity; enzyme mechanism; enzyme structure; nuclear magnetic resonance spectroscopy; phosphatidylinositols; phosphodiesterases; phospholipase C; phosphotransferases

Phosphatidylinositol-specific phospholipase C enzymes catalyze the cleavage of phosphoinositides to diacylglycerol (a second messenger activator of protein kinase C) and inositol phosphates. The mechanism is sequential with a phosphotransferase step forming the intermediate inositol 1,2-(cyclic) phosphate (cIP) followed by a cyclic phosphodiesterase reaction to hydrolyze the cIP to inositol phosphate (I-I- P). The mammalian enzymes, as key regulators of PI-signaling have critical roles in cell growth and proliferation. Since PI-PLC enzymes are water-soluble and their normal substrates, phosphoinositides, are localized in membranes, their activity can also be modulated by lipid interfaces and other membrane localized signaling components. We are interested in identifying and characterizing the mechanisms by which interfaces activate these enzymes. To that end we have concentrated on the cyclic phosphodiesterase reaction and developed phosphotransferase assay systems with soluble substrates (glycerol phosphoinositol phosphates or short-chain PI) that do not partition into interfaces. Monitoring how non-substrate interfaces affect these soluble reactions allows separation of allosteric effects on the enzyme from alterations of the interfacial substrate. The primary PI-PLC enzymes to be studied include a small (35 kDa) bacterial PI-PLC from Bacillus thuringiensis and the smallest (85 kDa) of the mammalian isozymes, PI-PLCdelta1. The active site module of this mammalian enzyme is structurally quite similar to the bacterial enzyme, and both exhibit unique kinetic features. A variety of physical techniques (NMR, fluorescence, isothermal titration calorimetry) will be used to characterize (1) the spatial relationship between functionally distinct phospholipid activator and catalytic sites; (2) the conformational changes in PI-PLC enzymes induced by lipid and solvent activators; (3) the effect of activators on bulk binding of the enzymes to bilayers; (4) why mammalian enzymes generate cIP and I-1-P in parallel; and (5) the effect of G protein alpha subunit on PLC-beta2 and PLC-beta3 cleavage on cIP. The results of these studies will provide a complete picture of the cyclic phosphodiesterase reaction for each of the major PI-PLC classes, allow comparison of this step involving a 'soluble' substrate with phosphoinositide cleavage, and provide a detailed picture of how different interfaces, include 'activating' phospholipids as well as other proteins, alter this chemistry. This information will be critical in finding ways to manipulate these enzymes in vivo.

磷脂酰肌醇特异的磷脂酶C酶催化磷脂酰肌醇裂解成二酰甘油(蛋白激酶C的第二信使激活剂)和肌醇磷酸盐。其机理是依次进行的，先是磷酸转移酶生成中间体1，2-(环)磷酸肌醇(CIP)，然后是环磷酸二酯酶反应，将CIP水解成肌醇磷酸(I-I-P)。哺乳动物的酶作为PI信号的关键调节因子，在细胞的生长和增殖中起着至关重要的作用。由于PI-PLC酶是水溶性的，其正常底物肌醇磷脂定位于膜上，其活性也可以受到脂类界面和其他膜定位信号成分的调节。我们感兴趣的是确定和表征界面激活这些酶的机制。为此，我们专注于环状磷酸二酯酶反应，并开发了具有不分界面的可溶性底物(甘油、磷酸肌醇或短链PI)的磷酸转移酶检测系统。监测非底物界面如何影响这些可溶反应，可以将变构对酶的影响与界面底物的变化分开。要研究的主要PI-PLC酶包括苏云金芽孢杆菌的一个小(35 KDa)细菌PI-PLC和哺乳动物同工酶中最小的(85 KDa)PI-PLCdelta1。这种哺乳动物酶的活性部位模块在结构上与细菌酶非常相似，都表现出独特的动力学特征。各种物理技术(核磁共振、荧光、等温滴定热法)将被用来表征(1)功能不同的磷脂激活剂和催化部位之间的空间关系；(2)脂类和溶剂激活剂诱导的PI-PLC酶的构象变化；(3)激活剂对酶与双层结合的影响；(4)哺乳动物酶为什么平行产生CIP和I-1-P；以及(5)G蛋白α亚基对PLC-β2和PLC-β3裂解的影响。这些研究的结果将提供每个主要PI-PLC类别的环状磷酸二酯酶反应的完整图景，允许比较这一步骤涉及的‘可溶性’底物和磷脂酰肌醇的切割，并提供不同的界面，包括‘激活’磷脂以及其他蛋白质，如何改变这种化学的详细图景。这些信息将是找到在体内操纵这些酶的方法的关键。