ACTIVATION OF PI-SPECIFIC PHOSPHOLIPASE C ENZYMES

PI 特异性磷脂酶 C 酶的激活

• 批准号: 6520134
• 负责人: Mary Fedarko Roberts
• 金额: $ 20.23万
• 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/6520134
• 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.

磷脂酰肌醇特异性磷脂酶C酶催化磷酸肌醇切割为二酰甘油（蛋白激酶C的第二信使激活剂）和磷酸肌醇。该机制依次为磷酸转移酶步骤，形成中间体肌醇1，2-（环状）磷酸（cIP），然后通过环状磷酸二酯酶反应将cIP水解为肌醇磷酸（I-I- P）。哺乳动物酶作为PI信号传导的关键调节因子，在细胞生长和增殖中发挥着关键作用。由于PI-PLC酶是水溶性的，并且它们的正常底物磷酸肌醇位于膜中，因此它们的活性也可以通过脂质界面和其他膜定位的信号传导组分来调节。我们感兴趣的是识别和表征接口激活这些酶的机制。为此，我们集中在环状磷酸二酯酶反应和开发磷酸转移酶测定系统与可溶性底物（甘油磷酸肌醇磷酸或短链PI），不分区到接口。监测非底物界面如何影响这些可溶性反应，允许从界面底物的改变中分离对酶的变构效应。待研究的主要PI-PLC酶包括来自苏云金芽孢杆菌的小（35 kDa）细菌PI-PLC和最小（85 kDa）的哺乳动物同工酶PI-PLC δ 1。这种哺乳动物酶的活性位点模块在结构上与细菌酶非常相似，并且都表现出独特的动力学特征。各种物理技术（NMR、荧光、等温滴定量热法）将用于表征（1）功能不同的磷脂活化剂和催化位点之间的空间关系;（2）由脂质和溶剂活化剂诱导的PI-PLC酶的构象变化;（3）活化剂对酶与双层的整体结合的影响;（4）为什么哺乳动物酶平行产生cIP和I-1-P;和（5）G蛋白α亚基对PLC-beta2和PLC-beta3切割cIP的影响。这些研究的结果将提供一个完整的图片的环状磷酸二酯酶反应的每一个主要的PI-PLC类，允许比较这一步骤涉及一个'可溶性'底物与磷酸肌醇裂解，并提供一个详细的图片如何不同的接口，包括'激活'磷脂以及其他蛋白质，改变这种化学。这些信息将是至关重要的，在寻找方法来操纵这些酶在体内。