REGULATION OF PHOSPHOINOSITIDE SPECIFIC PHOSPHOLIPASE C

磷酸肌醇特异性磷脂酶 C 的调节

• 批准号: 6179707
• 负责人: Mario J Rebecchi
• 金额: $ 18.66万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179707
• 关键词: calcium binding protein; chimeric proteins; conformation; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate analog; fluorescence spectrometry; gene targeting; guanine nucleotide binding protein; isozymes; membrane activity; membrane lipids; molecular site; nucleic acid sequence; phosphatidylinositols; phospholipase C; polymerase chain reaction; protein engineering; protein sequence; site directed mutagenesis

Phosphoinositide-specific phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to yield inositol 1,4,5-trisphosphate and diacylglycerol, two intracellular second messengers that are active in all eukaryotes. Operating through calcium and protein kinases, these signaling molecules trigger a broad range of bioligical responses that include hormone and neurotransmitter secretion, smooth muscle contraction and mitosis. Although the broad outlines of PLC regulation are known, the molecular basis for its activation is not. Contributing to this lack of progress is the misunderstood role of the membrane surface, which is both a barrier to substrate access and an actiation cofactor. The goals of this project are (1) to identify the regions of PLC that facilitate phospholipid binding and access to substrate, (2) to determine how calcium ions and GTP-binding proteins control catalysis and (3) to test the idea that penetration of the membrane surface activates the enzyme by an 'induced fit' mechanism. PLC-delta1 and beta2, isozymes with distinct modes of regulation, will be studies in parallel to gain insight into the activation process. A combination of genetic and biophysical approaches will be used. The modular regions found in the PLC isozymes (and a host of other signaling proteins), including the pleckstrin homology (PH), EF-hand and C-2 domains, will be expressed as isolated fusion proteins. Function in the intact enzyme will be further tested by subjecting critical residues to site-directed mutagenesis and by exchanging each region of PLC-delta1 with the corresponding module found in PLC-beta2. The binding properties and catalytic behaviors of these engineered proteins will be studied in membrane bilayers, monolayers, and detergent/phospholipid mixed micelles, and in solution with monomeric substrate analogs. High pressure fluorescence spectrosopy will be used to examine the relationships between emebrane penetration, catalysis and the enzyme's conformational state.

磷酸肌醇特异性磷脂酶C（PLC）水解 磷脂酰肌醇4,5-双磷酸（PI（4,5）P2）产生肌醇 1,4,5-三磷酸和二酰基甘油，两个细胞内第二 活跃于所有真核生物的使者。 操作 钙和蛋白激酶，这些信号分子触发了宽阔的 包括激素和神经递质在内的生物原始反应范围 分泌，平滑肌收缩和有丝分裂。 虽然很广泛 PLC调节的轮廓是已知的，这是其分子基础 激活不是。 缺乏进步的原因是 误解了膜表面的角色，这既是障碍物 底物访问和作用辅助因子。 这个项目的目标 是（1）识别促进磷脂的PLC区域 结合和访问底物，（2）确定钙离子和如何 GTP结合蛋白控制催化和（3）测试以下想法 膜表面的渗透会激活酶 “诱发拟合”机制。 plc-delta1和beta2，同工酶具有独特的同工酶 调节模式将是并行研究，以洞悉 激活过程。 遗传和生物物理的结合 将使用方法。 PLC中发现的模块化区域 同工酶（以及许多其他信号蛋白），包括 PLECKSTRIN同源性（pH），EF手和C-2域，将是 表示为孤立的融合蛋白。 完整酶的功能 将通过将关键残留物对定向进行进一步测试 诱变并通过将PLC-DELTA1的每个区域与 在PLC-BETA2中发现的相应模块。 结合特性 这些工程蛋白的催化行为将在 膜双层，单层和洗涤剂/磷脂混合 胶束，在具有单体底物类似物的溶液中。 高的 压力荧光光谱将用于检查 艾米布兰渗透，催化与酶之间的关系 构象状态。