Regulatory Mechanisms of Secretory Phospholipases A2

描述(申请者描述)：我们的长期目标是确定 分泌型磷脂酶A2(PLA2)激活的结构基础 与聚集的底物结合(界面活化)。PLA2水解物 磷脂来释放脂肪酸和溶质脂，从而启动 二十烷基类化合物和血小板激活因子的生物合成 炎症、过敏、细胞凋亡和肿瘤发生。该项目的重点是 分泌PLA2，包括人胰腺和滑膜PLA2，因为这些 酶具有重要的生物学和临床意义及其调节作用 人们对这一机制知之甚少。我们的具体目标是：1.确定角色 膜静电性、膜结合强度、取向 膜结合的PLA2和膜诱导的PLA2激活的结构变化。 为了验证我们的假设，膜表面特性和膜诱导 PLA2的结构变化在PLA2的激活中起协同作用，a 膜表面电势之间的关系将被建立， 膜结合PLA2，酶的结构变化和PLA2活性。 磷脂酶A2对膜表面性质的相互作用及其作用 对PLA2活化过程中的脂肪酸和溶菌脂进行了研究。3D 膜结合PLA2在活化前后的取向将是 基于未标记和片段-13C标记的红外二色性测定 PLA2来验证PLA2在膜表面重新定向的假设 可能与PLA2的激活有关。2.表征的构象变化 在膜结合和激活过程中，不同组的PLA2分泌。至 解决由表面吸附引起的PLA2的构象变化， 我们将研究PLA2的次级和动态结构变化 时间分辨FTIR、核磁共振与磷脂膜或胶束结合的研究 荧光光谱和圆二色谱。通过使用FTIR和核磁共振技术，我们 将检验我们的假设，即PLA2的膜结合诱导 标准的αI螺旋到不太稳定的αII螺旋。我们将通过FTIR进行检查 光谱学阿尔法-阿尔法-阿尔法II转变是否能被高反转 压力或低温。3.确定PLA2的特定区域 磷脂酶A2与膜结合后发生结构变化并参与其 激活。多学科方法，使用节段性同位素标记 PLA2，FTIR，核磁共振，荧光光谱和酰胺氢交换，将 应用于实现这一目标。中的全局和站点特定的更改 PLA2在界面活化过程中的二级和动态结构 经~(13)C同位素编辑的FTIR和异核核磁共振测定。FAST动态 将测量PLA2主链在其界面活化过程中的 使用15N核弛豫技术。总体而言，我们的结果将有助于 开发调控PLA2的新策略，这是一种深刻的生物医学酶 重要性。

Description (applicant's description): Our long-term goal is to identify the structural basis for the activation of secretory phospholipases A2 (PLA2) upon binding to the aggregated substrate (interfacial activation). PLA2s hydrolyze phospholipids to free fatty acids and lysolipids and thus initiate the biosynthesis of eicosanoids and platelet activating factor, potent mediators of inflammation, allergy, apoptosis and tumorigenesis. The project is focused on secretory PLA2s, including human pancreatic and synovial PLA2s, because these enzymes are of great biological and clinical importance and their regulatory mechanisms are poorly understood. Our specific aims are: 1. Identify the roles of membrane electrostatics, membrane binding strength, orientation of membrane-bound PLA2 and membrane-induced structural changes in PLA2 activation. To test our hypothesis that membrane surface properties and membrane-induced structural changes in PLA2 act synergistically in activation of PLA2, a relationship will be established between the membrane surface potential, membrane binding of PLA2, structural changes in the enzyme and PLA2 activity. The reciprocal effects of PLA2 on membrane surface properties and the roles of the fatty acid and lysolipid in PLA2 activation will be studied. The 3D orientation of membrane-bound PLA2 before and after activation will be determined based on the infrared dichroism of unlabeled and segment-13C-labeled PLA2 to test the hypothesis that reorientation of PLA2 at the membrane surface may contribute to PLA2 activation. 2. Characterize conformational changes in secretory PLA2s of different groups during membrane binding and activation. To resolve conformational changes in PLA2s that are induced by surface adsorption, we will study the secondary and dynamic structural changes in PLA2s upon binding to phospholipid membranes or micelles using FTIR, NMR, time-resolved fluorescence spectroscopy and circular dichroism. By using FTIR and NMR, we will test our hypothesis that membrane binding of PLA2 induces a conversion of standard alphaI helices to less stable alphaII helices. We will check by FTIR spectroscopy whether alphaI-alphaII transition can be reverted by high pressures or low temperatures. 3. Identify the specific regions of PLA2 that undergo structural changes upon membrane binding of PLA2 and contribute to its activation. A multidisciplinary approach, using segmental isotopic labeling of PLA2s, FTIR, NMR, fluorescence spectroscopy and amide hydrogen exchange, will be applied to accomplish this aim. Global and site-specific changes in the secondary and dynamic structure of PLA2 during interfacial activation will be determined by 13C-isotope-edited FTIR and heteronuclear NMR. The fast dynamics of the main chain of PLA2 during its interfacial activation will be measured using 15N nuclear relaxation techniques. Collectively, our results will help develop new strategies for regulation of PLA2s, enzymes of profound biomedical importance.