Regulatory Mechanisms of Secretory Phospholipases A2

描述（申请人的描述）：我们的长期目标是确定 分泌型磷脂酶A2（PLA 2）激活的结构基础， 与聚集的基质结合（界面活化）。PLA 2水解 磷脂转化为游离脂肪酸和溶血脂，从而引发 类花生酸和血小板活化因子的生物合成， 炎症、过敏、细胞凋亡和肿瘤发生。该项目着重 分泌型PLA 2，包括人胰腺和滑膜PLA 2，因为这些 酶具有重要的生物学和临床意义， 机制知之甚少。我们的具体目标是：1.确定角色 膜的静电、膜结合强度、膜的取向 膜结合的PLA 2和膜诱导的PLA 2活化的结构变化。 为了验证我们的假设，即膜表面性质和膜诱导的 PLA 2的结构变化在PLA 2的活化中协同作用， 将在膜表面电位之间建立关系， PLA 2的膜结合、酶的结构变化和PLA 2活性。 磷脂酶A_2对膜表面性质的交互作用及磷脂酶A_2在膜表面性质中的作用 将研究PLA 2活化中的脂肪酸和溶血脂质。的3D 在活化之前和之后，膜结合的PLA 2的取向将是 基于未标记和片段-13C-标记的红外二色性测定 PLA 2的重定向，以检验PLA 2在膜表面的重定向 可能有助于PLA 2激活。2.表征以下物质的构象变化： 在膜结合和激活过程中，不同群体的分泌型PLA 2。到 解决由表面吸附引起的PLA 2构象变化， 我们将研究PLA 2的二级和动态结构变化， 结合磷脂膜或胶束使用FTIR，NMR，时间分辨 荧光光谱和圆二色性。通过FTIR和NMR， 将检验我们的假设，即PLA 2的膜结合诱导了 标准α I螺旋到较不稳定的α II螺旋。我们将通过FTIR检查 α I-α II跃迁是否可以被高 压力或低温。3.确定PLA 2的特定区域， 在PLA 2的膜结合后发生结构变化，并有助于其 activation.一种多学科的方法，使用节段性同位素标记， PLA 2s、FTIR、NMR、荧光光谱和酰胺氢交换，将 应用于实现这一目标。全球和特定地点的变化 界面活化过程中PLA 2的二级和动态结构将被 通过13 C-同位素编辑的FTIR和杂波NMR测定。快速动态 将测量PLA 2的主链在其界面活化期间的 使用15 N核弛豫技术。总的来说，我们的结果将有助于 开发新的策略，用于调节PLA 2s，具有深远生物医学意义的酶 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.