Blood Coagulation Protein - Metal Ion - Lipid Interactions

描述（由申请人提供）：在超过30年的持续资助期间，本研究计划的总体目标是确定维生素K依赖性凝血蛋白的体外和体内结构-功能关系，特别关注g-羧基谷氨酸（Gla）结构域（GD）与金属离子、膜和受体的相互作用。我们已经解决了这些问题，通过使用蛋白质化学，酶学，结构生物学，分子/细胞生物学和基因靶向策略。在最近的过去，该基金集中在GD模拟神经活性肽，conantokin，特别是它们的结构-功能关系方面的阳离子结合，N-甲基-D-天冬氨酸受体（NMDAR）结合，和他们的生物学特性的变构抑制离子通道开放和随之而来的调节Ca 2+稳态的神经元细胞和其他类型的细胞转染NMDAR亚基组合。在此更新申请中，努力将集中于将这些小的神经活性GD模拟肽的结构与它们与神经元NMDAR的不同亚基组合的功能相互作用的特异性以及它们的细胞信号传导特性相关联，所述神经元NMDAR在脑中是时间和空间可变的。为了利用化学（目的1）和细胞生物学（目的2）结果与体内使用基于伴丝蛋白的药物之间的关系，我们将使用大鼠闭塞性中风模型（目的3），其导致脑细胞的下游缺氧和随后的NMDAR离子通道的失调，导致细胞中钙水平异常，对宿主生物体具有有害后果（例如，缺血性中风后导致细胞死亡的神经元凋亡）。提出了三个高度相关的具体目标：1）描绘与芋螺毒素相互作用所需的NR 1和NR 2亚基的细胞外区域的组分，并鉴定芋螺毒素内决定NMDAR选择性的结构元件，测试芋螺毒素可以被工程化以实现其NMDAR亚基选择性的假设。2)在NMDAR亚基转染的HEK 293细胞和原代神经元中，研究芋头蛋白在调节细胞信号传导中ERK 1/2的NMDAR依赖性活化中的作用，检验NR 2B特异性芋头蛋白的下游神经保护作用是通过调节ERK 1/2途径的步骤实现的假设。3)采用大鼠闭塞性中风的体内模型来研究天然和变异的芋螺毒素对下游缺氧诱导的脑细胞凋亡的影响，检验芋螺毒素的NMDAR亚基特异性可用于减弱发生的凋亡过程的假设。该提议的总体假设是，结构不同的芋螺蛋白在不同的NMDAR亚基组合下显示出不同的效力和功效，这是理解许多神经疾病的分子基础的重要考虑因素，例如，缺血性中风相关的细胞死亡。 公共卫生相关性：通过激活的N-甲基-D-天冬氨酸受体（NMDAR）通道进入神经元细胞的异常钙离子流是多种神经病的全部或部分原因，包括缺血性中风后神经元炎驱动的细胞死亡。在这种情况下，需要通过NMDAR治疗性抑制钙流动。存在直接NMDAR离子通道阻断药物，但与其应用相关的拟精神副作用限制了这些药物的使用。来自锥螺的含有维生素K依赖性γ-羧基谷氨酸（Gla）的天然肽代表了潜在的重要治疗类别的NMDAR抑制剂，其对离子通道具有变构性，并通过与直接通道阻断无关的过程抑制钙流动。该提议涉及芋螺毒素在体外和体内的复杂作用机制，并将这些特性与其在闭塞性中风模型中的神经保护作用联系起来。

DESCRIPTION (provided by applicant): The overall goals of this research program during a more than a 30-year period of continual funding are to define structure-function relationships of vitamin K-dependent coagulation proteins, both in vitro and in vivo, with specific attention paid to interactions of the g-carboxyglutamic acid (Gla) domains (GD) with metal ions, membranes, and receptors. We have addressed these issues by use of protein chemistry, enzymology, structural biology, molecular/cell biology, and gene targeting strategies. During the recent past, this grant has centered on GD-mimetic neuroactive peptides, the conantokins, especially their structure-function relationships with regard to cation binding, N-methyl-D-aspartate receptor (NMDAR) binding, and their biological properties of allosteric inhibition of ion channel opening and consequent regulation of Ca2+ homeostasis in neuronal cells and in other cell types transfected with NMDAR subunit combinations. In this renewal application, efforts will be focused on relating the structures of these small neuroactive GD mimetic peptides to their specificities for functional interactions with different subunit combinations of the neuronal NMDAR, which are temporally and spatially variable in the brain, and with their cell signaling properties. To exploit the relationships between the chemical (aim 1) and cell biology (aim 2) results with the in vivo use of conantokin-based drugs, we will use a model of occlusive stroke in rats (aim 3), which results in downstream hypoxia of brain cells and consequent dysregulation of the NMDAR ion channel, leading to abnormal levels of calcium in cells, with deleterious consequences to the host organism (e.g., neuronal apoptosis resulting in cell death after ischemic stroke). Three highly interrelated specific aims are proposed: 1) to delineate the components of the extracellular regions of NR1 and NR2 subunits required for interaction with conantokins and to identify structural elements within the conantokins that dictate NMDAR selectivity, testing the hypothesis that conantokins can be engineered to enable their NMDAR subunit- selectivity. 2) to study the role of conantokins in modulating the NMDAR-dependent activation of ERK1/2 in cell signaling, in both NMDAR subunit-transfected HEK293 cells and in primary neurons, testing the hypothesis that the downstream neuroprotective effects of NR2B-specific conantokins are achieved by modulation of steps of the the ERK1/2 pathway. 3) to employ an in vivo model of occlusive stroke in rats to investigate the effects of native and variant conantokins on downstream hypoxia-induced brain cell apoptosis, examining the hypothesis that NMDAR subunit specificity of the conantokins can be employed to attenuate the apoptotic processes that occur. The overall hypothesis of this proposal is that conantokins that differ with respect to their structures display varying potency and efficacy at different NMDAR subunit combinations, an important consideration for understanding the molecular bases for many neurological diseases, e.g., ischemic stroke-related cell death. PUBLIC HEALTH RELEVANCE: Abnormal calcium ion flow into neuronal cells through activated N-methyl-D-aspartate receptor (NMDAR) channels is wholly or partially responsible for a variety of neuropathies, including neuronal apoptotis-driven cell death consequent to ischemic stroke. In such cases, therapeutic inhibition of calcium flow by the NMDAR is desirable. Direct NMDAR ion channel blockade drugs exist, but psychomimetic side-effects associated with their application limit the use of these agents. Vitamin K-dependent gamma-carboxyglutamate (Gla)- containing natural peptides from cone snails represent a potentially significant therapeutic class of NMDAR inhibitors that are allosteric to the ion channel and inhibit calcium flow by processes not associated with direct channel blocking. This proposal is concerned with the complex mechanisms of action of the conantokins in vitro and in vivo and relating these properties to their neuroprotective effects in a model of occlusive stroke.