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，本研究项目的总体目标是确定体外和体内维生素k依赖性凝血蛋白的结构-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。我们利用蛋白质化学、酶学、结构生物学、分子/细胞生物学和基因靶向策略来解决这些问题。在最近的过去，该资助集中在gd -模拟神经活性肽，conantokins，特别是它们在阳离子结合方面的结构-功能关系，n -甲基- d -天冬氨酸受体（NMDAR）结合，以及它们在神经细胞和其他转染了NMDAR亚基组合的细胞类型中对离子通道开放的变构抑制和随后的Ca2+稳态调节的生物学特性。在这个更新的应用中，努力将集中在这些小的神经活性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，这些亚基组合在大脑中是时间和空间可变的，以及它们的细胞信号传导特性。为了探索化学（目的1）和细胞生物学（目的2）结果与体内使用基于conantokin的药物之间的关系，我们将使用大鼠闭塞性卒中模型（目的3），该模型导致脑细胞下游缺氧和随后的NMDAR离子通道失调，导致细胞内钙水平异常，对宿主生物有害（例如，缺血性卒中后神经元凋亡导致细胞死亡）。提出了三个高度相关的具体目标：1)描述与conantokins相互作用所需的NR1和NR2亚基的细胞外区域的成分，并确定conantokins中决定NMDAR选择性的结构元件，测试conantokins可以被设计以使其具有NMDAR亚基选择性的假设。2)在NMDAR亚单位转染的HEK293细胞和原代神经元中，研究conantokins在调节NMDAR依赖的ERK1/2在细胞信号传导中的激活中的作用，验证nr2g特异性conantokins通过调节ERK1/2通路的步骤来实现下游神经保护作用的假设。3)采用大鼠缺血性卒中的体内模型，研究天然和变异conantokins对下游缺氧诱导的脑细胞凋亡的影响，验证conantokins的NMDAR亚基特异性可以减弱发生的凋亡过程的假设。该建议的总体假设是，结构不同的康泛素在不同的NMDAR亚基组合中表现出不同的效力和功效，这是理解许多神经系统疾病（例如缺血性卒中相关细胞死亡）的分子基础的重要考虑因素。公共卫生相关性：异常钙离子通过激活的n -甲基-d -天冬氨酸受体（NMDAR）通道进入神经元细胞，是多种神经病变的全部或部分原因，包括缺血性中风引起的神经元凋亡驱动的细胞死亡。在这种情况下，通过NMDAR治疗性抑制钙流是可取的。存在直接的NMDAR离子通道阻断药物，但其应用相关的拟精神副作用限制了这些药物的使用。来自锥体蜗牛的含维生素k依赖性γ -羧谷氨酸（Gla）的天然肽代表了一种潜在的重要治疗类NMDAR抑制剂，它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。这一建议涉及康妥酮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卒中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.