Blood Coagulation Protein - Metal Ion - Lipid Interactions

凝血蛋白-金属离子-脂质相互作用

• 批准号: 7819188
• 负责人: FRANCIS J CASTELLINO
• 金额: $ 3.48万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-08 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7819188
• 关键词: 3-Dimensional; Acids; Address; Adverse effects; Agonist; Amino Acid Sequence; Apoptosis; Apoptotic; Area; Attention; Attenuated; Behavior; Binding; Biochemical; Biological; Blood coagulation; Brain; Brain Ischemia; CREB1 gene; Calcium; Calcium ion; Cations; Cell Death; Cells; Cellular biology; Chemicals; Chimeric Proteins; Coagulation Process; Complex; Crystallography; Dimerization; Disease; Divalent Cations; Drug Design; Edema; Electrophysiology (science); Elements; Engineering; Enzymatic Biochemistry; Epitopes; Evaluation; Event; Functional disorder; Funding; Gene Targeting; Generations; Glean; Goals; Grant; Health; Hemostatic Agents; Hemostatic function; Heparin Binding; Hippocampus (Brain); Homeostasis; Hypoxia; In Vitro; Individual; Infarction; Inflammatory Response; Ion Channel; Ions; Ischemic Stroke; Knockout Mice; Kringles; Label; Laboratories; Lead; Lipids; Maintenance; Mediating; Membrane; Metals; Methodology; Methods; Middle Cerebral Artery Occlusion; Modeling; Molecular; Mus; N-Methyl-D-Aspartate Receptors; N-terminal; NR1 gene; Nature; Neurologic; Neuronal Hypoxia; Neurons; Neuropathy; Neurosciences; Organism; Pathway interactions; Pattern; Peptides; Pharmaceutical Preparations; Phospholipids; Physiological; Plasminogen; Process; Property; Protein C; Protein Chemistry; Proteins; Rattus; Reaction; Recombinants; Regulation; Reporting; Research; Retinal Cone; Roentgen Rays; Role; Science; Signal Pathway; Signal Transduction; Snails; Source; Specificity; Stroke; Structure; Structure-Activity Relationship; Synapses; System; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Thrombin; Time; Variant; Vitamin K; Work; analytical ultracentrifugation; attenuation; base; biological systems; brain cell; cell type; conformational conversion; design; excitotoxicity; extracellular; gamma Carboxyglutamate; in vivo; in vivo Model; inhibitor/antagonist; interest; microcalorimetry; mimetics; mouse model; nervous system disorder; neuron apoptosis; neuropathology; new technology; novel strategies; peptide structure; polypeptide; programs; protein function; protein structure; public health relevance; receptor; receptor binding; response; small molecule; structural biology

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.

描述（由申请人提供）：在超过30年的持续资金期间，该研究计划的总体目标是在体外和体内定义维生素K依赖性凝血蛋白的结构 - 功能关系，并特别注意G-carbarboxyglutamic Acid（GLA）（GD）（GD）与金属的相互作用，并受到金属，受体，味道，并受众。我们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来解决这些问题。 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亚基组合。在此续签应用中，将努力将这些小型神经活性GD模拟肽的结构与它们的特异性联系起来，以与神经元NMDAR的不同亚基组合的功能相互作用，它们在大脑中是时间和空间上的变化，以及其细胞信号传导特性。 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.,神经元细胞凋亡导致缺血性中风后细胞死亡）。提出了三个高度相互关联的特定目的：1）描述与蛋白酶相互作用所需的NR1和NR2亚基的细胞外区域的组成部分，并确定在蛋白酶中识别决定NMDAR选择性的结构元素，以测试依antokins可以通过工程化来启用其nMdar subunitientive conantokins的假设。 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）在大鼠中采用闭塞性中风的体内模型，以研究天然和变体康纳蛋白对下游低氧诱导的脑细胞凋亡的影响，以研究康纳氏蛋白的NMDAR亚基特异性可用于消除发生的副作用过程。该提案的总体假设是，在不同NMDAR亚基组合中，与其结构不同的结构构成蛋白表现出不同的效力和功效，这是理解许多神经疾病的分子碱基的重要考虑因素，例如，缺血性中风相关细胞死亡。 公共卫生相关性：通过活化的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动完全或部分负责各种神经性神经病，包括神经蛋白popoptotis驱动的细胞死亡，导致缺血性中断。在这种情况下，希望通过NMDAR对钙流的治疗抑制。存在直接的NMDAR离子通道阻断药物，但是与应用程序的使用限制了这些药物的使用相关的心理副作用。维生素K依赖性的γ-羧谷氨酸（GLA） - 含有锥蜗牛的天然肽代表了一种潜在的重要治疗性NMDAR抑制剂类别，它们对离子通道变构并通过与直接通道相关的过程抑制钙流。该提议与conantokins在体外和体内的复杂作用机理有关，并将这些特性与其神经保护作用相关联在闭塞性中风模型中。