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NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

缺氧期间 NMDA 受体的无介导修饰

基本信息

批准号：
7675059
负责人：
OM P MISHRA
金额：
$ 31.88万
依托单位：
DREXEL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-06-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7675059
关键词：
7-nitroindazole Active Sites Address Animals Arginine Binding Biotin Brain Caspase Cell Death Cell membrane Cerebral Hypoxia Cerebral cortex Cerebrum Cessation of life Characteristics Charge Cyclic AMP-Responsive DNA-Binding Protein Cysteine DNA DNA Fragmentation DNA Nucleotidylexotransferase DNTT gene Data Development Diethylamines Energy Metabolism Enzymes Epidermal Growth Factor Receptor Fluorescence Spectrometry Free Radicals Functional disorder Generations Genetic Transcription Hypoxia Hypoxic Neuronal Death Immunoblotting In Situ Nick-End Labeling In Vitro Label Lead Mass Spectrum Analysis Measurement Mediating Membrane Modeling Modification Molecular N-Methyl-D-Aspartate Receptors Neurons Newborn Infant Nitric Oxide Nitric Oxide Donors Nitric Oxide Synthase Nitric Oxide Synthase Type I Nuclear Numbers Object Attachment Pathway interactions Peroxonitrite Phosphocreatine Phosphorylation Phosphotransferases Prevention strategy Protein Dephosphorylation Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Protocols documentation SH protein-tyrosine phosphatase Sodium Techniques Testing Tissues Tyrosine Tyrosine Phosphorylation apoptotic protease-activating factor 1 base calmodulin-dependent protein kinase IV caspase-3 caspase-9 design diethylamine enzyme activity indexing inhibitor/antagonist inorganic phosphate kinase inhibitor novel pro-caspase-3 pro-caspase-9 protein expression protein tyrosine phosphatase 1B src-Family Kinases

项目摘要

The proposed studies will investigate nitric oxide (NO)-mediated mechanism of procaspase-9 activation that results in hypoxic neuronal death by transcription-independent and transcription-dependent pathways in the newborn. We will test the hypothesis that NO produced during hypoxia leads to increased phosphorylation at tyrosine 153 (Tyr153) of procaspase-9 and increased phosphorylation at tyrosine 24 (Tyr24) of apoptotic protease activating factor-1 (Apaf-1) resulting in tyrosine phosphorylation-dependent activation of procaspase-9.. We propose that tyrosine phosphorylation at specific residues during hypoxia leads to increased binding of procaspase-9 with Apaf-1 due to increased charge-charge interaction (increased number of H-bonds) between their prodomains and results in increased activation of procaspase-9. We propose that NO free radicals generated during hypoxia lead to inactivation of protein tyrosine phosphatases (PTP, SH-PTP-1 and SH-PTP-2) and result in increased activation of protein tyrosine kinases (PTK, EGFR kinase and Src kinase) and subsequently increased tyrosine phosphorylation of procaspase-9 and Apaf-1. The degree of cerebral tissue hypoxia will be determined by measurement of high energy phosphate compounds. Experimental protocols will be carried out on newborn piglets investigating: (1) the effect of hypoxia on Tyr153 phosphorylation of procaspase-9 and Tyr24 phosphorylation of Apaf-1, expression and activation of procaspase-9 and relate these to the degree of cerebral hypoxia; and perform these studies with the administration of highly selective nNOS inhibitors to demonstrate that the hypoxia-induced increased Tyr153 phosphorylation of procaspase-9, Tyr24 phosphorylation of Apaf-1, expression and increased activation of procaspase-9 are mediated by nNOS-derived NO; (2) the effect of inhibitors of EGFR kinase and Src kinase on increased phosphorylation of procaspase-9 at Tyr153 and Apaf-1 at Tyr24, activation of procaspase- 9 and procaspase-3, nuclear DNA fragmentation and immunocytochemical and morphological indices of neuronal death, to demonstrate that the mechanism of procaspase-9 activation that results in hypoxic neuronal death is dependent on EGFR and Src kinase activity; (3) the effect of tyrosine phosphorylation and dephosphorylation of procaspase-9 in vitro on caspase-9 activity to demonstrate that tyrosine phosphorylation regulates procaspase-9 activation; (4) the effect of NO donor (diethylamine NONOate) and peroxynitrite on the activity of SH-PTP-1 and SH-PTP-2 to demonstrate that NO-free radicals generated during hypoxia lead to inactivation of SH-PTP-1 and SH-PTP-2 resulting in increased activation of EGFR kinase and Src kinase and subsequently increased tyrosine phosphorylation of procaspase-9 and Apaf-1 by a protein tyrosine kinase-dependent mechanism. The proposed studies will establish a novel mechanism of NO-mediated EGFR kinase and Src kinasedependent tyrosine phosphorylation of procaspase-9 at Tyr153 and Apaf-1 at Tyr24 leading to increased activation of procaspase-9 that results in hypoxic neuronal death. The elucidation of NO-mediated tyrosine phosphorylation-dependent molecular mechanism of procaspase-9 activation during hypoxia will aid in the development of novel tyrosine-kinase inhibition based preventive strategies for hypoxia-induced brain dysfunction in the newborn.

拟议的研究将调查一氧化氮 (NO) 介导的 procaspase-9 激活机制通过转录独立和转录依赖性途径导致缺氧神经元死亡新生。我们将检验以下假设：缺氧期间产生的 NO 会导致磷酸化增加 procaspase-9 的酪氨酸 153 (Tyr153) 和凋亡细胞酪氨酸 24 (Tyr24) 的磷酸化增加蛋白酶激活因子-1 (Apaf-1) 导致酪氨酸磷酸化依赖性激活 procaspase-9..我们提出缺氧期间特定残基的酪氨酸磷酸化导致由于电荷-电荷相互作用的增加，procaspase-9 与 Apaf-1 的结合增加（增加前结构域之间的氢键数量），导致 procaspase-9 的激活增加。我们提出缺氧时产生的NO自由基导致蛋白酪氨酸磷酸酶失活（PTP、SH-PTP-1 和 SH-PTP-2）并导致蛋白酪氨酸激酶（PTK、EGFR 激酶和 Src 激酶），随后增加了 procaspase-9 和 Apaf-1 的酪氨酸磷酸化。通过测定高能磷酸盐来判断脑组织缺氧程度化合物。实验方案将在新生仔猪上进行，调查：（1）缺氧对 procaspase-9 的 Tyr153 磷酸化和 Apaf-1 的 Tyr24 磷酸化、表达和激活 procaspase-9 并将其与脑缺氧程度相关；并进行这些研究通过施用高度选择性的 nNOS 抑制剂来证明缺氧诱导的增加 procaspase-9 的 Tyr153 磷酸化、Apaf-1 的 Tyr24 磷酸化、表达和激活增加 procaspase-9 由 nNOS 衍生的 NO 介导； (2)EGFR激酶和Src抑制剂的作用激酶增加 procaspase-9 在 Tyr153 和 Apaf-1 在 Tyr24 的磷酸化，激活 procaspase- 9和procaspase-3、核DNA片段化以及免疫细胞化学和形态学指标神经元死亡，以证明 procaspase-9 激活导致缺氧的机制神经元死亡取决于 EGFR 和 Src 激酶活性； (3)酪氨酸磷酸化的影响体外 procaspase-9 去磷酸化对 caspase-9 活性的影响，以证明酪氨酸磷酸化调节 procaspase-9 激活； (4)NO供体(二乙胺NONOate)的作用和过氧亚硝酸盐对 SH-PTP-1 和 SH-PTP-2 活性的影响，证明 NO 自由基的产生缺氧期间导致 SH-PTP-1 和 SH-PTP-2 失活，导致 EGFR 激活增加激酶和 Src 激酶，随后通过增加 procaspase-9 和 Apaf-1 的酪氨酸磷酸化蛋白酪氨酸激酶依赖性机制。拟议的研究将建立 NO 介导的 EGFR 激酶和 Src 激酶依赖性的新机制 procaspase-9 在 Tyr153 处和 Apaf-1 在 Tyr24 处的酪氨酸磷酸化导致增加激活 procaspase-9，导致神经元缺氧死亡。 NO介导的酪氨酸的阐明缺氧期间 procaspase-9 激活的磷酸化依赖性分子机制将有助于开发基于酪氨酸激酶抑制的新型脑缺氧预防策略新生儿功能障碍。