NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

拟议的研究将探讨一氧化氮(NO)介导的前天冬氨酸酶-9激活机制， 通过转录非依赖性和转录依赖性途径导致缺氧性神经元死亡 刚出生的。我们将检验这样一种假设，即缺氧时产生的NO会导致磷酸化增加 原天冬氨酸酶-9的酪氨酸153(Tyr153)和酪氨酸24(Tyr24)的磷酸化增加 蛋白水解酶激活因子-1(APAF-1)导致酪氨酸磷酸化激活 Proaspase-9..我们认为，低氧时特定残基的酪氨酸磷酸化导致 由于电荷相互作用增加，原天冬氨酸氨基转移酶-9与APAF-1的结合增加(增加 它们的原结构域之间的氢键数量)，并导致原天冬氨酸氨基转移酶-9的激活增加。我们 提出低氧时产生的NO自由基可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)的激活增加 激酶和Src激酶)，随后增加原天冬氨酸氨基转移酶-9和APAF-1的酪氨酸磷酸化。 脑组织缺氧的程度将通过测量高能磷酸盐来确定 化合物。实验方案将在新生仔猪上进行，调查：(1) 低氧对前天冬氨酸氨基转移酶-9的Tyr153磷酸化和APAF-1的Tyr24磷酸化的影响 原天冬氨酸氨基转移酶-9的激活及其与脑缺氧程度的关系；并进行这些研究 给予高度选择性的nNOS抑制剂以证明缺氧诱导的 前天冬氨酸氨基转移酶-9的Tyr153磷酸化，APAF-1的Tyr24磷酸化，表达和激活增加 原天冬氨酸氨基转移酶-9的表达是由nNOS产生的NO介导的；(2)EGFR激酶和Src抑制剂的作用 酪氨酸天冬氨酸氨基转移酶原-9在Tyr153处和APAF-1在Tyr24处的磷酸化增加，激活原天冬氨酸氨基转移酶-2 9和原天冬氨酸氨基转移酶-3、细胞核DNA片段化及免疫细胞化学和形态指标 神经元死亡，以证明导致缺氧的原天冬氨酸酶-9激活的机制 神经元的死亡依赖于EGFR和Src激酶的活性；(3)酪氨酸磷酸化和 体外去磷酸化前天冬氨酸酶-9对半胱氨酸天冬氨酸酶-9活性的影响 磷酸化调节前天冬氨酸酶-9的激活；(4)NO供体(二乙胺NOate)和 过氧亚硝酸根对SH-PTP-1和SH-PTP-2活性的影响 缺氧导致SH-PTP-1和SH-PTP-2失活，导致EGFR活性增加 蛋白酪氨酸酶原-9和蛋白天冬氨酸氨基转移酶-1的酪氨酸磷酸化水平 蛋白酪氨酸激酶依赖的机制。 建议的研究将建立一种新的机制，即NO介导的EGFR激酶和Src激酶依赖 Tyr153位的前天冬氨酸氨基转移酶-9和24位的APAF-1的酪氨酸磷酸化导致 激活前天冬氨酸氨基转移酶-9，导致缺氧性神经元死亡。一氧化氮介导的酪氨酸的研究进展 低氧时原天冬氨酸酶-9激活依赖于磷酸化的分子机制将有助于 基于酪氨酸激酶抑制的低氧脑损伤预防策略的研究进展 新生儿的功能障碍。

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.