NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

拟开展的研究将探讨一氧化氮（NO）介导的caspase-9活化机制， 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。 新生儿我们将检验这一假设，即缺氧时产生的NO导致磷酸化水平增加， 酪氨酸153（Tyr 153）和增加的酪氨酸24（Tyr 24）磷酸化的凋亡蛋白。 蛋白酶激活因子-1（Apaf-1）导致酪氨酸磷酸化依赖性激活 半胱天冬酶原-9我们认为，在缺氧过程中，特定残基的酪氨酸磷酸化导致 由于电荷-电荷相互作用增加（增加），半胱天冬酶原-9与Apaf-1的结合增加 氢键数目），并导致半胱天冬酶原-9的活化增加。我们 提出在缺氧过程中产生的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）的活化增加 激酶和Src激酶），并随后增加半胱天冬酶原-9和Apaf-1的酪氨酸磷酸化。 脑组织缺氧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，研究：（1） 缺氧对半胱氨酸天冬氨酸蛋白酶原-9的Tyr 153磷酸化和Apaf-1的Tyr 24磷酸化、表达和 procaspase-9的激活，并将其与脑缺氧的程度联系起来;并进行这些研究 给予高选择性nNOS抑制剂，以证明缺氧诱导的 半胱氨酸天冬氨酸蛋白酶原-9的Tyr 153磷酸化，Apaf-1的Tyr 24磷酸化，表达和活化增加 nNOS源性NO介导的caspase-9前体蛋白的表达;（2）EGFR激酶和Src抑制剂的作用 激酶对半胱氨酸蛋白酶原-9（procaspase-9）Tyr 153和Apaf-1（Tyr 24）磷酸化的影响，半胱氨酸蛋白酶原-9（procaspase-9）的活化， 9和半胱天冬酶原-3，核DNA片段化和免疫细胞化学和形态学指标， 神经元死亡，以证明导致缺氧的半胱氨酸天冬氨酸蛋白酶原-9活化的机制， 神经元死亡依赖于EGFR和Src激酶活性;（3）酪氨酸磷酸化的作用， 体外胱天蛋白酶原-9去磷酸化对胱天蛋白酶-9活性的影响，以证明酪氨酸 磷酸化调节蛋白酶原-9活化;（4）NO供体（二乙胺NONOate）和 过氧亚硝酸盐对SH-PTP-1和SH-PTP-2活性的影响，以证明NO-自由基产生 缺氧导致SH-PTP-1和SH-PTP-2失活，导致EGFR活化增加 激酶和Src激酶，并随后增加蛋白酶原-9和Apaf-1的酪氨酸磷酸化， 一种蛋白酪氨酸激酶依赖性机制。 本研究将建立一种新的NO介导的EGFR激酶和Src激酶依赖的机制 蛋白酶原-9在Tyr 153和Apaf-1在Tyr 24的酪氨酸磷酸化导致增加的 导致缺氧性神经元死亡的半胱氨酸天冬氨酸蛋白酶原-9的活化。一氧化氮介导的酪氨酸 缺氧时半胱氨酸天冬氨酸蛋白酶原-9活化的磷酸化依赖性分子机制将有助于 基于酪氨酸激酶抑制剂的缺氧性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.