14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide
14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide
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DOI：
10.1074/jbc.m111.271973
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发表时间：
2011-12-09
影响因子：
4.8
通讯作者：
Gladwin, Mark T.
中科院分区：
文献类型：
--
作者：
Jayaraman, Thottala;Tejero, Jesus;Gladwin, Mark T.
神经球蛋白在体外和体内保护神经元免受缺氧影响；然而，人们对这种效应的根本机制仍知之甚少。大多数神经球蛋白以六配位状态存在，血红素袋中的近端和远端组氨酸直接与血红素铁结合。在平衡状态下，五坐标神经红蛋白的浓度仍然很低（0.1-5%）。最近的研究表明，神经球蛋白表面硫醇二硫化物形成的翻译后氧化还原调节增加了血红素口袋的开放概率，并允许亚硝酸盐结合和反应形成 NO。我们假设六配位态和五配位态之间的平衡以及与亚硝酸盐形成NO的次级反应可以通过其他缺氧依赖性翻译后修饰来调节。蛋白质序列模型确定了 14-3-3 结合和磷酸化的候选位点。在体外实验和暴露于缺氧和葡萄糖剥夺的人类 SH-SY5Y 神经元细胞中，我们观察到：1）在缺氧和代谢应激期间，神经球蛋白磷酸化以及与 14-3-3 的蛋白质-蛋白质相互作用增加； 2) 神经球蛋白与 14-3-3 结合可稳定并增加磷酸化的半衰期； 3) 磷酸化增加了血红素口袋的开放概率，从而增加了配体结合（CO 和亚硝酸盐）并加速了厌氧亚硝酸盐还原形成 NO 的速率。这些数据揭示了神经球蛋白的一系列缺氧依赖性翻译后修饰，调节六到五个血红素口袋平衡和血红素与配体的接触。亚硝酸盐和神经球蛋白的缺氧调节反应可能有助于细胞对缺氧的适应。
Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1-5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six-and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.