The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid.
The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid.
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硝基共轭亚油酸（一种保护性细胞信号脂质）中硫醇加成的化学基础。
DOI：
10.1074/jbc.m116.756288
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Schopfer,FranciscoJ
中科院分区：
文献类型：
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作者：
Turell,Lucía;Vitturi,DaríoA;Coitiño,ELaura;Lebrato,Lourdes;Möller,MatíasN;Sagasti,Camila;Salvatore,SoniaR;Woodcock,StevenR;Alvarez,Beatriz;Schopfer,FranciscoJ
硝基烯烃脂肪酸在体内形成，并通过关键信号通路中的含硫醇蛋白质的可逆迈克尔加成发挥保护和抗炎作用。硝基共轭亚油酸 (NO2-CLA) 优先形成，构成人体中最丰富的硝化脂肪酸，并含有两个可能与硫醇反应的碳，调节信号传导作用和水平。在这项工作中，我们研究了 NO2-CLA 与低分子量硫醇（谷胱甘肽、半胱氨酸、同型半胱氨酸、半胱氨酰甘氨酸和 β-巯基乙醇）和人血清白蛋白的反应。反应遵循可逆双相动力学，这与 NO2-CLA 中相对于硝基位于 β 碳和 δ 碳上存在的两个亲电子中心一致。通过电子结构的计算建模证实了差异反应性。对于不同的硫醇，确定了两个反应的速率（konandkoff）和平衡常数。 LC-UV-Visible和LC-MS分析表明，快速反应对应于β-加合物的形成（动力学产物），而慢速反应对应于δ-加合物（热力学产物）的形成。速率常数的 pH 依赖性、固有反应性和硫醇 pKa 之间的相关性以及氘溶剂动力学同位素效应的缺失表明硫醇盐对 NO2-CLA 的攻击作为速率控制步骤的逐步机制。计算模型支持了该机制，并揭示了过渡态、阴离子中间体和最终中性产物的其他特征。重要的是，人尿液中半胱氨酸-δ-加合物的检测为该反应的生物学相关性提供了证据。最后，发现人血清白蛋白既能非共价结合 NO2-CLA，又能在 Cys-34 处形成共价加合物，这表明了全身分布的潜在模式。这些结果为 NO2-CLA 信号传导作用的化学基础提供了新的见解。
Nitroalkene fatty acids are formedin vivoand exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (konandkoff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.