CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology, via Amendola 165/A, 70126 Bari, Italy.
Department DiBEST (Biologia, Ecologia, Scienze della Terra), Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
Biochim Biophys Acta Bioenerg. 2017 Jul;1858(7):475-482. doi: 10.1016/j.bbabio.2017.04.002. Epub 2017 Apr 22.
S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71μM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.
已经在天然蛋白和重组蛋白（在脂质体中重新构建）以及完整线粒体上研究了线粒体肉碱/酰基辅酶 A 载体（CACT）的 S-亚硝基化。广泛使用的 NO 释放化合物 GSNO 强烈抑制了用来自大鼠肝线粒体的天然 CACT 或在大肠杆菌中过表达的重组大鼠 CACT 重新构建的脂质体中的反转运。还原剂二硫苏糖醇可逆转抑制作用，表明抑制机制基于 CACT 半胱氨酸残基的亚硝基化。天然和重组蛋白的半抑制常数（IC50）非常相似，分别为 74 和 71μM。抑制作用对底物肉碱具有竞争性。NO 也与 NEM 竞争，这与先前的数据很好地相关，表明 NEM 干扰了底物转运途径。使用重组 CACT 的半胱氨酸残基的定点突变方法，确定了 NO 的靶标。C136 在反应机制中起主要作用。用 GSNO 处理后，在完整的线粒体中通过免疫沉淀和用特异性抗 NO-Cys 抗体进行免疫染色，证明了 S-亚硝基化的发生。在平行样本中，GSNO 处理后，完整线粒体中 CACT 的转运活性受到强烈抑制。讨论了 CACT 的翻译后修饰的可能生理和病理意义。
