Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid Peroxides
Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid Peroxides
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DOI：
10.1021/tx700108w
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发表时间：
2007-10-01
影响因子：
4.1
通讯作者：
Schoeneich, Christian
中科院分区：
文献类型：
--
作者：
Drernina, Elena S.;Sharov, Victor S.;Schoeneich, Christian
蛋白质的氧化修饰在广泛的病理过程和衰老中发挥着重要作用。蛋白质被许多生物氧化剂修饰，包括过氧化氢、过氧亚硝酸盐、单线态氧以及以氧和氮为中心的自由基。最近，又鉴定出了一类生理上重要的氧化剂，即肽和蛋白质过氧化物。后者与蛋白质半胱氨酸残基反应相当迅速且有选择性。肌浆/内质网 Ca-ATP 酶 (SERCA) 通过特定半胱氨酸残基的 NO 依赖性 S-谷胱甘肽化进行可逆调节。因此，这些半胱氨酸残基的不可逆氧化可能会损害一氧化氮依赖性肌肉松弛。在这里，我们发现特定的蛋白质衍生（氨基酸）过氧化物选择性地与SERCA1的22个还原半胱氨酸残基的子集发生反应，包括含有肽的Cys（674）和Cys（675），其中Cys（674）（在SERCA2中）代表NO依赖性S-谷胱甘肽化的靶标之一。在 11 种测试的氨基酸、肽和蛋白质过氧化物中，源自游离色氨酸和游离酪氨酸的过氧化物对 SERCA 显示出最高的反应性，而在类似的实验条件下通过过氧化氢未检测到氧化。在色氨酸的过氧化物中，与 N 端和 C 端封闭的色氨酸的过氧化物相比，游离色氨酸的过氧化物显示出显着更高的反应性。定量 HPLC-MS/MS 分析表明，观察到色氨酸衍生的过氧化物的最高反应性为 Cys774 和 Cys938（半胱氨酸残基），它们嵌入 SERCA1 的跨膜结构域中。跨膜结构域的这种不寻常的反应性不能仅通过氧化剂的疏水性来合理解释，因为与源自 DL-色氨酸的过氧化物相比，来自 DL-色氨酸的过氧化物显示出相当高的反应性N-乙酰基色氨酸甲酯。我们的数据证明肽和蛋白质衍生的过氧化物作为体内氧化应激的重要介质的潜在作用，这可能导致半胱氨酸残基的选择性氧化，从而导致膜蛋白失活。
The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys(674) and Cys(675), where Cys(674) (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1 This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from DL-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein-derived peroxides as important mediators of oxidative stress in vivo, which may cause a selective oxidation of Cys residues leading to inactivation of membrane proteins.