Martin Daniel R, Dutta Priyanka, Mahajan Shikha, Varma Sameer, Stevens Stanley M
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
Sci Rep. 2016 Apr 1;6:23658. doi: 10.1038/srep23658.
Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H2O2). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H2O2 exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H2O2-induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.
磷酸组氨酸磷酸酶1（PHPT1）是哺乳动物中唯一已知的磷酸组氨酸磷酸酶，它调节多种蛋白质的磷酸组氨酸水平，这些蛋白质包括参与信号传导、脂质代谢和钾离子转运的蛋白质。虽然人类PHPT1（hPHPT1）的高分辨率结构是已知 的，并且已经报道了对底物结合和催化活性重要的残基，但对于调节hPHPT1活性的翻译后修饰知之甚少。在这里，我们描述了hPHPT1暴露于活性氧过氧化氢（H2O2）后氧化对其结构和功能的影响。具体来说，我们使用液相色谱-串联质谱法定量hPHPT1氧化还原敏感残基的位点特异性氧化。这项研究的结果表明，H2O2暴露会诱导hPHPT1在Met95处发生选择性氧化，Met95是底物结合区域内的一个残基。然而，显式溶剂分子动力学模拟预测，Met95氧化对hPHPT1催化位点的无配体状态的结构和动力学只有轻微影响，这表明如果Met95氧化改变了hPHPT1的活性，那么它将通过改变中间状态的稳定性来实现。采用一种基于质谱的新方法，我们确定H2O2诱导的氧化不会对hPHPT1功能产生负面影响；这一结果与通常认为蛋白质氧化通常是功能丧失修饰的观点相反。
