Post-translational modifications (PTMs) of protein tyrosine (Tyr) residues can serve as a molecular fingerprint of exposure to distinct oxidative pathways and are observed in abnormally high abundance in the majority of human inflammatory pathologies. Reactive oxidants generated during inflammation include hypohalous acids and nitric oxide-derived oxidants, which oxidatively modify protein Tyr residues via halogenation and nitration, respectively, forming 3-chloroTyr, 3-bromoTyr, and 3-nitroTyr. Traditional methods for generating oxidized or halogenated proteins involve nonspecific chemical reactions that result in complex protein mixtures, making it difficult to ascribe observed functional changes to a site-specific PTM or to generate antibodies sensitive to site-specific oxidative PTMs. To overcome these challenges, we generated a system to efficiently and site-specifically incorporate chloroTyr, bromoTyr, and iodoTyr, and to a lesser extent nitroTyr, into proteins in both bacterial and eukaryotic expression systems, relying on a novel amber stop codon-suppressing mutant synthetase (haloTyrRS)/tRNA pair derived from the Methanosarcina barkeri pyrrolysine synthetase system. We used this system to study the effects of oxidation on HDL-associated protein paraoxonase 1 (PON1), an enzyme with important antiatherosclerosis and antioxidant functions. PON1 forms a ternary complex with HDL and myeloperoxidase (MPO) in vivo. MPO oxidizes PON1 at tyrosine 71 (Tyr71), resulting in a loss of PON1 enzymatic function, but the extent to which chlorination or nitration of Tyr71 contributes to this loss of activity is unclear. To better understand this biological process and to demonstrate the utility of our GCE system, we generated PON1 site-specifically modified at Tyr71 with chloroTyr and nitroTyr in Escherichia coli and mammalian cells. We demonstrate that either chlorination or nitration of Tyr71 significantly reduces PON1 enzymatic activity. This tool for site-specific incorporation of halotyrosine will be critical to understanding how exposure of proteins to hypohalous acids at sites of inflammation alters protein function and cellular physiology. In addition, it will serve as a powerful tool for generating antibodies that can recognize site-specific oxidative PTMs.

中文翻译：

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卤代酪氨酸氨基酸到蛋白质的位点特异性原核和真核有效结合。

蛋白酪氨酸（Tyr）残基的翻译后修饰（PTM）可以作为暴露于不同氧化途径的分子指纹，并且在大多数人类炎症性疾病中以异常高的丰度观察到。炎症过程中产生的反应性氧化剂包括次卤酸和一氧化氮衍生的氧化剂，它们分别通过卤化和硝化作用氧化修饰蛋白质Tyr残基，形成3-chloroTyr，3-bromoTyr和3-nitroTyr。产生氧化或卤化蛋白质的传统方法涉及非特异性化学反应，该反应会导致复杂的蛋白质混合物，从而难以将观察到的功能变化归因于位点特异性PTM或生成对位点特异性氧化PTM敏感的抗体。为了克服这些挑战，我们建立了一个系统，可依靠新型琥珀色终止密码子抑制突变体合成酶（haloTyrRS）/，有效地和定点地将chloroTyr，bromoTyr和iodoTyr以及较小程度的nitroTyr掺入细菌和真核表达系统的蛋白质中。衍生自巴氏甲烷八叠球菌酪氨酸合成酶系统的tRNA对。我们使用该系统研究了氧化对HDL相关蛋白对氧磷酶1（PON1）的影响，该酶具有重要的抗动脉粥样硬化和抗氧化功能。PON1在体内与HDL和髓过氧化物酶（MPO）形成三元复合物。MPO在酪氨酸71（Tyr71）上氧化PON1，导致PON1酶功能丧失，但是尚不清楚Tyr71氯化或硝化导致这种活性降低的程度。为了更好地理解该生物学过程并证明我们的GCE系统的实用性，我们在大肠杆菌和哺乳动物细胞中生成了在yr71上被chloroTyr和nitroTyr特异性修饰的PON1。我们证明，Tyr71的氯化或硝化可显着降低PON1的酶促活性。该工具用于卤代酪氨酸的位点特异性结合对于了解蛋白质在炎症位点暴露于次卤酸如何改变蛋白质功能和细胞生理学至关重要。此外，它将用作生成可识别位点特异性氧化PTM的抗体的强大工具。我们证明，Tyr71的氯化或硝化可显着降低PON1的酶促活性。该工具用于卤代酪氨酸的位点特异性结合对于了解蛋白质在炎症位点暴露于次卤酸如何改变蛋白质功能和细胞生理学至关重要。此外，它将用作生成可识别位点特异性氧化PTM的抗体的强大工具。我们证明，Tyr71的氯化或硝化可显着降低PON1的酶促活性。该工具用于卤代酪氨酸的位点特异性结合对于了解蛋白质在炎症位点暴露于次卤酸如何改变蛋白质功能和细胞生理学至关重要。此外，它将用作生成可识别位点特异性氧化PTM的抗体的强大工具。