Department of Biochemistry and Biophysics , Oregon State University , Corvallis , Oregon 97331 , United States.
Department of Cardiovascular & Metabolic Sciences , Lerner Research Institute, Cleveland Clinic , Cleveland , Ohio 44195 , United States.
ACS Chem Biol. 2020 Feb 21;15(2):562-574. doi: 10.1021/acschembio.9b01026. Epub 2020 Feb 10.
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 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) . 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 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.
蛋白质酪氨酸（Tyr）残基的翻译后修饰（PTMs）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病理学中以异常高的丰度存在。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氧化修饰蛋白质 Tyr 残基，形成 3-氯 Tyr、3-溴 Tyr 和 3-硝 Tyr。生成氧化或卤化蛋白质的传统方法涉及非特异性化学反应，导致产生复杂的蛋白质混合物，使得难以将观察到的功能变化归因于特定位点的 PTM，或者生成对特定位点氧化 PTM 敏感的抗体。为了克服这些挑战，我们开发了一种系统，该系统可以在细菌和真核表达系统中高效且特异性地将氯 Tyr、溴 Tyr 和碘 Tyr（以及程度较小的硝 Tyr）掺入蛋白质中，该系统依赖于一种新型琥珀终止密码子抑制突变合成酶（haloTyrRS）/tRNA 对，源自吡咯赖氨酸合成酶系统。我们使用该系统研究了氧化对高密度脂蛋白相关蛋白对氧磷酶 1（PON1）的影响，PON1 是一种具有重要抗动脉粥样硬化和抗氧化功能的酶。PON1 与高密度脂蛋白和髓过氧化物酶（MPO）形成三元复合物。MPO 在 Tyr71 处氧化 PON1，导致 PON1 酶活性丧失，但 Tyr71 的氯化或硝化对这种活性丧失的贡献程度尚不清楚。为了更好地理解这一生物学过程，并展示我们的 GCE 系统的实用性，我们在细菌和哺乳动物细胞中生成了 Tyr71 处被氯 Tyr 和硝 Tyr 特异性修饰的 PON1。我们证明 Tyr71 的氯化或硝化显著降低了 PON1 的酶活性。该工具可用于在特定位置掺入卤代酪氨酸，这对于理解蛋白质在炎症部位暴露于次卤酸时如何改变蛋白质功能和细胞生理学至关重要。此外，它将成为生成可识别特定位点氧化 PTM 的抗体的有力工具。
