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ACS Chem Biol. 2020 Feb 21;15(2):562-574. doi: 10.1021/acschembio.9b01026. Epub 2020 Feb 10.

Efficient Site-Specific Prokaryotic and Eukaryotic Incorporation of Halotyrosine Amino Acids into Proteins.

Author information

1
Department of Biochemistry and Biophysics , Oregon State University , Corvallis , Oregon 97331 , United States.
2
Department of Cardiovascular & Metabolic Sciences , Lerner Research Institute, Cleveland Clinic , Cleveland , Ohio 44195 , United States.
3
Center for Microbiome & Human Health , Lerner Research Institute, Cleveland Clinic , Cleveland , Ohio 44195 , United States.
4
Department of Cardiovascular Medicine, Heart and Vascular Institute , Cleveland Clinic , Cleveland , Ohio 44195 , United States.

Abstract

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.

PMID:
31994864
DOI:
10.1021/acschembio.9b01026

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