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

Research output: Contribution to journalArticle

Authors

  • Hyo Sang Jang
  • Xiaodong Gu
  • Richard B. Cooley
  • Joseph J. Porter
  • Rachel L. Henson
  • Taylor Willi
  • Joseph A. Didonato
  • Stanley L. Hazen
  • Ryan A. Mehl

External Institution(s)

  • Oregon State University
  • Cleveland Clinic Foundation

Details

Original languageEnglish (US)
Pages (from-to)562-574
Number of pages13
JournalACS chemical biology
Volume15
Issue number2
StatusPublished - Feb 21 2020
Peer-reviewedYes

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.

Citation formats

APA

Jang, H. S., Gu, X., Cooley, R. B., Porter, J. J., Henson, R. L., Willi, T., ... Mehl, R. A. (2020). Efficient Site-Specific Prokaryotic and Eukaryotic Incorporation of Halotyrosine Amino Acids into Proteins. ACS chemical biology, 15(2), 562-574. https://doi.org/10.1021/acschembio.9b01026

Harvard

Jang, HS, Gu, X, Cooley, RB, Porter, JJ, Henson, RL, Willi, T, Didonato, JA, Hazen, SL & Mehl, RA 2020, 'Efficient Site-Specific Prokaryotic and Eukaryotic Incorporation of Halotyrosine Amino Acids into Proteins', ACS chemical biology, vol. 15, no. 2, pp. 562-574. https://doi.org/10.1021/acschembio.9b01026