The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid*♦

  1. Francisco J. Schopfer4
  1. From the Laboratorios de ‡Enzimología,
  2. ‖Química Teórica y Computacional, and
  3. **Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, and
  4. §Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11400, Uruguay and
  5. the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
  1. ↵3 To whom correspondence may be addressed: Laboratorio de Enzimología, Facultad de Ciencias. Iguá 4225, Montevideo 11400, Uruguay. Tel.: 598-2-525-0749; E-mail: beatriz.alvarez{at}fcien.edu.uy.
  2. ↵4 To whom correspondence may be addressed: Dept. of Pharmacology and Chemical Biology, Thomas E. Starzl Biomedical Science Tower E1340, 200 Lothrop St., University of Pittsburgh, Pittsburgh, PA 15213. Tel.: 412-648-0193; Fax: 412-648-2229; E-mail: fjs2{at}pitt.edu.

  1. ↵1 Both authors contributed equally to this work.

  2. Edited by F. Peter Guengerich

Abstract

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.

Footnotes

  • ↵2 Active members of the National System of Researchers and of PEDECIBA (Uruguay).

  • ↵* This work was supported by grants and fellowships from Comisión Sectorial de Investigación Científica (Universidad de la República, Uruguay) (to L. T., M. N. M., and B. A.), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) (to L. L.), National Institutes of Health Grants K01-HL133331 (to D. A. V.) and R01-AT006822 (to F. J. S.), and American Heart Association Grant in Aid 14GRNT20170024 (to F. J. S.). D. A. V., S. R. W., and F. J. S. acknowledge financial interest in Complexa Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • ↵♦ This article was selected as one of our Editors' Picks.

  • ↵Graphic This article contains supplemental Figs. S1 and S2 and Table S1.

  • Received August 30, 2016.
  • Revision received November 23, 2016.
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  1. First Published on December 6, 2016 doi: 10.1074/jbc.M116.756288 The Journal of Biological Chemistry 292, 1145-1159.
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