Peroxynitrite preferentially oxidizes the dithiol redox motifs of protein-disulfide isomerase

Author(s)

Peixoto, Albert Souza ^[1] ; Ryan Geyer, R. ^[1] ; Iqbal, Asif ^[1] ; Truzzi, Daniela R. ^[1] ; Soares Moretti, Ana I. ^[2] ; Laurindo, Francisco R. M. ^[2] ; Augusto, Ohara ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Quim, Dept Bioquim, Ave Lineu Prestes 748, BR-05508000 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Heart Inst InCor, Sch Med, Vasc Biol Lab, BR-05403000 Sao Paulo - Brazil

• Document type: Journal article
• Source: Journal of Biological Chemistry; v. 293, n. 4, p. 1450-1465, JAN 26 2018.
• Web of Science Citations: 5

Abstract

Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cys(53) and Cys(397)) by hydrogen peroxide (k = 17.3 +/- 1.3 m(-1) s(-1) at pH 7.4 and 25 degrees C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (k = (6.9 +/- 0.2) x 10(4) m(-1) s(-1)), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides (i.e. Cys(53)-Cys(56) and Cys(397)-Cys(400)). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active-site residues (Trp(52), Trp(396), and Tyr(393)). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI. (AU)

• FAPESP's process: 08/57721-3 - Redoxome
• Grantee: Ohara Augusto
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC