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Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

ABSTRACT: Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO₂-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO₂-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO₂-OA increased intracellular GSSG in an oxidative stress-independent manner. NO₂-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with k_on of (3.45 ± 0.04)?×?10³ M^-1 s^-1, k_off of (4.4 ± 0.4)?×?10^-4 s^-1, and K_eq of (1.3 ± 0.1)?×?10^-7 M. Akin to NO₂-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.

SUBMITTER: Jobbagy S

PROVIDER: S-EPMC6348771 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

Jobbagy Soma S Vitturi Dario A DA Salvatore Sonia R SR Turell Lucía L Pires Maria F MF Kansanen Emilia E Batthyany Carlos C Lancaster Jack R JR Freeman Bruce A BA Schopfer Francisco J FJ

Redox biology 20181122

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO<sub>2</sub>-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effec ...[more]

PMID: 30654300

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Project description:In American Tegumentary Leishmaniasis (ATL) production of cytokines, reactive oxygen species (ROS) and nitric oxide (•NO) by host’ macrophages normally lead to parasite death. However, some Leishmania (Viannia) braziliensis strains evade those microbicidal mechanisms, exhibiting a resistance profile. L. braziliensis NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible strains, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analysed the effect of pro- and antioxidant molecules on the infectivity in vitro of L. braziliensis strains exhibiting polar phenotypes of resistance or susceptibility to •NO. In addition, we conducted an unbiased and comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. In vitro infection showed that NO-resistant parasites were more infective to peritoneal macrophages even in the presence of high levels of ROS and RNS, suggesting that parasite’s responses to oxidative and nitrosative stress are differentially regulated in resistant and susceptible strains. Principal component analysis of the absolute protein concentration values demonstrated a clear differentiation between NO-resistant and NO-susceptible parasites supporting the hypothesis that there are natural intrinsic differences at molecular level among L. braziliensis strains. Besides, upon •NO exposure, there is a rapid modulation of resistant parasites’ proteome involving a significant increase in their total protein content. Accordingly, after •NO stimulus, NO-resistant parasites increased glutathione (GSH) metabolism by upregulating enzymes involved in GSH biosynthesis and activity, as well as by increasing the absolute concentration of proteins with S-nitrosoglutathione reductase activity, whereas NO-susceptible did not. In addition, NO-resistant parasites showed increased glucose consumption, and elevated concentration levels of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) abundance after nitrosative challenge, all of which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, the upregulation of glucose consumption and GSH-mediated redox capability may explain the natural resistance of L. braziliensis against •NO-mediated damage. Data are available via ProteomeXchange with identifier

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Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

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Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

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