Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish

Abstract

Nitro-fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities. We recently demonstrated that nitro-fatty acids activate the Keap1-Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear. A number of chemical compounds with different structures have been reported to activate the Keap1-Nrf2 system, which can be categorized into at least six classes based on their sensing pathways. In this study, we showed that nitrooleic acid (OA-NO2), one of major nitro-fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators. In transgenic embryos, GFP was induced in the whole body by treatment with OA-NO2, 15d-PGJ2 or diethylmaleate (DEM), but not with hydrogen peroxide (H2O2), when exogenous Nrf2 and Keap1 were co-overexpressed. Induction by OA-NO2 or 15d-PGJ2 but not DEM was observed, even when a C151S mutation was introduced in Keap1. Our results support the contention that OA-NO2 and 15d-PGJ2 share an analogous cysteine code as electrophiles and also have similar anti-inflammatory roles.

Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

“…Although several in vitro experiments have shown that Cys151, Cys273, and Cys288 are highly reactive to many electrophiles (10)(11)(12)(13)(14)(15)(16)(17), evaluation of the sensor function of Cys273 and Cys288 has not been technically feasible, since replacements of Cys273 and Cys288 with serine or alanine render Keap1 unable to ubiquitinate and repress Nrf2 (18,19,21). To overcome this problem, we attempted a systemic analysis by introducing amino acid substitutions into Cys273 and Cys288 sites of human KEAP1 (Fig.…”

“…6D) and 4-HNE (Fig. 6E), both of which were reported to exploit Cys273 and Cys288 to modify Keap1 activity (15,16,34), resulted in the accumulation of Nrf2 in both wild-type and Keap1 C288E/C288E macrophages. To our surprise, PGA 2 induced Nrf2 accumulation in Keap1 C288E/C288E macrophages (Fig.…”

“…Indeed, Cys273 and Cys288 residues have been suggested to be reactive to 15d-PGJ 2 , prostaglandin A 2 (PGA 2 ), 9-OA-NO 2 and dexamethasone 21-mesylate (Dex-Mes) (10,(14)(15)(16). However, there exists a technical difficulty to validate this notion.…”

“…Keap1 is a cysteine-rich protein possessing 27 and 25 cysteine residues in the human and mouse proteins, respectively. A number of in vitro labeling and mass spectrometry studies have detected covalent modifications of some of the cysteine residues upon exposures of Keap1 to electrophiles (10)(11)(12)(13)(14)(15)(16)(17). The functional significance of these cysteine residues has been examined in experimental systems exploiting site-directed mutagenesis of Keap1.…”

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

“…Urinary Kelch-like ECH-associated protein 1 (Keap1) regulator of nuclear factor (erythroid-derived-2)-like 2 (Nrf2), heat shock factor-1 (HSF-1), peroxisome proliferator-activated receptor-␥ (PPAR ␥ ), and histone deacetylases (HDAC) ( 7 ). These transcriptional regulatory proteins contribute to the control of the expression of hundreds of genes that include cytokines, antioxidant enzymes, heat shock response proteins, and enzymes of intermediary metabolism ( 4,5,(8)(9)(10)(11)(12)(13)(14).…”