Fig 2: In vivo assessment of novel intra-protein disulfide in sEH. (A) Reducing Western blots showing that angiotensin II induced sEH expression over 7 days. The lower GAPDH blot confirms equal protein loading. (B) Angiotensin II treatment significantly increased sEH expression in mouse hearts. (C) Angiotensin II treatment significantly increased sEH activity in mouse hearts. (D) Correcting hydrolase activity for the increased sEH protein content revealed that hydrolase activity was still significantly increased, consistent with oxidant-induced activation of the hydrolase. (E-H) There was a significant decrease in the EET/DHET ratio for 14,15-EET/DHET and 11,12-EET/DHET as compared to saline treatment. No significant differences were observed for the 8,9- or 5,6-EET/DHET ratios. (I-J) Non-reducing blot showing there was a downward band shift in sEH in the hearts from angiotensin II treated mice, with quantification shown below, consistent with oxidant–induced disulfide formation. (K) H2O2 induced constriction of mouse aortas, consistent with sEH activation. (L-M) H2O2 pre-treatment potentiated 15d-PGJ2-induced arterial dilation, as indicated by the dose-response curve being shifted to the left as compared to the 15d-PGJ2 alone and by the decreased EC50. (N) Mouse aortas were assessed for intra-protein disulfide bond formation following peroxide treatment. There was a subtle downwards shift to the faster migrating oxidized form of sEH as shown in the Western blot.

Fig 3: sEH oxidation results in the formation of a disulfide bond. (A) An LC-MS/MS spectra identifying a peptide with an m/z of 744.59914+ (-0.4 ppm) was assigned to the peptide 250LHFVELGSGPAVCLCHGFPESWYSWR275 in the air oxidized protein samples. A disulfide bond was detected between the two cysteine residues, C262 and C264, as shown in red. The peptide was confidently assigned through the identification of its b and y ions as shown. (B) A peptide with an m/z of 808.90364+ (9.9 ppm) was identified and assigned to peptide 248VRLHFVELGSGPAVCLCHGFPESWYSWR275, indicating the absence of a disulfide bond as is anticipated when the reductant DTT was present. (C) Analysis with biotin-PAA confirmed disulfide bond formation via vicinal thiols within purified recombinant sEH. Following peroxide treatment, biotin-PAA was unable to adduct to sEH, however in reduced samples a band corresponding to sEH was observed when probed with streptavidin-HRP. Western blot below showing that WT sEH protein was present in all lanes following biotin-PAA treatment. This non-reducing blot also showed there was a downwards band shift in sEH when it was exposed to oxidizing conditions. (D-E) In separate experiments, recombinant sEH protein samples, which had been exposed to reducing or oxidizing conditions, were probed to monitor for the downwards band shift in sEH. There was a downwards band shift in WT sEH after oxidant exposure, consistent with disulfide formation. In contrast, C262S sEH did not as it does not have the thiols needed to form the disulfide that mediates the shift. (F) A C35A Trx trapping mutant was used to identify proteins that form disulfide bonds. Western blot indicating there was equal amounts of sEH in the input for the immunoprecipitation. (G) Western blot showing sEH immunoprecipitated from C35A Trx tapping mutant. Following auranofin treatment, a significant amount of WT sEH was captured by the C35A Trx trapping mutant. However, when C262S sEH is used there is significantly less sEH captured by the C35A Trx trapping mutant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: hsEH oxidation might induce conformational rearrangements. (A) sEH is a dimer with a swapped-domain architecture, where the C-terminal domain of one subunit interacts with both the C-terminal domain and the NTD of the other monomeric unit. (B) The panel reports the cartoon representation of the structure of hsEH C-terminal domain and its 170° rotation. The ‘L-shaped’ active site is depicted in surface mode. The F267 pocket, vertex and W334 niche are coloured respectively in magenta, blue and cyan. The cysteine residues involved in the disulfide bond are reported as orange sticks. The residues of the dimerization interface are depicted by green sticks. (C) AlloSigMA analysis of the perturbation of C262 and C264. The panel shows a cartoon model of sEH C-terminal domain (same orientations as in B), coloured according to the predicted ?gi. The darker the shade of red or blue, the greater the destabilization or gain in rigidity, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: sEH protein levels remain elevated in GR+/- rats under high salt diet. (A) Representative Western blot analysis and quantification of sEH protein expression under standard diet and (B) under high salt diet; (C) representative Western blot analysis and quantification of sEH protein expression in the adrenal glands of GR+/+ rats (n = 6) under standard and high salt diet (upper panel) and representative Western blot analysis and its quantification of sEH protein expression in the adrenal glands of GR+/- rats (n = 6) under standard and high salt diet; (D) enzymatic activity of sEH in the adrenal glands of GR+/+ (n = 6) and GR+/- rats (n = 6) under standard diet; (E) enzymatic activity of sEH in the adrenal glands of GR+/+ (n = 6) and GR+/- rats (n = 6) under high salt diet; (F) sEH activity was estimated indirectly by the molar ratios of individual and total DHET/(EET + DHET) for arachidonic acids and (G) for linoleic acids. Values are indicated as mean ± SEM, and data were evaluated by unpaired two-tailed t test and differences assessed at * p < 0.05, ** p < 0.01.