The endocannabinoid, 2-arachidonoylglycerol (2-AG), is a selective substrate for the inducible isoform of prostaglandin H synthase (PGHS), PGHS-2. Its turnover leads to the formation of glyceryl esters of prostaglandins (PG-Gs), a subset of which elicit agonism at unique, as yet unidentified, receptors. The kcat/Kms for oxygenation of arachidonic acid (AA) and 2-AG by PGHS-2 are very similar, but the sensitivities of the two substrates to peroxide-dependent activation have not been compared. 15-Hydroperoxy derivatives of AA and 2-AG were found to be comparable in their ability to serve as substrates for the peroxidase activities of PGHS-2, PGHS-1, and glutathione peroxidase (GPx). They also were comparable in the activation of AA oxygenation by cyanide-inhibited PGHS-2. However, oxygenation of 2-AG was significantly suppressed relative to AA by the presence of GPx and GSH. Furthermore, 2-AG oxygenation by peroxidase-deficient H388YmPGHS-2 was much less efficient than AA oxygenation. Wild-type rates of 2-AG oxygenation were restored by treatment of H388YmPGHS-2 with hydroperoxide derivatives of AA or 2-AG. RNAi silencing of phospholipid hydroperoxide-specific GPx (GPx4) in NIH3T3 cells led to increases in cellular peroxidation and in the levels of the isoprostane product, 8-epi-PGF2α. GPx4 silencing led to two- to four-fold increases in PG-G formation but no change in PG formation. Thus cellular peroxide tone may be an important determinant of the extent of endocannabinoid oxygenation by PGHS-2.
"Vertebrates have two distinct PGHS isoforms: PGHS-1 and -2 (Kulmacz et al. 2003; Rouzer and Marnett 2009; Simmons et al. 2004; Smith et al. 2000b). The majority of the studies associated with mammalian PGHSs have been conducted with native or recombinant ovine PGHS-1 and recombinant human or murine PGHS-2 (Mbonye et al. 2006; Musee and Marnett 2012; Nemeth et al. 2001; Vecchio and Malkowski 2011; Vecchio et al. 2012). Thus there is little experimental data on human PGHSs (hPGHSs), particularly concerning hPGHS-1. "
[Show abstract][Hide abstract] ABSTRACT: Prostaglandin H synthases (PGHSs) are N-glycosylated membrane proteins that catalyse the committed step in prostaglandin synthesis. Unlike PGHS-2, the production of recombinant PGHS-1 in non-mammalian expression systems is complicated. The majority of the heterologous enzyme is inactive due to misfolding. Correct N-glycosylation is proposed to be obligatory for proper folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Accumulation of non-glycosylated hPGHSs was not observed in the crude lysate of the yeast cells. The N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. The isoforms exhibited similar N-glycosylation site occupancy. The results indicate that there are more complex grounds for the inactivity of the recombinant hPGHS-1 produced in yeast.
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[Show abstract][Hide abstract] ABSTRACT: Several lines of investigation are being developed to assess the impact of polyunsaturated fatty acids, namely those of the omega 3 series, intake on oxidative stress. Keeping in mind that there might be a dose-response relation, in vivo and in vitro data strongly suggest that omega 3 fatty acids might act as anti- rather than pro-oxidant in several cells such as vascular cells, hence diminishing inflammation, oxidative stress, and, in turn, the risk of atherosclerosis and degenerative disorders such as cardiovascular disease.
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