Prostaglandin H synthase-2-catalyzed oxygenation of 2-arachidonoylglycerol is more sensitive to peroxide tone than oxygenation of arachidonic acid
ABSTRACT 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.
- SourceAvailable from: Sergo Kasvandik
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- "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. "
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. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-436) contains supplementary material, which is available to authorized users.SpringerPlus 08/2014; 3(436). DOI:10.1186/2193-1801-3-436
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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.Prostaglandins Leukotrienes and Essential Fatty Acids 12/2013; 90(1). DOI:10.1016/j.plefa.2013.11.002 · 1.98 Impact Factor