Characterization of human glutathione-dependent microsomal prostaglandin E synthase-1

摘要: Prostaglandins (PGs) are lipid mediators, which act as local hormones. PGs formed in most cells and synthesized de novo from membrane-released arachidonic acid (AA) upon cell activation. Prostaglandin H synthase (PGHS) –1 or 2, also referred to COX-1 COX-2, metabolize AA PGH2, is subsequently converted a cell-specific manner by downstream enzymes biologically active prostanoids, i.e. PGE2, PGD2, PGF2α, PGI2 TXA2. PGHS-1 constitutively expressed many mainly involved housekeeping functions, such vascular homeostasis, whereas PGHS-2 can be induced proinflammatory cytokines at sites of inflammation. E (PGES) specifically catalyzes the conversion PGH2 potent prostaglandin several pathological conditions; including pain, fever, inflammation possibly some forms cancers neurodegenerative diseases. mPGES-1 was initially identified homologue microsomal glutathione transferase-1 (MGST1) with 37% identity on amino sequence level MGST1-like 1 (MGST1L1). Based properties MGST1-L1, regarding size, sequence, hydropathy membrane localization, protein member MAPEG-superfamily (membraneassociated proteins eicosanoid metabolism). The superfamily consists 16-18 kDa, integral typical profiles diverse functions. MAPEG family comprises six human members, addition are; 5-lipoxygenase activating (FLAP), leukotriene C4 (LTC4S), MGST1, MGST2 MGST3. MGST1 -2 -3 transferases well glutathione-dependent peroxidases, while FLAP LTC4S crucial for biosynthesis. Human cloned characterized 16 inducible GSH-dependent PGE synthase. Northern dot blot analysis mRNA demonstrated low expression tissues, medium reproductive organs high two cancer lines (A549 HeLa). A549 had been used earlier model system study induction cytokine IL-1β these cells. A similar size detected microsomes sheep vesicular glands, known contain highly efficient PGES, indicating that long-sought bound PGES. Furthermore, time revealed coordinate enzymes, correlated increased PGES activity fraction. Tumor necrosis factor-α (TNF-α) dexamethasone found counteract effect induction. method based RP-HPLC UV-detection developed efficiently quantify activity. small set potential inhibitors were tested NS-398, Sulindac sulfide LTC4 inhibit IC50-values 20 μM, 80 μM 5 respectively. gene structure investigated. span region approximately 15 kb, divided into three exons localized chromosome 9q34.3. 682 bp fragment directly upstream translation start site exhibited promoter when transfected putative GC-rich, lacks TATA box functional contains numerous transcription factor binding-sites. Two GC-boxes, tandem Barbie-boxes an aryl hydrocarbon response element identified. transcriptionally reporter constructs regulated and phenobarbital. investigated synovial tissues patients suffering rheumatoid arthritis (RA). Primary obtained RA treated TNF-α. Both induce basal levels maximum after 24 hours inhibited dose-dependent manner. linear increase up 72 h. In contrast, peak (4-8 h). biochemical experiments any significant contribution cytosolic other non-inducible synthases ruled out. purification protocol developed. histidine tag Eschericha coli, solubilized Triton X-100 purified combination hydroxyapatite immobilized metal affinity chromatography. catalyzed rapid PGE2 (170 μmol/min mg). enzyme, displayed against PGG2, forming 15-hydroperoxy (250 addition, possessed activities; peroxidase towards cumene hydroperoxide, 5-HpETE 15hydroperoxy-PGE2, conjugation 1-chloro-2,4-dinitrobenzene (CDNB) GSH. These activities likely reflect relationship enzymes. Two-dimensional crystals 10 projection map determined electron crystallography. Hydrodynamic studies performed mPGES-1-Triton complex investigate oligomeric state protein. Electron crystallography hydrodynamic independently trimeric organization mPGES-1. Together published date, has verified drug target next step this validation process requires specific animal disease models.