Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-Δ(12,14)-prostaglandin J(2)

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.     

A new mouse model for studying EGFR-dependent gastric polyps

Hyperactivation of the epidermal growth factor receptor (EGFR) in gastric cells due to excess of its ligand transforming growth factor-α (TGFA) is associated with hyperplastic lesions in Ménétrier's disease patients and in transgenic mice. Other EGFR ligands, however, have never been associated with stomach diseases. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) results in a severe, age-dependent hyperplasia of foveolar epithelium. The stomach weight of affected mice reached up to 3g representing more than 10% of total body weight. The preexisting corpus mucosa was severely depleted, and both parietal and chief cells were replaced by proliferating foveolar epithelium. The lesions were more severe in male as compared to female transgenic mice, and partially regressed in the former after castration-mediated androgen removal. The gastric hyperplasia fully disappeared when BTC-tg mice were crossed into the Egfr(Wa5) background expressing a dominant-negative EGFR, indicating that the phenotype is EGFR-dependent. This is, to our knowledge, the first report of hyperplastic gastric lesions due to the overexpression of an EGFR ligand other than TGFA. BTC-tg mice are therefore a new, promising model for studying EGFR-dependent gastric polyps.     

Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis

Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.     

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.     

Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.     

Reduced hexokinase II impairs muscle function 2 wk after ischemia-reperfusion through increased cell necrosis and fibrosis

We previously demonstrated that hexokinase (HK) II plays a key role in the pathophysiology of ischemia-reperfusion (I/R) injury of the heart (Smeele et al. Circ Res 108: 1165-1169, 2011; Wu et al. Circ Res 108: 60-69, 2011). However, it is unknown whether HKII also plays a key role in I/R injury and healing thereafter in skeletal muscle, and if so, through which mechanisms. We used male wild-type (WT) and heterozygous HKII knockout mice (HKII(+/-)) and performed in vivo unilateral skeletal muscle I/R, executed by 90 min hindlimb occlusion using orthodontic rubber bands followed by 1 h, 1 day, or 14 days reperfusion. The contralateral (CON) limb was used as internal control. No difference was observed in muscle glycogen turnover between genotypes at 1 h reperfusion. At 1 day reperfusion, the model resulted in 36% initial cell necrosis in WT gastrocnemius medialis (GM) muscle that was doubled (76% cell necrosis) in the HKII(+/-) mice. I/R-induced apoptosis (29%) was similar between genotypes. HKII reduction eliminated I/R-induced mitochondrial Bax translocation and oxidative stress at 1 day reperfusion. At 14 days recovery, the tetanic force deficit of the reperfused GM (relative to control GM) was 35% for WT, which was doubled (70%) in HKII(+/-) mice, mirroring the initial damage observed for these muscles. I/R increased muscle fatigue resistance equally in GM of both genotypes. The number of regenerating fibers in WT muscle (17%) was also approximately doubled in HKII(+/-) I/R muscle (44%), thus again mirroring the increased cell death in HKII(+/-) mice at day 1 and suggesting that HKII does not significantly affect muscle regeneration capacity. Reduced HKII was also associated with doubling of I/R-induced fibrosis. In conclusion, reduced muscle HKII protein content results in impaired muscle functionality during recovery from I/R. The impaired recovery seems to be mainly a result of a greater susceptibility of HKII(+/-) mice to the initial I/R-induced necrosis (not apoptosis), and not a HKII-related deficiency in muscle regeneration.     

The depth-specific significance and relative abundance of anaerobic ammonium-oxidizing bacteria in estuarine sediments (Medway Estuary, UK)

Variations in the overall and depth-specific significance of anammox were measured using (15) N isotope experiments in both bioirrigated and undisturbed sediments of the Medway Estuary, UK. This was performed over two surveys, alongside FISH experiments, to identify and track shifts in the relative abundance of anammox organisms with depth. In Survey 1 (initially screening for the presence of anammox), the potential for anammox (ra) decreased from 32% upstream to 6% downstream. In Survey 2, depth-specific values of ra varied between a maximum of 37% upstream and a minimum of 4% downstream. This was linked to a small population of anammox organisms accounting for 相似文献   

Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys?3?, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys?3?. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys2??, was identified. Furthermore, the active-site Cys?3? was found to be located on top of a loop structure, formed by the two flanking residues Cys?2? and Cys?3?, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys?2? and Cys?3? are within disulfide bond distance and that a persulfide transfer from Cys?3? to Cys2?? is indeed possible.     

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.     

The effect of prolyl oligopeptidase inhibition on extracellular acetylcholine and dopamine levels in the rat striatum

Prolyl oligopeptidase (PREP, EC 3.4.21.26) inhibitors have potential as cognition enhancers, but the mechanism of action behind the cognitive effects remains unclear. Since acetylcholine (ACh) and dopamine (DA) are known to be associated with the regulation of cognitive processes, we investigated the effects of two PREP inhibitors on the extracellular levels of ACh and DA in the rat striatum using in vivo microdialysis. KYP-2047 and JTP-4819 were administered either as a single systemic dose (50 μmol/kg～17 mg/kg i.p.) or directly into the striatum by retrodialysis via the microdialysis probe (12.5, 37.5 or 125 μM at 1.5 μl/min for 60 min). PREP inhibitors had no significant effect on striatal DA levels after systemic administration. JTP-4819 significantly decreased ACh levels both after systemic (by ～25%) and intrastriatal (by ～30-50%) administration. KYP-2047 decreased ACh levels only after intrastriatal administration by retrodialysis (by ～40-50%) when higher drug levels were reached, indicating that higher brain drug levels are needed to modulate ACh levels than to inhibit PREP. This result does not support the earlier hypothesis that the positive cognitive effects of PREP inhibitors in rodents would be mediated through the cholinergic system. In vitro specificity studies did not reveal any obvious off-targets that could explain the observed effect of KYP-2047 and JTP-4819 on ACh levels, instead confirming the concept that these compounds have a high selectivity towards PREP.