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81.
Melet A Marques-Soares C Schoch GA Macherey AC Jaouen M Dansette PM Sari MA Johnson EF Mansuy D 《Biochemistry》2004,43(49):15379-15392
The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'. 相似文献
82.
Inactivation and intracellular retention of the human I183N mutated melanocortin 3 receptor associated with obesity 总被引:2,自引:0,他引:2
Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G protein-coupled receptors (GPCRs) by stimulating adenylate cyclase. The melanocortin 3 receptor (MC3R) in the melanocortin receptor (MCR) family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. Until now, only one heterozygous mutation (I183N) has been identified in the coding region of this receptor in two obese patients of the same family. In this study, we reported the functional characterization of the I183N mutated MC3R compared with that of the wild-type MC3R after transfection in HEK293 cells. Our results showed that the I183N mutation totally abolished the activity of the mutated receptor to generate intracellular cAMP. Furthermore, confocal microscopy observation revealed that the mutation induced an intracellular retention of the mutated receptor. Moreover, we demonstrated for the first time by co-transfection studies that the mutated receptor could reduce the wild-type receptor activity through a dominant negative effect. 相似文献
83.
Methyl-beta-cyclodextrin (MbetaCD) was used to explore a role for cholesterol-enriched plasma membrane microdomains in coupling lysophosphatidic acid (LPA) stimulation to phosphoinositide 3-kinase (PI3K) activation. Cholesterol depletion strongly inhibited the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in Vero cells stimulated with LPA. In agreement, the phosphorylation of Akt/protein kinase B, but not of Erk kinases, was suppressed by MbetaCD. MbetaCD did not interfere with the overall phospholipid metabolism, and its effects were reversed in cholesterol add-back experiments. Finally, PI3K was detected in lipid rafts prepared from control but not MbetaCD-treated cells, suggesting that these microdomains contribute to LPA signalling by compartmentalising component(s) of the PI3K pathway. 相似文献
84.
Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth 下载免费PDF全文
Lessard C Cochu A Lemay JD Roy D Vaillancourt K Frenette M Moineau S Vadeboncoeur C 《Journal of bacteriology》2003,185(23):6764-6772
The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose. 相似文献
85.
Maron PA Coeur C Pink C Clays-Josserand A Lensi R Richaume A Potier P 《Journal of microbiological methods》2003,53(1):87-95
In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested. 相似文献
86.
Melet A Assrir N Jean P Pilar Lopez-Garcia M Marques-Soares C Jaouen M Dansette PM Sari MA Mansuy D 《Archives of biochemistry and biophysics》2003,409(1):80-91
A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9. 相似文献
87.
88.
The role of Helicobacter pylori infection is explored in more and more extragastric diseases without definite proof in most of the studies, except possibly some hematologic diseases. In cardiovascular diseases, including stroke, the presence of CagA positive strains may be involved. The possible role of helicobacters in hepatobiliary diseases goes beyond that of H. pylori to involve enterohepatic helicobacters. New Helicobacter species are regularly described and molecular methods are developed to improve their detection. Helicobacter felis remains the major species to be used in animal models of Helicobacter infection. 相似文献
89.
Génin E Huebner A Jaillard C Faure A Halaby G Saka N Clark AJ Durand P Bégeot M Naville D 《Human genetics》2002,111(4-5):428-434
In several cases of familial glucocorticoid deficiency (FGD), referred to as FGD type 1, mutations have been described in the coding exon of the adrenocorticotropin receptor (melanonocortin receptor type 2, MC2R) gene. However, for the majority of cases (FGD type 2), no mutations were found in this gene. In the more informative families, the involvement of the MC2R locus could be excluded by linkage or sequencing analysis and, as there was no obvious candidate gene, a genome linkage scan was performed. Fourteen families were studied in this report. Evidence of linkage was found with markers on chromosome 8q in three out of the 14 families (maximum heterogeneity LOD score of 2.81 at D8S1763). These three families were consanguineous and the gene could be located by homozygosity mapping between markers D8S285 and D8S1718 in a 8.8-cM region. No potential candidate genes were apparent in the region. Linkage to this region could be excluded in some families from our sample giving highly negative LOD scores with the markers of the region. This result suggests that at least one other gene, located on a different region, must be responsible for FGD in these families and provides new evidence of genetic heterogeneity of this disorder. 相似文献
90.
The CD3 complex is an essential component of the T-cell receptor (TCR) implicated in T-cell maturation and activation. This TCR has been identified in both cartilaginous and bony vertebrates. In different studies where the CD3 chains were cloned and sequenced, it appeared that the CD3 complex is composed of several chains, all susceptible to phosphorylation and able to transduce signals. Here, by an approach combining degenerative oligonucleotide primers and RACE-PCR, we report the cloning and sequencing of a CD3 cDNA from the salamander Pleurodeles waltl, highly homologous to the Xenopus and chicken CD3 gamma/delta cDNAs. Using semi-quantitative PCR and Northern blot analysis, we found the highest CD3 gamma/delta mRNA expression in the thymus; weaker expression was observed in the spleen and blood, followed by the intestine, therefore confirming the tissue and lymphoid specificities of this mRNA. The signals in the spleen, blood and intestine represented 55%, 33% and 16%, respectively, of the signal detected in the thymus. During the embryonic and larval stages of Pleurodeles waltl development, CD3 gamma/delta mRNA expression begins early at the neurula stage (stage 15, 69 h after laying), increases up to stage 33 (9 days after laying) and afterwards remains stable, at least until the larval stage 42 (28 days after laying). As the thymus primordium appears much later, the question of the formation and maturation of the first T-cell precursors outside this organ is posed. 相似文献