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Introduction In the human body, the adrenal cortex constitutes a major site for the biosynthesis and secretionofa varietyof steroid hormones including pregnenolone, dehydroepiandrosterone (DHEA), and androstenedione (Turcu et al. 2014). [...], The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of DHEA as well as some other hydroxysteroids such aspregnenolone. The present study was designedtoinvestigate how genetic polymorphismsof the human SULT2A1 gene may affect the sulfation of DHEA and pregnenolone. Online databases were systematically searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non-synonymous coding SNPs identified, seven were selected for further investigation. Site-directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 Escherichia coli cells and purified by glutathione-Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward both DHEA and pregnenolone. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild-type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes. Key words: single nucleotide polymorphisms, cytosolic sulfotransferase, SULT, SULT2A1, sulfation, dehydroepiandrosterone, DHEA, pregnenolone. La sulfotransferase (SULT) cytosolique SULT2A1 est connue pour catalyser la sulfatation de la dehydroepiandrosterone (DHEA) de meme que d'autres hydroxysteroides comme la pregnenolone. La presente etude a ete concue pour examiner comment les polymorphismes genetiques du gene SULT2A1 humain peuvent affecter la sulfatation de la DHEA et de la pregnenolone. Des bases de donnees en ligne ont ete systematiquement fouillees afin d'identifier des polymorphismes mononucleotidiques (SNP) deSULT2A1 humain. Des 98 SNP codants non-synonymes de SULT2A1 identifies, sept ont ete choisis pour des etudes plus approfondies.La mutagenese dirigee aete utilisee pour generer des ADNc codant ces sept allozymes de SULT2A1, qui ont ete exprimees chez Escherichia coli BL21 et purifiees par chromatographie d'affinite sur glutathion-Sepharose. Les dosages enzymatiques ont revele que les allozymes de SULT2A1 purifiees presentaient des activites de sulfatation differentielles envers la DHEA et la pregnenolone. Les analyses cinetiques ont en outre montre la differenciation de l'efficacite catalytique et de l'affinite pour les substrats des allozymes de SULT2A1, comparativement a SULT2A1 sauvage. Ces resultats ont fourni une information utile en ce qui concerne les effets des polymorphismes genetiques sur l'activite de sulfatation des allozymes de SULT2A1. [Traduit par la Redaction] Mots-cles : polymorphismes mononucleotidiques, sulfotransferase cytosolique, SULT, SULT2A1, sulfatation, dehydroepiandrosterone, DHEA, pregnenolone.

Introduction Naproxen is a widely used non-steroidal anti-inflammatory drug (Duggan et al. 2010). It exerts its antipyretic, anti-inflammatory, and analgesic activity via reversible inhibition of COX-1 and COX-2 enzymes, and [...], In this study, we aimed to obtain a comprehensive account of the human cytosolic sulfotransferases (SULTs) that are capable of sulfating 6-0-desmethylnaproxen (0-DMN), a major metabolite of naproxen. Of the 13 known human SULTs tested, 7 (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT1E1) displayed 0-DMN-sulfating activity, when analyzed using an elevated substrate concentration (500 [micro]mol x [L.sup.-1]) together with 14[micro] mol x [L.sup.-1] of the sulfate donor, 3'-phosphoadenosine-5'phosphosulfate (PAPS). At 10 [micro]mol x [L.sup.-1] 0-DMN concentration, however, only SULT1A1 and SULT1A3 displayed detectable activity, with the former being nearly 2 orders of magnitude more active than the latter. A pH-dependence study indicated that SULT1A1 exhibited a broad pH optimum spanning pH 5.5-7. Kinetic parameters of the sulfation of 0-DMN by SULT1A1 were determined. The production and release of sulfated 0-DMN was demonstrated using cultured human HepG2 hepatoma cells and Caco-2 colon carcinoma cells. Moreover, assays using human organ specimens revealed that the 0-DMN-sulfating activities present in the cyto-sols of liver and small intestine (at 502.5 and 497.2 [micro]mol x [min.sup.-1-][(mg protein).sup.1], respectively) were much higher than those detected for the cyto-sols of lung and kidney. Taken together, these results provided relevant information concerning the sulfation of 0-DMN both in vitro and in vivo. Key words: naproxen, 6-0-desmethylnaproxen, sulfation, cytosolic sulfotransferase, SULTs. Dans cette etude, nous visions a obtenir un registre detaille des sulfotransferases (SULT) cytosoliques humaines, qui permettent de sulfater le 6-0-demethyle naproxene (0-DMN), un des principaux metabolites du naproxene. Des 13 SULT humains connus analyses, la SULT1A1, la SULT1A2, la SULT1A3, la SULT1B1, la SULT1C2, la SULT1C4 et la SULT1E1 ont presente une activite 0-DMN sulfatante a l'analyse de concentrations elevees de substrat (500 [micro]mol x [L.sup.-1]) avec le donneur de sulfates PAPS (pour 3'-phosphoadenosine 5'-phosphosulfate) a 14 [micro]mol x [L.sup.-1]. Ce-pendant, avecle 0-DMN a 10 [micro]mol x [L.sup.-1], seules l'activite de la SULT1A1 et l'activite de la SULT1A3 etaient detectables, et la premiere etait presque deux fois plus elevee que la seconde. Une etude de dependance au pH a montre que l'expression de la SULT1A1 etait optimale a l'interieur d'une large plage de pH (de 5,5 a 7). Nous avons etabli les parametres cinetiques de la sulfatation du 0-DMN par la SULT1A1. Nous avons montre la production et la liberation d'0-DMN sulfate a l'aide de cellules humaines d'hepatome (HepG2) et de cancer du colon (Caco-2) en culture. De surcroit, des analyses sur des echantillons d'organes humains ont revele que l'activite sulfatante de l'0-DMN presente dans le cytosol de cellules de foie et d'intestin grele (502,5 et 497,2 [micro]mol x [min.sup.-1]-[(mg de proteines).sup.-1], respectivement) etait beaucoup plus elevee que dans le cytosol de cellules de poumon et de rein. En somme, ces resultats ont permis d'obtenir des renseignements pertinents quant a la sulfatation de l'0-DMN, tant in vitro qu'in vivo. [Traduit par la Redaction] Mots-cles: naproxene, 6-0-demethyle naproxene, sulfatation, sulfotransferase cytosolique, SULT.

This study aimed to identify human cytosolic sulfotransferases (SULTs) that are capable of mediating hyperoside sulfation and examine the impact of genetic polymorphisms on their sulfating activity. Of the thirteen known human SULTs analyzed, five (1A1, 1A2, 1A3, 1C2, and 1C4) displayed sulfating activity toward hyperoside. Kinetic parameters of SULT1C4 that showed the strongest sulfating activity were determined. Ten SULT1C4 allozymes previously prepared were shown to display differential sulfating activities toward hyperoside, revealing clearly the functional impact of SULT1C4 genetic polymorphisms. These findings provided a robust biochemical foundation for further studies on the metabolism of hyperoside by sulfation. [ABSTRACT FROM AUTHOR]

Posttranscriptional regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK), a 50-kDa uPAR mRNA binding protein. PGK catalyzes a reversible transfer of a phosphoryl group from 1,3-biphosphoglycerate to ADP in the glycolytic pathway. Our previous studies showed that overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells results in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression through destabilization of the mRNA. In order to determine the role of PGK enzymatic activity on uPAR mRNA stability we mutated PGK by changing amino acid P204H and amino acid D219A. The mutant proteins were expressed in Epicurian coli BL21 cells, and the purified proteins were analyzed for PGK activity. We found that mutation of amino acid P204H and D219A reduced PGK activity by 99 and 83%, respectively. By gel mobility shift and Northwestern assay, we found that the mutant proteins were able to bind to uPAR mRNA as effectively as wild-type PGK. Overexpression of mutant, inactive PGK in H157 cells reduced cell surface uPAR protein as well as uPAR mRNA expression. Run-on transcription analysis indicated that overexpression of mutant PGKs fails to alter the rate of synthesis of uPAR mRNA, whereas transcription chase experiments demonstrated that both mutants and wild-type PGK reduce the stability of the uPAR mRNA transcripts to a similar extent. Overexpression of mutant PGK also inhibited the rate of DNA synthesis and the invasion-migration ratio. These results demonstrate that uPAR mRNA binding activity as well as PGK-mediated regulation of uPAR mRNA are independent of PGK enzymatic activity. messenger ribonucleic acid stability; messenger ribonucleic acid binding proteins