The intracellular localization of the mineralocorticoid receptor is regulated by 11beta-hydroxysteroid dehydrogenase type 2

11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11beta-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11beta-HSD2, we coexpressed epitope-tagged MR and 11beta-HSD2 in HEK-293 cells lacking 11beta-HSD2 activity and analyzed their subcellular localization by fluorescence microscopy. When expressed alone in the absence of hormones, the MR was both cytoplasmic and nuclear. However, when coexpressed with 11beta-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11beta-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11beta-HSD2, but not with 11beta-HSD1 or other steroid-metabolizing enzymes. Aldosterone induced rapid nuclear translocation of the MR, whereas moderate cortisol concentrations (10-200 nm) did not activate the receptor, due to 11beta-HSD2-dependent oxidation to cortisone. Compromised 11beta-HSD2 activity (due to genetic mutations, the presence of inhibitors, or saturating cortisol concentrations) led to cortisol-induced nuclear accumulation of the MR. Surprisingly, the 11beta-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11beta-HSD2-dependent mechanism. Our results provide evidence that 11beta-HSD2, besides inactivating 11beta-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation.     

13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides

UDP-galactose 4-epimerase contains the coenzyme NAD+ bound tightly at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+ may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+ bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm to 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 153 to Met results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD+ in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+ in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes the positive electrostatic field and attenuates the reactivity of NAD+. Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD+. The effects of binding the substrate analogue P1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.     

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site

A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ). From the model, two groups of active site residues were identified. The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH). These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are discussed within the context of the 4,6-dehydratase active site model and chemical mechanism.     

Characterization of the transition-state structure of the reaction of kanamycin nucleotidyltransferase by heavy-atom kinetic isotope effects

The transition-state structure for the reaction catalyzed by kanamycin nucleotidyltransferase has been determined from kinetic isotope effects. The primary (18)O isotope effects at pH 5.7 (close to the optimum pH) and at pH 7.7 (away from the optimum pH) are respectively 1.016 +/- 0.003 and 1.014 +/- 0.002. Secondary (18)O isotope effects of 1.0033 +/- 0.0004 and 1.0024 +/- 0.0002 for both nonbridge oxygen atoms were measured respectively at pH 5.7 and 7.7. These isotope effects are consistent with a concerted reaction with a slightly associative transition-state structure.     

Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.     

Synergy between ascorbate and alpha-tocopherol on fibroblasts in culture

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and alpha-tocopherol in cells is difficult to detect as both ascorbate and alpha-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and alpha-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and alpha-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and alpha-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of alpha-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected alpha-tocopherol but only in presence of cells. Cell viability was preserved by alpha-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and alpha-tocopherol corresponds to a consumption of ascorbate which spares alpha-tocopherol but only in presence of cells.     

Production of the fungal biocontrol agent Ulocladium atrum by submerged fermentation: accumulation of endogenous reserves and shelf-life studies

A method was developed for the induction of submerged conidiation of Ulocladium atrum Preuss (isolate 385) for the first time, using an oatmeal extract broth. Two inoculum types were produced by this process: spores and mycelial fragments. Spore production was stimulated by reducing the broth water potential (psi) to -2.1 MPa and adding 20 mM calcium chloride. In contrast, mycelial fragments were dominant at -7.0 MPa psi. Maximum total inoculum (mycelial fragments and conidia) yields were approximately 2 x 10(7) ml(-1) after 9 days incubation at 25 degrees C at 100 rpm. Biomass from liquid cultures responded to water-stress by accumulating increased concentrations of endogenous sugar alcohols (polyols), particularly glycerol. Long-term shelf-life studies showed that submerged inoculum from cultures subjected to an intermediate water-stress (-2.1 MPa psi) and containing enhanced levels of glycerol (> 300 mg g(-1) freeze-dried material) retained viability significantly better (P < 0.05) than that from unstressed cultures, when assessed on agar with fully available water. This level of viability was comparable to that of aerial U. atrum spores from a 4-week solid-substrate fermentation on oat grains. However, in contrast to aerial spores, the ability of submerged biomass to germinate in drier conditions declined significantly after 6 months.     

Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast antioxidant functional in pathogen defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.