Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.

Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities

Vitamin A is a nutrient with remarkable effects on adipose tissue and skeletal muscles, and plays a role in controlling energy balance. Retinoic acid (RA), the carboxylic form of vitamin A, has been associated with improved glucose tolerance and insulin sensitivity. In contrast, elevated glucocorticoids have been implicated in the development of insulin resistance and impaired glucose tolerance. Here, we investigated whether RA might counteract glucocorticoid effects in skeletal muscle cells by lowering 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local glucocorticoid activation and/or activation of glucocorticoid receptor (GR). We found a dose-dependent down-regulation of 11beta-HSD1 mRNA expression and activity upon incubation of fully differentiated mouse C2C12 myotubes with RA. In addition, RA inhibited GR transactivation by an 11beta-HSD1-independent mechanism. The presence of RA during myogenesis did not prevent myotube formation but resulted in relatively glucocorticoid-resistant myotubes, exhibiting very low 11beta-HSD1 expression and GR activity. The use of selective retinoic acid receptor (RAR) and retinoid X receptor ligands provided evidence that these effects were mediated through RARgamma. Importantly, short hairpin RNA against RARgamma abolished the effect of RA on 11beta-HSD1 and GR. In conclusion, we provide evidence for an important role of RA in the control of glucocorticoid activity during myogenesis and in myotubes. Disturbances of the nutrient and hormonal regulation of glucocorticoid action in skeletal muscles might be relevant for metabolic diseases.

Distamycin-NA: A DNA Analog with an Aromatic Heterocyclic Polyamide Backbone. Part 1. Synthesis and structural analysis of monomers and dimers containing the nucleobase uracil

The synthesis of the monomeric building block 13 and its constitutional isomer 12 of a new type of DNA analog, distamycin-NA, is presented (Schemes 1 and 2). This building block consists of a uracil base attached to a thiophene core unit via a biaryl-like axis. Next to the biaryl-like axis on the thiophene chromophore, a carboxy and an amino substituent are located allowing for oligomerization via peptide coupling. The proof of constitution and the conformational preferences about the biaryl-like axis were established by means of X-ray analyses of the corresponding nitro derivatives 10 and 11. Thus, the uracil bases are propeller-twisted relative to the thiophene core, and bidentate H-bonds occur between two uracil bases in the crystals. The two amino-acid building blocks 12 and 13 were coupled to give the dimers 15 and 16 using dicyclohexylcarbodiimide (DCC) in THF/LiCl and DMF, respectively. While the dimer 15 showed no atropisomerism on the NMR time scale at room temperature, its isomer 16 occurred as distinct diastereoisomers due to the hindered rotation around its biaryl-like axis. Variable-temperature 1H-NMR experiments allowed to determine a rotational barrier of 19 ± 1 kcal/mol in 16. The experimental data were complemented by AM1 calculations.

Distamycin-NA: A DNA analog with an aromatic heterocyclic polyamide backbone. Part 2. Solid-phase synthesis of distamycin-NAs containing the nucleobase uracil: Unexpected solvent participation in the coupling step

10.1002/hlca.19980810512.abs The synthesis of the Fmoc-protected amino acid 2 is presented. First attempts of amide-bond formation to the homodimer 4 in solution showed only poor coupling yields indicative for the low reactivity of the amino and carboxy groups in the building blocks 1 and 2, respectively (Scheme 1). Best coupling yields were found using dicyclohexylcarbodiimide (DCC) without any additive. The oligomerization of building block 2 adopting the Fmoc ((9H-fluoren-9-ylmethoxy)carbonyl) solid-phase synthesis yielded a mixture of N-terminal-modified distamycin-NA derivatives. By combined HPLC and MALDI-TOF-MS analysis, the N-terminal functional groups could be identified as acetamide and N,N-dimethylformamidine functions, arising from coupling of the N-terminus of the growing chain with residual AcOH or DCC-activated solvent DMF. An improved preparation of building block 2 and coupling protocol led to the prevention of the N-terminal acetylation. However, ‘amidination’ could not be circumvented. A thus isolated tetramer of 2, containing a lysine unit at the C-terminus and a N,N-dimethylformamidine-modified N-terminus, not unexpectedly, showed no complementary base pairing to DNA and RNA, as determined by standard UV-melting-curve analysis.