Thiazolidinediones (PPARgamma agonists) but not PPARalpha agonists increase IRS-2 gene expression in 3T3-L1 and human adipocytes.

Thiazolidinediones (TZD) improve insulin sensitivity in human as well as in different animal models of insulin resistance and Type 2 diabetes. However, no clear link to the insulin signaling events has been identified. Using differentiated 3T3-L1 adipocytes, we found that TZD rapidly and markedly increased IRS-2 gene expression. This effect was specific for PPARgamma agonists and was not seen with PPARalpha agonists. It was rapidly induced (within 4 h) and maintained throughout the observation period of 48 h. It was also concentration dependent (EC50 approximately 50 nM) and not inhibited by cycloheximide, suggesting a direct effect on the IRS-2 promoter. There was no evidence that TZD altered IRS-2 mRNA stability, supporting that the increased mRNA levels were due to an increased gene transcription. IRS-2 protein expression was increased approximately 30% after 48 h and approximately 50% after 96 h. No effects of TZD were seen on IRS-1, PKB/Akt, or GLUT4 gene expression. TZD also increased IRS-2 mRNA levels in cultured human adipose tissue. These data show the first direct link between TZD and a critical molecule in insulin's signaling cascade in both 3T3-L1 and human adipocytes, and indicate a novel mode of action of these compounds.

Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

Three-dimensional structure prediction of the NAD binding site of proton-pumping transhydrogenase from Escherichia coli.

A three-dimensional structure of the NAD site of Escherichia coli transhydrogenase has been predicted. The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions. The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the alpha-subunit. The proposed structure spans residues alpha 145 to alpha 287, and it includes five beta-strands and five alpha-helices oriented in a typical open twisted alpha/beta conformation. The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues alpha 172 to alpha 177) correlates exactly to a typical fingerprint region for ADP binding beta alpha beta folds in dinucleotide binding enzymes. In the model, aspartic acid alpha 195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety. Threonine alpha 196 and alanine alpha 256, located at the end of beta B and beta D, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside. The nicotinamide part is located in a hydrophobic cleft between alpha A and beta E. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support the predicted structure, and no residue crucial for proton pumping was detected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump.

Prediction and site-specific mutagenesis of residues in transmembrane alpha-helices of proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli and bovine heart mitochondria.

Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg). The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E. coli showed an identity of 52%, indicating similarly folded structures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit. Various conserved amino acid residues of the E. coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis. Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit. beta Glu82 was replaced by Lys or Gln and beta Asp213 by Asn or His. However, the catalytic as well as the proton pumping activity was retained. In contrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping. Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue. The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted alpha-helix 10 of the E. coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.

Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase.

Nicotinamide nucleotide transhydrogenase (E.C.1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds to the alpha subunit and whose C-terminal region corresponds to the beta subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit. In addition, tyrosine 315 of the beta subunit is of interest since mutation of an adjacent residue (glycine 314) leads to inactivation [Ahmad, S., Glavas, N. A., & Bragg, P. D. (1992) Eur. J. Biochem. 207, 733-739]. In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bacteriol. 162, 367-373]. Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH. These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or quinacrine fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis.

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.

Expression of spinach plastocyanin in E. coli.

An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fermentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.