I am a biologist working on sulfur amino acid metabolism and associated pathways. My research expands from structural studies to pathological changes associated with this essential part of cellular metabolism.
The intermediate filament protein vimentin constitutes a critical sensor for electrophiles and ox... more The intermediate filament protein vimentin constitutes a critical sensor for electrophiles and oxidative stress in mesenchymal cells. The structure of the vimentin network structure is altered by lipoxidation, although the mechanisms involved are not completely understood. Here, we have studied the response of vimentin to oxidation and lipoxidation, both in vitro and in cells. Electrophilic lipids including 15d-PGJ2 and HNE bind to isolated vimentin in vitro, whereas the oxidant diamide induces disulfide bond formation. Requirement for the presence of the cysteine residue differs among these modifications. Interestingly, pre-incubation of vimentin with these agents disrupts NaCl-induced filament formation, whereas NaCl polymerized-vimentin appears to be more resistant to filament disruption by electrophiles and oxidants. These observations suggest that (lip)oxidative modifications of soluble vimentin subunits would be more deleterious than modification of polymerized vimentin. There...
Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased re... more Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased reactive oxygen or electrophilic species can play a pathogenic role. Aldose reductase (AKR1B1) is involved in aldehyde detoxification and redox balance. Recent evidence from animal models points to a role of AKR1B1 in asthma and rhinitis, but its involvement in human allergy has not been addressed. Here, the putative association of allergic rhinitis and asthma with AKR1B1 variants has been explored by analysis of single-strand variants on the AKR1B1 gene sequence in 526 healthy subjects and 515 patients with allergic rhinitis, 366 of whom also had asthma. We found that the rs2229542 variant, introducing the p.Lys90Glu mutation, was significantly more frequent in allergic patients than in healthy subjects. Additionally, in cells transfected with expression vectors carrying the wild-type or the p.Lys90Glu variant of AKR1B1, the mutant consistently attained lower protein levels than the wild-...
The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in ... more The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC p...
The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a pho... more The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.
Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetas... more Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribo...
Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homo... more Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit....
<p>The figure shows representative titration experiments carried out with the wild type reg... more <p>The figure shows representative titration experiments carried out with the wild type regulatory β subunit to characterize binary and tertiary binding with α2 subunits and/or NADP<sup>+</sup>. Panel A depicts titration of α2 (13.4 µM in the cell) and β subunits (190 µM in the syringe). Panel B shows titration of the β subunit (9.8 µM in the cell) and NADP<sup>+</sup> (155 µM in the syringe); the insets show titrations using the β subunit (20 µM) and 300 µM NAD<sup>+</sup> (top) and the Y159F/K163A-β subunit (20 µM) and 330 µM NADP<sup>+</sup> (bottom). The very low affinity observed for the mutant precluded a precise estimation of the binding affinity, and hence only a lower limit for the dissociation constant could be determined. Panel C illustrates titration of α2 (4.3 µM in the cell) and β subunits (62.4 µM in the syringe) in the presence of NADP<sup>+</sup> (300 µM in both the cell and the syringe). All measurements were performed at 25°C as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050329#s2" target="_blank">Materials and Methods</a> section.</p
The intermediate filament protein vimentin constitutes a critical sensor for electrophiles and ox... more The intermediate filament protein vimentin constitutes a critical sensor for electrophiles and oxidative stress in mesenchymal cells. The structure of the vimentin network structure is altered by lipoxidation, although the mechanisms involved are not completely understood. Here, we have studied the response of vimentin to oxidation and lipoxidation, both in vitro and in cells. Electrophilic lipids including 15d-PGJ2 and HNE bind to isolated vimentin in vitro, whereas the oxidant diamide induces disulfide bond formation. Requirement for the presence of the cysteine residue differs among these modifications. Interestingly, pre-incubation of vimentin with these agents disrupts NaCl-induced filament formation, whereas NaCl polymerized-vimentin appears to be more resistant to filament disruption by electrophiles and oxidants. These observations suggest that (lip)oxidative modifications of soluble vimentin subunits would be more deleterious than modification of polymerized vimentin. There...
Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased re... more Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased reactive oxygen or electrophilic species can play a pathogenic role. Aldose reductase (AKR1B1) is involved in aldehyde detoxification and redox balance. Recent evidence from animal models points to a role of AKR1B1 in asthma and rhinitis, but its involvement in human allergy has not been addressed. Here, the putative association of allergic rhinitis and asthma with AKR1B1 variants has been explored by analysis of single-strand variants on the AKR1B1 gene sequence in 526 healthy subjects and 515 patients with allergic rhinitis, 366 of whom also had asthma. We found that the rs2229542 variant, introducing the p.Lys90Glu mutation, was significantly more frequent in allergic patients than in healthy subjects. Additionally, in cells transfected with expression vectors carrying the wild-type or the p.Lys90Glu variant of AKR1B1, the mutant consistently attained lower protein levels than the wild-...
The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in ... more The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC p...
The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a pho... more The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.
Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetas... more Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribo...
Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homo... more Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit....
<p>The figure shows representative titration experiments carried out with the wild type reg... more <p>The figure shows representative titration experiments carried out with the wild type regulatory β subunit to characterize binary and tertiary binding with α2 subunits and/or NADP<sup>+</sup>. Panel A depicts titration of α2 (13.4 µM in the cell) and β subunits (190 µM in the syringe). Panel B shows titration of the β subunit (9.8 µM in the cell) and NADP<sup>+</sup> (155 µM in the syringe); the insets show titrations using the β subunit (20 µM) and 300 µM NAD<sup>+</sup> (top) and the Y159F/K163A-β subunit (20 µM) and 330 µM NADP<sup>+</sup> (bottom). The very low affinity observed for the mutant precluded a precise estimation of the binding affinity, and hence only a lower limit for the dissociation constant could be determined. Panel C illustrates titration of α2 (4.3 µM in the cell) and β subunits (62.4 µM in the syringe) in the presence of NADP<sup>+</sup> (300 µM in both the cell and the syringe). All measurements were performed at 25°C as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050329#s2" target="_blank">Materials and Methods</a> section.</p
The Liver: Oxidative stress and dietary antioxidants, 2018
The methionine cycle synthesizes S-adenosylmethionine, the main cellular methyl donor.
Regulatio... more The methionine cycle synthesizes S-adenosylmethionine, the main cellular methyl donor.
Regulation of the methionine cycle is exerted at several levels from transcription up to subcellular localization.
The liver is the major organ for methionine consumption and metabolization.
The methionine cycle also produces S-adenosylhomocysteine and homocysteine.
Reduced S-adenosylmethionine levels, as well as increased plasma homocysteine concentrations associate with a variety of diseases.
Dozens of reactions use S-adenosylmethionine and among them epigenetic modifications.
Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic... more Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.
Uploads
Papers by Maria Angeles A Pajares
Regulation of the methionine cycle is exerted at several levels from transcription up to subcellular localization.
The liver is the major organ for methionine consumption and metabolization.
The methionine cycle also produces S-adenosylhomocysteine and homocysteine.
Reduced S-adenosylmethionine levels, as well as increased plasma homocysteine concentrations associate with a variety of diseases.
Dozens of reactions use S-adenosylmethionine and among them epigenetic modifications.