Biomedical subjects

Ana V Coelho

Publications and source records attributed to Ana V Coelho.

8 recordsLinked to original sources

PubMed2006

Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins.

Protein glycation by methylglyoxal is a nonenzymatic post-translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end-products. Methylglyoxal-derived advanced glycation end-products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI-TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolase's activity loss, in a glycation-dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end-products were determined by MALDI-TOF. Yeast cells experienced selective pressure towards efficient use of d-glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).

Amino Acid Sequence↗

PubMed2006

Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Amino Acid Sequence↗

PubMed2006

Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease.

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.

Adolescent↗

PubMed2005

Hydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenase.

The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H(2) production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S](2+/1+) cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe](1) Hase, with the Type I cytochrome c (3) as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.

Amino Acid Sequence↗

PubMed2005

Liquid chromatography-diode array detection-electrospray ionisation mass spectrometry/nuclear magnetic resonance analyses of the anti-hyperglycemic flavonoid extract of Genista tenera. Structure elucidation of a flavonoid-C-glycoside.

The anti-hyperglycemic flavonoid extract obtained from Genista tenera was first studied by liquid chromatography (LC)-diode array detection (DAD) which showed the presence of two major compounds. One of them was identified as genistein-7-O-glucoside. Luteolin-7-O-glucoside was detected as a minor constituent, while luteolin-7,3'-di-O-glucoside and rutin were found in trace amounts. LC-DAD-ESI-MS and NMR were used to confirm the structure of these compounds and allowed the elucidation of the structure of the unknown major compound, which is the flavonoid 5,7,4'-trihydroxyisoflavone-8-C-glucoside.

Carbohydrate Conformation↗

PubMed2004

Effect of the manganese ion on human alpha3/4 fucosyltransferase III activity.

The effect of manganese and other divalent cations on the activity of a soluble recombinant form of human alpha3/4 fucosyltransferase III (SFT3) expressed in Spodoptera frugiperda (Sf9) insect cells was studied. SFT3 was active in the absence of divalent cations with an optimum pH of 4.5. In the absence of Mn2+ increasing the pH from 4.5 to 7.0 caused a decrease in the affinity of SFT3 for the acceptor Galbeta3GlcNAcO(CH2)3NHCO(CH2)5NH-biotin, as monitored by the 4-fold increase in the apparent KM value (0.9 to 3.3 mM). At pH 7.0, the addition of Mn2+ activated the enzyme and caused an increase in the affinity of SFT3 for the acceptor, as monitored by the 5-fold decrease of the apparent KM value (3.3 to 0.7 mM). In solution, a complex between GDP-Fuc donor and the divalent cation Mn2+ was observed by electrospray ionization mass spectrometry, in a 1:1 stoichiometry. These results indicated that Mn2+ bound the enzyme and increased its affinity for the acceptor; one possible functional role of manganese in catalysis could be as an electrophilic catalyst, co-ordinating the negative charges of the phosphate groups of the GDP-Fuc donor and promoting Fuc transfer. At low pH values such role would be played by the proton.

Amino Acid Sequence↗

PubMed2002

A novel iron centre in the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774.

The facultative sulfate/nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 harbours a split-Soret cytochrome c. This cytochrome is a homodimeric protein, having two bis-histidinyl c-type haems per monomer. It has an unique architecture at the haem domain: each haem has one of the coordinating histidines provided by the other monomer, and in each monomer the haems are parallel to each other, almost in van der Waals contact. This work reports the cloning and sequencing of the gene encoding for this cytochrome and shows, by transcriptional analysis, that it is more expressed in nitrate-grown cells than in sulfate-grown ones. In addition, the gene-deduced amino acid sequence revealed two new cysteine residues that could be involved in the binding of a non-haem iron centre. Indeed, the presence of a novel type of an iron-sulfur centre (possibly of the [2Fe-2S] type) was demonstrated by EPR spectroscopy, and putative models for its localization and structure in the cytochrome molecule are proposed on the basis of the so-far-known 3D crystallographic structure of the aerobically purified split-Soret cytochrome, which lacks this centre.

Amino Acid Sequence↗

PubMed2002

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer.

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.

Amino Acid Motifs↗