RESUMO
Epidemiological studies have documented a reduced prevalence of Alzheimer's disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Abeta42 peptide (the 42-residue isoform of the amyloid-beta peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant beta-amyloid precursor protein (APP) lowered their brain levels of Abeta42. In cultured cells, the decrease in Abeta42 secretion was accompanied by an increase in the Abeta(1-38) isoform, indicating that NSAIDs subtly alter gamma-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Abeta42 peptide levels independently of COX activity and that this Abeta42-lowering activity could be optimized to selectively target the pathogenic Abeta42 species.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Fragmentos de Peptídeos/metabolismo , Sulindaco/análogos & derivados , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/etiologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Ácido Aspártico Endopeptidases , Encéfalo/metabolismo , Células CHO , Cricetinae , Modelos Animais de Doenças , Endopeptidases/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Ibuprofeno/farmacologia , Indometacina/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulindaco/farmacologia , Células Tumorais CultivadasRESUMO
The enzymatic oxidation of uridine 5'-diphospho-alpha-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-alpha-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-epsilon-amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-alpha-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-N-acetyl-alpha-D-galactosamine (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases beta3Gal-T5, beta4Gal-T1, and beta4Gal-T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin-p-aminophenyl N-acetyl-beta-D-glucosaminide (BSA-(GlcNAc)17) and ovalbumin. The detection of the biotin tag transferred by beta3Gal-T5 onto BSA-(GlcNAc)17 with streptavidin-enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree of Gal-biotin tag transfer onto agalactosylated hybrid N-glycans present at the single glycosylation site of ovalbumin was dependent on the Gal-T used (either beta3Gal-T5, beta4Gal-T4, or beta4Gal-T1), which indicates that the acceptor specificity may direct the transfer of the Gal-biotin tag. The potential of this biotinylated UDP-Gal as a novel donor substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under-galactosylated glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.
Assuntos
Biotina/análogos & derivados , Galactose Oxidase/química , Galactosiltransferases/metabolismo , Glicosiltransferases/metabolismo , Uridina Difosfato Galactose/química , Uridina Difosfato N-Acetilglicosamina/química , Biotina/química , Biotina/metabolismo , Biotinilação , Western Blotting , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/química , Espectroscopia de Ressonância Magnética , Ovalbumina/química , Estreptavidina/químicaRESUMO
Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.
Assuntos
Enzimas/química , Enzimas/genética , Mutagênese Sítio-Dirigida , Oxigênio/química , Proteínas de Bactérias/química , Carbono/química , DNA Polimerase Dirigida por DNA/genética , Biblioteca de Peptídeos , Software , TemperaturaRESUMO
The present review gives a survey on the biosynthetic pathways of nucleotide sugars which are important for the in vitro synthesis of mammalian glycoconjugates. With respect to the use of these enzymes in glycotechnology the availability as recombinant enzymes from different sources, the large-scale synthesis of nucleotide sugars and their in situ regeneration in combination with glycosyltransferases are summarized and evaluated.