title: Leishmania donovani: proteasome-mediated down-regulation of methionine adenosyltransferase.
abstract: Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and 'mock-transfected' leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.
authors.affiliation: Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n; 24071 León, Spain. myperp@unileon.es
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Pérez-Pertejo
authors.forename: Raquel
authors.initials: R
authors.lastname: Alvarez-Velilla
authors.forename: Carlos García
authors.initials: CG
authors.lastname: Estrada
authors.forename: Rafael
authors.initials: R
authors.lastname: Balaña-Fouce
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Reguera
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects*; D047468:Immunoprecipitation; D007700:Kinetics; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D007976:Leupeptins / Q000494:pharmacology; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010957:Plasmids; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D007976:Leupeptins; D009842:Oligopeptides; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011994:Recombinant Proteins; D025801:Ubiquitin; C089486:carbobenzoxy-leucyl-leucyl-norvalinal; C067713:lactacystin; D012436:S-Adenosylmethionine; D003513:Cycloheximide; D008716:Methionine Adenosyltransferase; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine; C078846:epoxomicin