RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Fragmentos de Péptidos/metabolismo , Sulindac/análogos & derivados , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/etiología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Ácido Aspártico Endopeptidasas , Encéfalo/metabolismo , Células CHO , Cricetinae , Modelos Animales de Enfermedad , Endopeptidasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Ibuprofeno/farmacología , Indometacina/farmacología , Espectrometría de Masas , Ratones , Ratones Transgénicos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sulindac/farmacología , Células Tumorales CultivadasRESUMEN
The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.
Asunto(s)
Glucurónidos/biosíntesis , Cetoprofeno/análogos & derivados , Riñón/metabolismo , Microsomas Hepáticos/metabolismo , Adulto , Androstano-3,17-diol/análogos & derivados , Androstano-3,17-diol/biosíntesis , Androstano-3,17-diol/metabolismo , Androstano-3,17-diol/farmacocinética , Animales , Perros , Femenino , Furosemida/análogos & derivados , Furosemida/metabolismo , Furosemida/farmacocinética , Gemfibrozilo/metabolismo , Gemfibrozilo/farmacocinética , Glucuronidasa/metabolismo , Humanos , Cetoprofeno/metabolismo , Cetoprofeno/farmacocinética , Riñón/enzimología , Cinética , Masculino , Microsomas/enzimología , Microsomas/metabolismo , Microsomas Hepáticos/enzimología , Persona de Mediana Edad , Modelos Animales , Propofol/metabolismo , Propofol/farmacocinética , Especificidad de la EspecieRESUMEN
The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/análisis , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Células CHO , Sistema Libre de Células , Cricetinae , Concentración de Iones de Hidrógeno , Modelos Biológicos , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , SolubilidadRESUMEN
Increased thyroid hormone glucuronidation in rats caused by exposure to xenobiotics has stimulated a search for the individual uridine diphosphate-glucuronosyltransferases (UGTs) catalyzing this reaction in rats and man. Microsomal preparations from Crigler-Najjar liver, normal human liver, and kidney have been used to try to identify the UGT isoforms responsible for glucuronidation of the thyroid hormones. The predominant thyroid hormone released from the thyroid gland, T4, and the inactive rT3 are glucuronidated by cloned expressed bilirubin UGT1A1 and also phenol UGT1A9. Results from Crigler-Najjar microsomal samples indicate that UGT1A1 is the main contributor to thyroid hormone glucuronidation in the liver, with rT3 being the preferential substrate. In kidney microsomes thyroid hormone glucuronidation is more complex, suggesting that more than just the UGT1A9 isoform may be involved. Bioactive T3 is not significantly glucuronidated by these isoforms and other UGTs, and sulfotransferases may be involved.
Asunto(s)
Síndrome de Crigler-Najjar/enzimología , Glucuronosiltransferasa/metabolismo , Riñón/enzimología , Microsomas Hepáticos/enzimología , Microsomas/enzimología , Tiroxina/metabolismo , Triyodotironina Inversa/metabolismo , Animales , Síndrome de Crigler-Najjar/genética , Exones , Glucuronosiltransferasa/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratas , Eliminación de Secuencia , Especificidad por Sustrato , TATA BoxRESUMEN
Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Células CHO , Dominio Catalítico , Línea Celular , Cricetinae , Endopeptidasas/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Presenilina-1RESUMEN
The effect of abundance and compartmentalization of antigenic epitopes expressed in Escherichia coli on phagocytic processing was studied by expressing fusion proteins containing the epitope from positions 52 to 61 of hen egg white lysozyme [HEL(52-61)], which binds the I-Ak murine major histocompatibility complex class II (MHC-II) molecule or the epitope from positions 257 to 264 of chicken egg ovalbumin [OVA(257-264]), which binds the Kb murine MHC-I molecule. Epitopes expressed as fusion proteins in the outer membrane protein LamB allowed exposure of the epitopes either at the bacterial surface, in the periplasmic space, or in the cytoplasm. Regardless of epitope compartmentalization within the bacterium, MHC-II-restricted or MHC-I-restricted presentation to T hybridoma cells occurred after macrophages phagocytosed bacteria producing the HEL(52-61) epitope or the OVA(257-264) epitope, respectively. Increased epitope abundance within a given microbial compartment resulted in increased processing and presentation to epitope-specific T hybridoma cells. Minor differences in the efficiency of epitope processing between the constructs was observed, and the HEL or OVA epitope exposed in the periplasmic space was processed most efficiently compared with the surface- or cytoplasm-localized epitopes. These differences could be overcome by increasing the amount of epitope per bacterium as little as two to five times. The minor differences in processing efficiency may be due to differing protein contexts of the epitope as well as differing epitope compartmentalizations within the bacteria. Thus, production of abundant epitope is the important parameter influencing processing of epitopes expressed in E. coli to induce T-cell responses rather than targeting of an epitope to a specific bacterial compartment.