Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/deficiência , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Sistema Nervoso Central/enzimologia , Endopeptidases , Camundongos , Camundongos Knockout/genética , Camundongos Knockout/metabolismo , Sistema Nervoso Periférico/enzimologia , Fenótipo , Placa Amiloide/enzimologiaRESUMO
Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase.
Assuntos
Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Glicoproteínas/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/anatomia & histologia , Endopeptidases/química , Glicoproteínas/metabolismo , Humanos , Hibridização In Situ , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Alinhamento de SequênciaRESUMO
Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Doença de Alzheimer/tratamento farmacológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , Endopeptidases , Endossomos/enzimologia , Expressão Gênica , Biblioteca Gênica , Complexo de Golgi/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
We have recently shown that the ability of some gram-negative bacteria to dissolve poorly soluble calcium phosphates (Mps+ phenotype) is the result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct oxidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline-quinone (PQQ) essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, these strains do not produce gluconic acid and are Mps-. Evidence is presented to show that expression of a single 396-base Pseudomonas cepacia open reading frame (designated gabY) in E. coli JM109 (a K-12 derivative) was sufficient to induce the Mps+ phenotype and production of gluconic acid. We present the nucleotide sequence of this open reading frame which coded for a protein (GabY) with a deduced M(r) of 14,235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) carrying gabY resulted in production of a protein with an M(r) of 14,750. Disruption of the open reading frame of gabY via site-directed mutagenesis changed the phenotype to Mps- and eliminated gluconic acid production. The deduced amino acid sequence of gabY has no apparent homology with those of previously cloned direct oxidation pathway genes but does share regions highly homologous with the histidine permease system membrane-bound protein HisQ as well as other proteins in this family. In the presence of 1 microM exogenous PQQ, both JM109(pSLY4) and JM109(pGAB1) produced 10 times as much gluconic acid as was seen with either the plasmid or exogenous PQQ alone.(ABSTRACT TRUNCATED AT 250 WORDS)
