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pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo.
Cells ; 8(5)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108937
ABSTRACT
ß-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and ß-amyloid (Aß) production, a critical step in the pathogenesis of Alzheimer's disease (AD). It is thus of considerable interest to investigate how BACE1 activity is regulated. BACE1 has its maximal activity at acidic pH and GFP variant-pHluorin-displays pH dependence. In light of these observations, we generated three tandem fluorescence-tagged BACE1 fusion proteins, named pHluorin-BACE1-mCherry, BACE1-mCherry-pHluorin and BACE1-mCherry-EGFP. Comparing the fluorescence characteristics of these proteins in response to intracellular pH changes induced by chloroquine or bafilomycin A1, we found that pHluorin-BACE1-mCherry is a better pH sensor for BACE1 because its fluorescence intensity responds to pH changes more dramatically and more quickly. Additionally, we found that (pro)renin receptor (PRR), a subunit of the v-ATPase complex, which is critical for maintaining vesicular pH, regulates pHluorin's fluorescence and BACE1 activity in pHluorin-BACE1-mCherry expressing cells. Finally, we found that the expression of Swedish mutant APP (APPswe) suppresses pHluorin fluorescence in pHluorin-BACE1-mCherry expressing cells in culture and in vivo, implicating APPswe not only as a substrate but also as an activator of BACE1. Taken together, these results suggest that the pHluorin-BACE1-mCherry fusion protein may serve as a useful tool for visualizing active/inactive BACE1 in culture and in vivo.
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Texto completo: Disponível Coleções: Bases de dados internacionais Base de dados: MEDLINE Assunto principal: Ácido Aspártico Endopeptidases / Proteínas de Fluorescência Verde / Secretases da Proteína Precursora do Amiloide / Proteínas Luminescentes Limite: Animais / Feminino / Humanos / Masculino Idioma: Inglês Ano de publicação: 2019 Tipo de documento: Artigo País de afiliação: China