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pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo

β-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and β-amyloid (Aβ) 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 a...

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Autores principales: Zhao, Lu, Zhao, Yang, Tang, Fu-Lei, Xiong, Lei, Su, Ce, Mei, Lin, Zhu, Xiao-Juan, Xiong, Wen-Cheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562731/
https://www.ncbi.nlm.nih.gov/pubmed/31108937
http://dx.doi.org/10.3390/cells8050474
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author Zhao, Lu
Zhao, Yang
Tang, Fu-Lei
Xiong, Lei
Su, Ce
Mei, Lin
Zhu, Xiao-Juan
Xiong, Wen-Cheng
author_facet Zhao, Lu
Zhao, Yang
Tang, Fu-Lei
Xiong, Lei
Su, Ce
Mei, Lin
Zhu, Xiao-Juan
Xiong, Wen-Cheng
author_sort Zhao, Lu
collection PubMed
description β-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and β-amyloid (Aβ) 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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spelling pubmed-65627312019-06-17 pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo Zhao, Lu Zhao, Yang Tang, Fu-Lei Xiong, Lei Su, Ce Mei, Lin Zhu, Xiao-Juan Xiong, Wen-Cheng Cells Article β-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and β-amyloid (Aβ) 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. MDPI 2019-05-17 /pmc/articles/PMC6562731/ /pubmed/31108937 http://dx.doi.org/10.3390/cells8050474 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhao, Lu
Zhao, Yang
Tang, Fu-Lei
Xiong, Lei
Su, Ce
Mei, Lin
Zhu, Xiao-Juan
Xiong, Wen-Cheng
pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title_full pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title_fullStr pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title_full_unstemmed pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title_short pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo
title_sort phluorin-bace1-mcherry acts as a reporter for the intracellular distribution of active bace1 in vitro and in vivo
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562731/
https://www.ncbi.nlm.nih.gov/pubmed/31108937
http://dx.doi.org/10.3390/cells8050474
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