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

1,2,3, 1,2, 3, 2,3, 1, 2,3, 1,* and 2,3,*
1
Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Cytology and Genetics, Northeast Normal University, Changchun 130024, China
2
Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA
3
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA
*
Authors to whom correspondence should be addressed.
Cells 2019, 8(5), 474; https://doi.org/10.3390/cells8050474
Received: 21 March 2019 / Revised: 9 May 2019 / Accepted: 15 May 2019 / Published: 17 May 2019
(This article belongs to the Special Issue Key Signalling Molecules in Aging and Neurodegeneration)
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Abstract

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. View Full-Text
Keywords: pHluorin-BACE1-mCherry; BACE1; intracellular trafficking; pH regulation; PRR; APP; Alzheimer disease pHluorin-BACE1-mCherry; BACE1; intracellular trafficking; pH regulation; PRR; APP; Alzheimer disease
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Zhao, L.; Zhao, Y.; Tang, F.-L.; Xiong, L.; Su, C.; Mei, L.; Zhu, X.-J.; Xiong, W.-C. pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo. Cells 2019, 8, 474.

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