| Alzheimer's disease (AD) is a progressive neurodegenerative disease that results in memory loss, global cognitive dysfunction, and functional impairments. It is one of the most common forms of dementia. Generation, aggregation, and deposition of amyloidβ-peptide (Aβ) in brains of AD patients are a prominent pathological feature of this devastating neurodegenerative disease, and play an important role in the AD pathogenesis. Cleavage of the amyloid precursor protein (APP) by the aspartyl proteaseβ-site APP-cleaving enzyme (BACE) is the first step in the generation of the Aβ. Therefore, BACE plays an important role in the generation of Aβand is believed to be a promising therapeutic target for the prevention and treatment of AD. It is crucial to exploit the monitoring technology of BACE activity and understand the structure of BACE.However, the traditional methods can not be used to monitor the activity and structure of BACE in the living cells under physiological conditions. Here, using the fluorescence resonance energy transfer ( FRET) technology , combining with the genetically encoded FRET probes and fluorescent proteins, we monitored the activity and structure of BACE in the living cells. The major results of this study are showed as following:1) Five genetically encoded FRET probes that based on green fluorescent protein (GFP) were constructed using bioengineering technique. The FRET sensors consist of a peptide linker sandwiched between monomeric yellow and cyan mutants of GFP. According to the sequence of the peptide linker, the FRET probes are used as a control probe (YcC) or four FRET probes for detecting the BACE activity: YβCwt, YβCSweS, YβCSweL, YβCNFEV, in those the peptide linkers are wild type BACE substrate site (BSS), shorter Sweden mutant BSS, longer Sweden mutant BSS, and'NFEV'mutant BSS, respectively. Fluorescence spectroscopic analysis showed that a strong FRET signal was recorded, demonstrating energy transfer from CFP to YFP in integral FRET probes. The fluorescence spectroscopic and SDS-PAGE analysis results showed that the control probe YcC can not be cleaved by BACE, and the other four probes that peptide linker are BSS can be cleaved by BACE, and the cleavage efficiency of YβCwt,YβCSweS,YβCSweL and YβCNFEV was orderly increased.2) Since BACE and APP are both secretory transmembrane proteins, the FRET probe is chosen to be constructed into a mammalian expression vector pDisplay, creating a FRET probe dYβC (displayed YβC) for detecting BACE activity and a control probe dYcC (displayed YcC). Confocal imaging showed that dYβC and dYcC could be directed into the secretory pathway and displayed on the cell surface, giving it the chance of being cleaved by BACE. The dYβC and dYcC in the living cells showed high FRET efficiency. The FRET efficiency of dYβC decreased significantly in present of BACE, and the FRET efficiency of dYcC was unchanged. The results indicate the FRET probe can be cleaved by BACE efficiency in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo. Furthermore, the activity of BACE in secretory pathway was detected using this FRET probe. The results suggest thatβ-secretase cleavage was initiated in the secretory pathway, as early as in the ER.3) The dimerization of BACE in intact living cells were monitered using confocal microscopy and acceptor photobleaching FRET. We constructed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) tagged BACE-FL and BACE-NT, respectively. The expression and location of BACE-FL and BACE-NT are observed by confocal microscopy and FRET between CFP- and YFP- tagged BACE was detected by acceptor photobleaching method. The results showed that the BACE-FL can be transported to the Golgi apparatus, plasma membrane and endosomes, however, the BACE-NT is retained in the ER. BACE-FL exists as a dimmer in living cells, but the BACE-NT is monomer. This results suggesting the transmembrane and C-terminus region is important for normal transport and location, and is necessary for the dimerization of BACE.
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