Detection of beta-secretase dimerization in living cells using acceptor photobleaching FRET

作者：Lu Jin Ling; Chu Jun; Yang Jie; Zeng Shao Qun; Luo Qing Ming; Zhang Zhi Hong^*

来源：Progress in Biochemistry and Biophysics, 2008, 35(3): 268-273.

• beta-secretase (BACE)
• dimerization
• Alzheimer'
• s disease (AD)
• fluorescence resonance energy transfer (FRET)
• acceptor photobleaching

Generation, aggregation, and deposition of amyloid beta-peptide (A beta) in the brains of Alzheimer';s disease (AD) patients are inherent pathological features of AD. Cleavage of the amyloid precursor protein (APP) by the beta-secretase (BACE) is the first step in the generation of A beta. Inhibition of BACE activity is a promising therapeutic strategy for the treatment of AD. Therefore, understanding the structure of BACE is very important. Using blue native gel electrophoresis it has been found that BACE exists as a homodimer. However, it is currently unknown whether in living cells BACE forms homodimer. To address this issue, the dimerization of BACE in intact living cells was monitored by using confocal microscopy and acceptor photobleaching FRET. Using bioengineering technique, fluorescence proteins (FPs)-tagged BACEs (BACE/FPs) was constructed. They are cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) tagged full length BACE (BACE-FL) and truncated BACE (BACE-NT). The CFP- and YFP- tagged BACE-FL (BACE-FL/FPs) or CFP- and YFP- tagged BACE-NT (BACE-NT/FPs) plasmids were transtected into HeLa cells, and the expression and location of BACE-FL and BACE-NT were observed by confocal microscopy. The dimerization of BACE was evaluated by the FRET between CFP- and YFP- tagged BACE which was detected by acceptor photobleaching method. The results show that the BACE-FL can be transported to the Golgi apparus, plasma membrane and endosomes. However, the BACE-NT is found in the ER 24 hours after transtection. The FRET efficiency of BACE-FL/FPs group is (12.90 +/- 0.73)%, significantly higher than that of control group (3.32 +/- 0.55%) (P < 0.05). The FRET efficiency of BACE-NT/FPs group is (3.80 +/- 0.74)%, which is not statistically different from that of control group. These results indicat that the BACE-FL exists as a dimmer in living cells, but the BACE-NT exists as a monomer, suggesting the transmembrane and C-terminus regions are important for normal transport and localization, and is necessary for the dimerization of BACE.

• 出版日期2008-3
• 单位华中科技大学