Exploring the Dimerization of BACE2-CFP Fusion Protein in HEK293 Cells through Fluorescence Microscopy Techniques

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder frequently characterized by the presence of senile amyloid plaques. These plaques are formed through the aggregation of the amyloid β (Aβ) peptide. Aβ is formed through the sequential proteolysis of amyloid precursor protein (APP) by the β-site APP cleaving enzyme-1 (BACE1) and γ-secretase. As the rate-limiting enzyme of Aβ formation, the amyloidogenic properties of BACE1 have been widely examined. Recently, there has been evidence that suggests BACE1 exists as a dimer that is significantly more catalytic than the monomeric form. However, as of yet, there have been no complimentary studies that have explored the dimerization properties of the BACE1 homolog, BACE2. The BACE1 and BACE2 enzymes make up a unique class of membrane-bound aspartyl proteases that share similar extracellular, transmembrane, and cytosolic domains. Interestingly, it is only the BACE1 enzyme that exhibits amyloidogenic properties. Contrarily, BACE2 demonstrates distinct anti-AD characteristics such as the non-amyloidogenic cleavage of APP at α- and θ-sites while also functioning as an efficient Aβ-degrading enzyme. Elucidating the dimerization of BACE2 can help us understand whether dimerization is an inherent characteristic of this unique class of proteases or, alternatively, if dimerization is an attribute required for the amyloidogenic properties of BACE1. In this study, cultured human embryonic kidney 293 (HEK293) cells were transfected with BACE2-CFP fusion protein and imaged using differential interference contrast (DIC) and confocal microscopy to examine sub-cellular localization of the fluorescent construct. Subsequently, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used to examine changes in the fluorescence lifetime distribution of transfected cells. The results of this study suggest that the BACE2 enzyme does not undergo dimerization in contrast to BACE1, as demonstrated in previous studies.