Background

Alzheimer’s disease (AD) is the most prevalent chronic neurodegenerative disease. AD is closely associated with the formation of amyloid plaques. These plaques mainly consist of aggregated amyloid β, which is generated by the cleavage of the C terminal 99 amino acids fragment (C99) of the amyloid precursor protein by γ-secretase. In spite of extensive efforts, it remains unknown how γ-secretase recognizes its substrates. The conventional Tango assay was designed to monitor the activation of GPCRs (Barnea et al., 2008) by engineering a TEV cleavage site and a transcription activator to the cytoplasmic C terminus to of GPCRs and a TEV protease linked to human β-arrestin2. Here we used endogenous or transfected γ-secretase to cleave the transmembrane portion of its substrates, i.e., the C terminus of C99 is fused with rTA to establish the γ-secretase Epsilon-cleavage assay (Figure 1). The assay was first established to investigate the initial cleavage of C99 by γ-secretase (Xu et al., 2016).


Figure 1. The principle of the γ-secretase epsilon-cleavage assay. Upon cleavage of the C99 (or other γ-secretase substrates) hybrid protein by γ-secretase, the rTA is released from the membrane and enters nucleus to bind tetO DNA-binding site to stimulate luciferase reporter gene activity as measurement for total cleavage, both by endogenous and by transfected γ-secretase variants. (Xu et al., 2016)

Here we use the Dual-luciferase reporter assay kit. The stably integrated luciferase-Firefly reads represent the γ-secretase cleavage activity, while the transfected Renilla luciferase reads serve as a normalization standard.