Visualization of PS/γ-Secretase Activity in Living Cells.

Polina Feschenko,Nicole M. Sekula,Masato Maesako,Anna Aristarkhova,Lauren C. Anderson,Oksana Berezovska

doi:10.1016/j.isci.2020.101139

Polina Feschenko, Nicole M. Sekula

Open Access

https://doi.org/10.1016/j.isci.2020.101139

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Abstract

SummaryA change in Presenilin (PS)/γ-secretase activity is linked to essential biological events as well as to the progression of many diseases. However, not much is known about how PS/γ-secretase activity is spatiotemporally regulated in cells. One of the limitations is lack of tools to directly monitor dynamic behavior of the PS/γ-secretase in intact/live cells. Here we present successful development and validation of the Förster resonance energy transfer (FRET)-based biosensors that enable quantitative monitoring of endogenous PS/γ-secretase activity in live cells longitudinally on a cell-by-cell basis. Using these FRET biosensors, we uncovered that PS/γ-secretase activity is heterogeneously regulated among live neurons.

Highlights

Presenilin (PS)/g-secretase is a membrane-embedded aspartic protease responsible for the proteolytic processing of a wide variety of membrane-associated proteins that include the amyloid precursor protein (APP) and Notch1 (De Strooper et al, 1998, 1999; Wolfe et al, 1999)
We present successful development and validation of the Forster resonance energy transfer (FRET)-based biosensors that enable quantitative monitoring of endogenous PS/g-secretase activity in live cells longitudinally on a cell-by-cell basis. Using these FRET biosensors, we uncovered that PS/g-secretase activity is heterogeneously regulated among live neurons
The C terminus of human APP C99 is tagged with RFP and EGFP is connected to the RFP with 20 amino acids (a.a.) SAGG-repeat linker (Komatsu et al, 2011)

Summary

Introduction

Presenilin (PS)/g-secretase is a membrane-embedded aspartic protease responsible for the proteolytic processing of a wide variety of membrane-associated proteins that include the amyloid precursor protein (APP) and Notch (De Strooper et al, 1998, 1999; Wolfe et al, 1999). The proteolytic cleavage of APP by BACE1/b-secretase releases the APP extracellular domain and generates the membrane-bound APP C99 fragment that is an immediate substrate of PS/g-secretase. The first step cleavage of APP C99 by PS/g-secretase, which is known as epsilon-cleavage, generates longer Ab48 or Ab49 peptides and the APP intracellular domain (AICD). In the case of Notch, the N-terminally truncated Notch by a furin-like protease and ADAMs/a-secretase is further processed by PS/g-secretase. This PS/g-secretase-mediated Notch processing generates Notch intracellular domain (NICD), resulting in transcriptional changes in the nucleus, which is known as Notch signaling (Schroeter et al, 1998)

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