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Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase proc...

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Autores principales: Houser, Mei C. Q., Turchyna, Yuliia, Perrin, Florian, Chibnik, Lori, Berezovska, Oksana, Maesako, Masato
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228125/
https://www.ncbi.nlm.nih.gov/pubmed/34073182
http://dx.doi.org/10.3390/bios11060169
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author Houser, Mei C. Q.
Turchyna, Yuliia
Perrin, Florian
Chibnik, Lori
Berezovska, Oksana
Maesako, Masato
author_facet Houser, Mei C. Q.
Turchyna, Yuliia
Perrin, Florian
Chibnik, Lori
Berezovska, Oksana
Maesako, Masato
author_sort Houser, Mei C. Q.
collection PubMed
description Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.
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spelling pubmed-82281252021-06-26 Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells Houser, Mei C. Q. Turchyna, Yuliia Perrin, Florian Chibnik, Lori Berezovska, Oksana Maesako, Masato Biosensors (Basel) Article Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells. MDPI 2021-05-26 /pmc/articles/PMC8228125/ /pubmed/34073182 http://dx.doi.org/10.3390/bios11060169 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Houser, Mei C. Q.
Turchyna, Yuliia
Perrin, Florian
Chibnik, Lori
Berezovska, Oksana
Maesako, Masato
Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title_full Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title_fullStr Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title_full_unstemmed Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title_short Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
title_sort limited substrate specificity of ps/γ-secretase is supported by novel multiplexed fret analysis in live cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228125/
https://www.ncbi.nlm.nih.gov/pubmed/34073182
http://dx.doi.org/10.3390/bios11060169
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