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Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER

The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases....

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Autores principales: Hoseki, Jun, Oishi, Asami, Fujimura, Takaaki, Sakai, Yasuyoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832336/
https://www.ncbi.nlm.nih.gov/pubmed/26934978
http://dx.doi.org/10.1042/BSR20160027
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author Hoseki, Jun
Oishi, Asami
Fujimura, Takaaki
Sakai, Yasuyoshi
author_facet Hoseki, Jun
Oishi, Asami
Fujimura, Takaaki
Sakai, Yasuyoshi
author_sort Hoseki, Jun
collection PubMed
description The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both in vitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER.
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spelling pubmed-48323362016-04-25 Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER Hoseki, Jun Oishi, Asami Fujimura, Takaaki Sakai, Yasuyoshi Biosci Rep Original Papers The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both in vitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER. Portland Press Ltd. 2016-04-15 /pmc/articles/PMC4832336/ /pubmed/26934978 http://dx.doi.org/10.1042/BSR20160027 Text en © 2016 The Author(s) http://creativecommons.org/licenses/by/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY) (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Original Papers
Hoseki, Jun
Oishi, Asami
Fujimura, Takaaki
Sakai, Yasuyoshi
Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title_full Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title_fullStr Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title_full_unstemmed Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title_short Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER
title_sort development of a stable errogfp variant suitable for monitoring redox dynamics in the er
topic Original Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832336/
https://www.ncbi.nlm.nih.gov/pubmed/26934978
http://dx.doi.org/10.1042/BSR20160027
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