PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate

Abstract The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of d...

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Autores principales:	Jan Lüddecke, Liliana Francois, Philipp Spät, Björn Watzer, Tomasz Chilczuk, Gernot Poschet, Rüdiger Hell, Bernhard Radlwimmer, Karl Forchhammer
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/59b27422404a4414a4cbbba02ae670d1
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spelling	oai:doaj.org-article:59b27422404a4414a4cbbba02ae670d12021-12-02T16:07:48ZPII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate10.1038/s41598-017-01440-w2045-2322https://doaj.org/article/59b27422404a4414a4cbbba02ae670d12017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01440-whttps://doaj.org/toc/2045-2322Abstract The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.Jan LüddeckeLiliana FrancoisPhilipp SpätBjörn WatzerTomasz ChilczukGernot PoschetRüdiger HellBernhard RadlwimmerKarl ForchhammerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jan Lüddecke Liliana Francois Philipp Spät Björn Watzer Tomasz Chilczuk Gernot Poschet Rüdiger Hell Bernhard Radlwimmer Karl Forchhammer PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
description	Abstract The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.
format	article
author	Jan Lüddecke Liliana Francois Philipp Spät Björn Watzer Tomasz Chilczuk Gernot Poschet Rüdiger Hell Bernhard Radlwimmer Karl Forchhammer
author_facet	Jan Lüddecke Liliana Francois Philipp Spät Björn Watzer Tomasz Chilczuk Gernot Poschet Rüdiger Hell Bernhard Radlwimmer Karl Forchhammer
author_sort	Jan Lüddecke
title	PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_short	PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_full	PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_fullStr	PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_full_unstemmed	PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_sort	pii protein-derived fret sensors for quantification and live-cell imaging of 2-oxoglutarate
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/59b27422404a4414a4cbbba02ae670d1
work_keys_str_mv	AT janluddecke piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT lilianafrancois piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT philippspat piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT bjornwatzer piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT tomaszchilczuk piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT gernotposchet piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT rudigerhell piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT bernhardradlwimmer piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate AT karlforchhammer piiproteinderivedfretsensorsforquantificationandlivecellimagingof2oxoglutarate
_version_	1718384717528563712

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate

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