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      Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells

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          Abstract

          Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1], [2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2O 2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •–) and H 2O 2 in the presence of NADH. Generation of the free radical O 2 •– and H 2O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

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          Highlights

          • Immature eGFP and TagRFP catalytically generate O 2 • – and H 2O 2 in the presence of NADH.

          • Cells expressing eGFP show gene expression associated with oxidative stress.

          • Experiments using fluorescent proteins should be alternatively validated.

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          Most cited references28

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            Creating new fluorescent probes for cell biology.

            Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.
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              Bright monomeric red fluorescent protein with an extended fluorescence lifetime.

              Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
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                Author and article information

                Contributors
                Journal
                Redox Biol
                Redox Biol
                Redox Biology
                Elsevier
                2213-2317
                10 March 2017
                August 2017
                10 March 2017
                : 12
                : 462-468
                Affiliations
                [a ]Free Radical Biology, Immunity, Inflammation & Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
                [b ]Stem Cell Toxicology Group, National Toxicology Program Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
                [c ]Protein Expression Core Facility, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
                Author notes
                [* ]Correspondence to: Free Radical Metabolites Group, Immunity, Inflammation & Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. mason4@ 123456niehs.nih.gov
                [1]

                Authors equally contributed to this work.

                [2]

                Current address: University of Copenhagen, Department of Biomedical Sciences, Cellular and Metabolic Research Section, Blegdamsvej 3, 2200 København N, 4.5, Copenhagen, Denmark.

                Article
                S2213-2317(17)30153-2
                10.1016/j.redox.2017.03.002
                5362137
                28334681
                cb003156-3fce-426e-8373-f2503add4109

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 28 February 2017
                : 3 March 2017
                Categories
                Research Paper

                gfp,h2o2 (hydrogen peroxide),o2•– (superoxide free radical anion),free radicals,oxidative stress,redox biology

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