'Reactive oxygen species' (ROS) is a generic term that defines a wide variety of oxidant molecules with vastly different properties and biological functions that range from signalling to causing cell ...damage. Consequently, the description of oxidants needs to be chemically precise to translate research on their biological effects into therapeutic benefit in redox medicine. This Expert Recommendation article pinpoints key issues associated with identifying the physiological roles of oxidants, focusing on H
O
and O
. The generic term ROS should not be used to describe specific molecular agents. We also advocate for greater precision in measurement of H
O
, O
and other oxidants, along with more specific identification of their signalling targets. Future work should also consider inter-organellar communication and the interactions of redox-sensitive signalling targets within organs and whole organisms, including the contribution of environmental exposures. To achieve these goals, development of tools that enable site-specific and real-time detection and quantification of individual oxidants in cells and model organisms are needed. We also stress that physiological O
levels should be maintained in cell culture to better mimic in vivo redox reactions associated with specific cell types. Use of precise definitions and analytical tools will help harmonize research among the many scientific disciplines working on the common goal of understanding redox biology.
Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these ...reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.
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•HyPer7 is an ultrasensitive, ultrafast, and pH-stable indicator for H2O2•Being controlled by the Trx system, H2O2 does not diffuse out from mitochondria•Intensity of the H2O2 gradient associates with the stability of cellular protrusions•H2O2 gradients can be visualized in wounded tissues with HyPer7
Pak et al. have developed HyPer7, a next-generation genetically encoded fluorescent probe for H2O2 detection. Importantly, HyPer7 is resistant to pH changes. Applying HyPer7 to study H2O2 diffusion from the mitochondrial matrix, the authors decipher the topology of H2O2 production by Complex I and visualize oxidant gradients in cell motility and wounded tissue.
HyPer Family Probes: State of the Art Bilan, Dmitry S; Belousov, Vsevolod V
Antioxidants & redox signaling,
05/2016, Letnik:
24, Številka:
13
Journal Article
Recenzirano
Hydrogen peroxide (H2O2) is not only a key mediator of oxidative stress but also one of the most important cellular second messengers. This small short-lived molecule is involved in the regulation of ...a wide range of different biological processes, including regulation of cellular signaling pathways. Studying the role of H2O2 in living systems would be challenging without modern approaches. A genetically encoded fluorescent biosensor, HyPer, is one of the most effective tools for this purpose.
HyPer has been used by many investigators of redox signaling in various models of different scales: from cytoplasmic subcompartments and single cells to tissues of whole organisms. In many studies, the results obtained using HyPer have enabled a better understanding of the roles of H2O2 in these biological processes. However, much remains to be learned.
In this review, we focus on the uses of HyPer. We provide a general description of HyPer and its improved versions. Separate chapters are devoted to the results obtained by various groups who have used this biosensor for their experiments in living cells and organisms.
HyPer is an effective tool for H2O2 imaging in living systems as indicated by the increasing numbers of publications each year since its development. However, this biosensor requires further improvements. In particular, much brighter and more pH-stable versions of HyPer are necessary for imaging in mammalian tissues. Antioxid. Redox Signal. 24, 731-751.
Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different ...compartments, to examine these processes in mammalian cells.
Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.
We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.
Genetically encoded fluorescent redox sensors Lukyanov, Konstantin A.; Belousov, Vsevolod V.
Biochimica et biophysica acta,
February 2014, 2014-Feb, 2014-02-00, Letnik:
1840, Številka:
2
Journal Article
Recenzirano
Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in ...living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.
In this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development.
Fluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H2O2, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP.
Genetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O2 and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
•We review genetically encoded fluorescent probes for intracellular redox events.•Probes for H2O2, GSH/GSSG and NAD+/NADH are discussed.•GFP redding as a redox-dependent process is highlighted.
Redox reactions play a key role in maintaining essential biological processes. Deviations in redox pathways result in the development of various pathologies at cellular and organismal levels. Until ...recently, studies on transformations in the intracellular redox state have been significantly hampered in living systems. The genetically encoded indicators, based on fluorescent proteins, have provided new opportunities in biomedical research. The existing indicators already enable monitoring of cellular redox parameters in different processes including embryogenesis, aging, inflammation, tissue regeneration, and pathogenesis of various diseases. In this review, we summarize information about all genetically encoded redox indicators developed to date. We provide the description of each indicator and discuss its advantages and limitations, as well as points that need to be considered when choosing an indicator for a particular experiment. One chapter is devoted to the important discoveries that have been made by using genetically encoded redox indicators.
•Genetically encoded indicators for redox biology research.•Detailed characteristics of each group of indicators.•Examples of studies using redox indicators.
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Hypoxia is an intensively investigated condition with profound effects on cell metabolism, migration, and angiogenesis during development and disease. Physiologically, hypoxia is linked to tissue ...homeostasis and maintenance of pluripotency. Hypoxia also contributes to pathologies including cardiovascular diseases and cancer. Despite its importance, microscopic visualization of hypoxia is largely restricted to the detection of reductively activated probes by immunostaining. Here, we describe a novel family of genetically encoded fluorescent sensors that detect the activation of HIF transcription factors reported by the oxygen‐independent fluorescent protein UnaG. It comprises sensors with different switching and memory behavior and combination sensors that allow the distinction of hypoxic and reoxygenated cells. We tested these sensors on orthotopically transplanted glioma cell lines. Using a cranial window, we could visualize hypoxia intravitally at cellular resolution. In tissue samples, sensor activity was detected in regions, which were largely devoid of blood vessels, correlated with HIF‐1α stabilization, and were highly heterogeneous at a cellular level. Frequently, we detected recently reoxygenated cells outside hypoxic areas in the proximity of blood vessels, suggestive of hypoxia‐promoted cell migration.
Synopsis
The Japanese eel green fluorescent protein UnaG, unlike GFP and RFP, matures independently of molecular oxygen, which makes it an ideal reporter to indicate the hypoxic state in vivo.
A hypoxia responsive promoter drives the expression of UnaG, which fluorescently marks hypoxic cells.
Through combination with different degrons, the kinetics of the sensor can be tuned, varying hypoxic memory.
A fusion sensor comprised of UnaG and mOrange identifies, in addition to hypoxic, recently reoxygenated cells.
RFP‐ and GFP‐based reporters require oxygen to mature. The Japanese eel green fluorescent protein UnaG matures independently of oxygen, which makes it an ideal reporter to reveal hypoxia.
Hydrogen peroxide (H
O
) is a key signaling molecule involved in the regulation of both physiological and pathological cellular processes. Genetically encoded HyPer probes are currently among the ...most effective approaches for monitoring H
O
dynamics in various biological systems because they can be easily targeted to specific cells and organelles. Since its development in 2006, HyPer has proved to be a robust and powerful tool in redox biology research. Recent Advances: HyPer probes were used in a variety of models to study the role of H
O
in various redox processes. HyPer has been increasingly used in the past few years for in vivo studies, which has already led to many important discoveries, for example, that H
O
plays a key role in the regulation of signaling cascades involved in development and aging, inflammation, regeneration, photosynthetic signaling, and other biological processes.
In this review, we focus on the main achievements in the field of redox biology that have been obtained from in vivo experiments using HyPer probes.
Further in vivo studies of the role of H
O
largely depend on the development of more suitable versions of HyPer for in vivo models: those having brighter fluorescence and a more stable signal in response to physiological changes in pH. Antioxid. Redox Signal. 29, 569-584.
Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to ...the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.
Cell metabolism heavily relies on the redox reactions that inevitably generate reactive oxygen species (ROS). It is now well established that ROS fluctuations near basal levels coordinate numerous ...physiological processes in living organisms, thus exhibiting regulatory functions. Hydrogen peroxide, the most long‐lived ROS, is a key contributor to ROS‐dependent signal transduction in the cell. H2O2 is known to impact various targets in the cell; therefore, the question of how H2O2 modulates physiological processes in a highly specific manner is central in redox biology. To resolve this question, novel genetic tools have recently been created for detecting H2O2 and emulating its generation in living organisms with unmatched spatiotemporal resolution. Here, we review H2O2‐sensitive genetically encoded fluorescent sensors and opto‐ and chemogenetic tools for controlled H2O2 generation.
Hydrogen peroxide (H2O2) is one of the most important signaling molecules, which coordinates many physiological processes ranging from development to wound healing. Identification of mechanisms underlying specificity of H2O2‐dependent signal transduction is a central issue in redox biology. A palette of genetically encoded tools for generation and detection of H2O2 with unprecedented spatiotemporal resolution has been recently created to resolve this issue. Here, we present an overview of these tools.
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