A genetically encoded sensor for H2O2 with expanded dynamic range

Markvicheva, Kseniya N.; Bilan, Dmitry S.; Mishina, Natalia M.; Gorokhovatsky, Andrey Yu.; Vinokurov, Leonid M.; Lukyanov, Sergey; Belousov, Vsevolod V.

doi:10.1016/j.bmc.2010.07.014

Cited by 164 publications

(141 citation statements)

References 17 publications

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“…After oxidation, slow reduction of HyPer due to disulfide reductase activity within the cell occurs, which leads to eventual decreases in the signal (Figure 1). The observation that oxidation occurs more rapidly than reduction is consistent with previous characterization of HyPer's signal in mammalian cells [24,25]. Because of its mechanism of action, HyPer can function as an intracellular, reversible sensor, but researchers should note that its signal reverses well after peroxide has been removed from the system by cellular antioxidants.…”

Section: Temporal Behavior Of Hyper's Signalsupporting

confidence: 88%

“…This suggests positive cooperativity in the reaction between HyPer and H 2 O 2 , which in turn suggests that oxidized HyPer may interact in some way with another HyPer molecule that is reduced, facilitating its oxidation by H 2 O 2 ; this would be consistent with the behavior of the OxyR transcription factor [27]. While purified HyPer has been characterized as a monomer, it is also known that it can exist as a mixture of dimers and monomers at higher concentrations [24,25].…”

Section: Effects Of Expression Host Strain On Dose-response Curves Anmentioning

confidence: 73%

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In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Lim

Barker

Huang

et al. 2014

Journal of Microbiological Methods

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Highlights• We characterized the signal of HyPer, a fluorescent probe for peroxide, in E. coli.• Each strain requires its own specific signal characterization.• HyPer's signal is reversible rather than real-time.• Expression of HyPer reduced the rate of peroxide scavenging by the expression host.• Careful, controlled use of HyPer facilitates quantitative comparisons across studies. AbstractGenetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H 2 O 2 ), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria. Here, we performed this characterization, specifically in Escherichia coli exposed to external H 2 O 2 . We found that the temporal behavior of the signal does not correspond exactly to peroxide concentration in the system as a function of time and expression of the sensor decreases the peroxide scavenging activity of the cell. We also determined the effects of cell number, both before and after normalization of externally added H 2 O 2 to the number of cells. Finally, we report quantitative characteristics of HyPer's signal in this context, including the dynamic range of the signal, the signal-to-noise ratio, and the half saturation constant. These parameters show statistically meaningful differences in signal between two commonly used strains of E. coli, demonstrating how signal can vary with strain. Taken together, our results establish a systematic, quantitative framework for researchers seeking to better understand the role of H 2 O 2 in the immunological response against bacteria, and for understanding potential differences in the details of HyPer's quantitative performance across studies.

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Section: Temporal Behavior Of Hyper's Signalsupporting

confidence: 88%

Section: Effects Of Expression Host Strain On Dose-response Curves Anmentioning

confidence: 73%

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Lim

Barker

Huang

et al. 2014

Journal of Microbiological Methods

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“…The HyPer2 H 2 O 2 biosensor yields an increase in fluorescence that is highly specific for H 2 O 2 . This biosensor has been characterized extensively in vitro and in cultured cells (29,(31)(32)(33). We isolated cardiac myocytes from mice after tail vein injection with the HyPer2 lentivirus and analyzed changes in cell-derived fluorescence after treating the cells with H 2 O 2 , Ang-II, or isoproterenol (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

Sartoretto

Kalwa

Pluth

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu 2 ðFL2EÞ] to study adult cardiac myocytes from wild-type, eNOS null , and nNOS null mice, we discovered that physiological concentrations of H 2 O 2 activate eNOS but not nNOS. H 2 O 2 -stimulated eNOS activation depends on phosphorylation of both the AMPactivated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H 2 O 2 biosensor HyPer2 show marked H 2 O 2 synthesis when stimulated by angiotensin II, but not following β-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H 2 O 2 , whereas β-adrenergic contractile responses occur independently of H 2 O 2 signaling. These studies establish differential roles for H 2 O 2 in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.

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“…21 While HyPer is predominantly monomeric, HyPer-2 forms a strong dimer. 15 Potentially, dimerization is a prerequisite for HyPer dynamic range enhancement. To evaluate the dimerization state of HyPer-3, we performed size-exclusion chromatography.…”

mentioning

confidence: 99%

HyPer-3: A Genetically Encoded H₂O₂ Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

et al. 2013

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High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H 2 O 2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H 2 O 2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for singlewavelength fluorescent lifetime imaging of H 2 O 2 levels both in vitro and in vivo. R eactive oxygen species (ROS) are products of incomplete molecular oxygen reduction. Among ROS, the superoxide anion radical O 2 * − and hydrogen peroxide H 2 O 2 are the most investigated in biology because they are produced by a wide range of enzymes, specifically or as side-products, and have a number of well-known biological effects.1 For a long time, ROS were viewed mostly in a context of their nonspecific damaging action on DNA, lipids, and proteins.2 This point of view was strongly supported by the discovery of antioxidant enzymes decomposing ROS and by the fact that phagocytes produce ROS when killing pathogens.1,3 Later, the ROS toxicity dogma was challenged after specialized O 2 * − and H 2 O 2 producing enzymes were found to be expressed in most cell types.4,5 This fact gave a new impulse to ROS investigations but, now in the context of their regulatory function, as signaling molecules. It was shown that H 2 O 2 acted as a second messenger selectively oxidizing those cysteine residues in proteins that were ionized (deprotonated) at physiological pH values. 6,7 Initially, protein tyrosine phosphatases were shown to be reversibly inactivated by H 2 O 2 . 8,9 Since then, the list of known redox regulated proteins grew exponentially. During the last years, proteomics and computational approaches added a lot of new members to the list.

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A genetically encoded sensor for H2O2 with expanded dynamic range

Cited by 164 publications

References 17 publications

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

HyPer-3: A Genetically Encoded H₂O₂ Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

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A genetically encoded sensor for H2O2 with expanded dynamic range

Cited by 164 publications

References 17 publications

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

HyPer-3: A Genetically Encoded H2O2 Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

Contact Info

Product

Resources

About

HyPer-3: A Genetically Encoded H₂O₂ Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging