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1.  Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings 
Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (EGSH) of more negative than −310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure EGSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between −280 and −290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about −238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of EGSH values that can be measured in vivo with roGFP2-based probes from about −320 mV for GRX1-roGFP2 down to about −210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an EGSH of about −260 mV in this mutant.
PMCID: PMC3863748  PMID: 24379821
glutathione; glutathione redox potential; GRX1-roGFP2; rml1; redox imaging
2.  Distinct Redox Regulation in Sub-Cellular Compartments in Response to Various Stress Conditions in Saccharomyces cerevisiae 
PLoS ONE  2013;8(6):e65240.
Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (EGSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. EGSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.
PMCID: PMC3676407  PMID: 23762325
3.  Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum 
PLoS Pathogens  2013;9(12):e1003782.
In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.
Author Summary
In the malaria parasite Plasmodium falciparum, cellular redox reactions play important roles not only in redox-regulatory processes and antioxidant defense but also in the mechanisms of drug action and drug resistance. The tripeptide glutathione is present in malaria parasites in millimolar concentrations and represents the most important low molecular weight antioxidant. In this work, we describe the application of a genetically encoded glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein. By targeting this probe to the parasites' cytosol, we were able to verify its stability and responsiveness to the redox environment. Using this probe in living parasites, we further studied the effects of various redox active compounds and antimalarial drugs on the cytosolic glutathione redox potential in short, medium, and long-term experiments. Interestingly, after 24 h most antimalarial drugs, including quinolines and artemisinin derivatives, induced pronounced changes in the redox potential in pharmacologically meaningful concentrations. The chloroquine-sensitive P. falciparum 3D7 strain was generally more susceptible to these changes than the resistant Dd2 strain. We propose the hGrx1-roGFP2 probe as a reliable and most valuable tool for studying redox metabolism in living malaria parasites.
PMCID: PMC3857819  PMID: 24348249
4.  Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells 
Environmental Health Perspectives  2012;121(3):312-317.
Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies.
Objective: We tested the utility of redox-sensitive green fluorescent protein (roGFP)–based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies.
Methods: roGFP2, a reporter of the glutathione redox potential (EGSH), was used to monitor EGSH in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15–1.0 ppm ozone (O3). Cells were imaged in real time using a custom-built O3 exposure system coupled to a confocal microscope.
Results: O3 exposure induced a dose- and time-dependent increase of the cytosolic EGSH. Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O3, and a Grx1-roGFP2 fusion protein responded more rapidly to O3 exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression–enhanced roGFP2 responsiveness to O3, suggesting that (hydro)peroxides are intermediates linking O3 exposure to glutathione oxidation.
Conclusion: Exposure to O3 induces a profound increase in the cytosolic EGSH of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties.
PMCID: PMC3621206  PMID: 23249900
glutathione; human airway epithelial cells; imaging; intracellular; oxidative stress; ozone; NADPH; redox; roGFP
5.  Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection 
PLoS Pathogens  2014;10(1):e1003902.
Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (EMSH) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial EMSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of EMSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in EMSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in EMSH, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied EMSH during infection and show that subpopulation with higher EMSH are susceptible to clinically relevant antibiotics, whereas lower EMSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence.
Author Summary
Approximately 30% of the global population is infected with Mycobacterium tuberculosis (Mtb). Persistence of Mtb in host phagocytes depends on its ability to resist oxidant-mediated antibacterial responses. Mycothiol (MSH) is the main antioxidant that provides an abundant source of reducing equivalent, which protects Mtb from oxidative stress encountered during infection. The majority of research into redox signaling in Mtb has relied on chemical analysis of MSH in whole cell extract, which creates oxidation artifacts and prohibits dynamic imaging of MSH redox state during infection. We have successfully developed a novel and noninvasive tool based on genetically encoded redox sensitive fluorescent probes to perform real-time measurement of mycothiol redox potential (EMSH) in Mtb during infection. For the first time we reveal the EMSH of virulent and avirulent mycobacterial strains, including drug-resistant clinical isolates. We used this technology and came to the surprising conclusion that within a single infected macrophage there is heterogeneity in the redox signature of individual Mtb bacilli. Importantly, we show that anti-TB drugs accelerate oxidative stress in Mtb within infected macrophages and redox heterogeneity can contribute to emergence of drug tolerant population. These findings have implications for mycobacterial persistence following treatment with anti-TB drugs.
PMCID: PMC3907381  PMID: 24497832
6.  Förster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment 
The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of −143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER glutathione pool may be more oxidized than is currently considered.
PMCID: PMC3415989  PMID: 22715429
live cell imaging; endoplasmic reticulum; redox-sensitive probe; green fluorescent protein; glutathione; Förster resonance energy transfer
7.  A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2 
Sensors (Basel, Switzerland)  2010;10(7):6290-6306.
A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10−7 (arsenite), 1.0 × 10−4 (naphthalene), 1.0 × 10−4 (Cu2+), 3.8 × 10−4 (H2O2), 1.0 × 10−3 (Cd2+), 1.0 × 10−3 (Zn2+), 1.0 × 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.
PMCID: PMC3231123  PMID: 22163550
oxidative biosensor; redox-sensitive GFP; ratiometric measurement; ROS; environmental stressors
Circulation research  2009;106(3):526-535.
Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation.
This study tested the hypothesis that hypoxia induces redox changes that differ among sub-cellular compartments in pulmonary (PASMC) and systemic (SASMC) smooth muscle cells.
Methods and Results
We employed a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMC and SASMC. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial inter-membrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMC or SASMC with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the over-expression of cytosolic catalase in PASMC.
These results indicate that hypoxia causes a decrease in non-specific ROS generation in the matrix compartment, while it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMC and SASMC.
PMCID: PMC2856085  PMID: 20019331
Mitochondria; reactive oxygen species; RoGFP; hypoxic pulmonary vasoconstriction
9.  Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine 
Redox Biology  2014;2:475-484.
Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.
Graphical abstract
•Sensitivity of TR1 and TR2 vs. PRXs to oxidative inactivation.•Role of Sec vs. Cys in the active site of TR in cell viability.•Reduction of roGFP as compared to PRX after oxidation.•Effect of TR1 and TR2 on oxidation state of cellular compartments.
PMCID: PMC3949094  PMID: 24624337
Redox signaling; Menadione; Hydrogen peroxide; Peroxiredoxins
10.  Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana 
Plant Signaling & Behavior  2013;8(7):e24781.
Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.
PMCID: PMC3912002  PMID: 23656871
Arabidopsis thaliana; ascorbate; Col-0; cytosolic roGFP1; glutathione; leaf redox status; mitochondrial roGFP1; recovery,vtc2; water stress
11.  Linking Oxidative Events to Inflammatory and Adaptive Gene Expression Induced by Exposure to an Organic Particulate Matter Component 
Environmental Health Perspectives  2011;120(2):267-274.
Background: Toxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ).
Objective: To elucidate the mechanisms involved in 1,2-NQ–induced inflammatory responses, we examined the role of oxidant stress in 1,2-NQ–induced expression of inflammatory and adaptive genes in a human airway epithelial cell line.
Methods: We measured cytosolic redox status and hydrogen peroxide (H2O2) in living cells using the genetically encoded green fluorescent protein (GFP)-based fluorescent indicators roGFP2 and HyPer, respectively. Expression of interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1) mRNA was measured in BEAS-2B cells exposed to 1,2-NQ for 1–4 hr. Catalase overexpression and metabolic inhibitors were used to determine the role of redox changes and H2O2 in 1,2-NQ–induced gene expression.
Results: Cells expressing roGFP2 and HyPer showed a rapid loss of redox potential and an increase in H2O2 of mitochondrial origin following exposure to 1,2-NQ. Overexpression of catalase diminished the H2O2-dependent signal but not the 1,2-NQ–induced loss of reducing potential. Catalase overexpression and inhibitors of mitochondrial respiration diminished elevations in IL-8 and COX-2 induced by exposure to 1,2-NQ, but potentiated HO-1 mRNA levels in BEAS cells.
Conclusion: These data show that 1,2-NQ exposure induces mitochondrial production of H2O2 that mediates the expression of inflammatory genes, but not the concurrent loss of reducing redox potential in BEAS cells. 1,2-NQ exposure also causes marked expression of HO-1 that appears to be enhanced by suppression of H2O2. These findings shed light into the oxidant-dependent events that underlie cellular responses to environmental electrophiles.
PMCID: PMC3279454  PMID: 21997482
confocal microscopy; hydrogen peroxide; mitochondrial dysfunction; oxidative stress; quinones; reactive oxygen species; real-time imaging; ROS
12.  Developmental Stage Oxidoreductive States of Chlamydia and Infected Host Cells 
mBio  2014;5(6):e01924-14.
A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer membrane proteins. The reduction-oxidation states of host cell compartments were monitored during the developmental cycle using live fluorescence microscopy. Organelle redox states were studied using redox-sensitive green fluorescent protein (roGFP1) expressed in CF15 epithelial cells and targeted to the cytosol, mitochondria, and endoplasmic reticulum (ER). The redox properties of chlamydiae and the inclusion were monitored using roGFP expressed by Chlamydia trachomatis following transformation. Despite the large morphological changes associated with chlamydial infection, redox potentials of the cytosol (Ψcyto [average, −320 mV]), mitochondria (Ψmito [average, −345 mV]), and the ER (ΨER [average, −258 mV]) and their characteristic redox regulatory abilities remained unchanged until the cells died, at which point Ψcyto and Ψmito became more oxidized and ΨER became more reduced. The redox status of the chamydial cytoplasm was measured following transformation and expression of the roGFP biosensor in C. trachomatis throughout the developmental cycle. The periplasmic and outer membrane redox states were assessed by the level of cysteine cross-linking of cysteine-rich envelope proteins. In both cases, the chlamydiae were highly reduced early in the developmental cycle and became oxidized late in the developmental cycle. The production of a late-developmental-stage oxidoreductase/isomerase, DsbJ, may play a key role in the regulation of the oxidoreductive developmental-stage-specific process.
Infectious Chlamydia organisms have highly oxidized and cysteine cross-linked membrane proteins that confer environmental stability when outside their host cells. Once these organisms infect a new host cell, the proteins become reduced and remain reduced during the active growth stage. These proteins become oxidized at the end of their growth cycle, wherein infectious organisms are produced and released to the environment. How chlamydiae mediate and regulate this key step in their pathogenesis is unknown. Using biosensors specifically targeted to different compartments within the infected host cell and for the chlamydial organisms themselves, the oxidoreductive states of these compartments were measured during the course of infection. We found that the host cell redox states are not changed by infection with C. trachomatis, whereas the state of the chlamydial organisms remains reduced during infection until the late developmental stages, wherein the organisms’ cytosol and periplasm become oxidized and they acquire environmental resistance and infectivity.
PMCID: PMC4217174  PMID: 25352618
13.  Developing a Redox-Sensitive Red Fluorescent Protein Biosensor 
Redox environments are of particular interest, especially in the mitochondria with its highly reducing environment and its role as the central processing unit of apoptosis. Monitoring of mitochondrial redox environments is crucial to the study of apoptotic disorders. Reporting of the thiol/disulfide status in live cells was made possible with the development of redox-sensitive green fluorescent protein (roGFP). We aim to develop a red version redox-sensitive fluorescent protein (roRFP). Expanding the array of redox-sensitive proteins with a red version will enable simultaneous visualization of multiple reducing intracellular compartments. mKeima is a monomeric red fluorescent protein that absorbs light maximally at 440nm and emits red light at 620nm. This large Stokes shift is dramatically decreased in acidic environments. By following protocol similar to that used in the development of roGFP, surface residues at key positions were changed to cysteines and random mutagenesis was performed on varying excitation species of mKeima. Mutants were screened and a ratiometric variant of mKeima was identified (roRFP2) which exhibits changes in its spectral properties as a result of changes in the thiol/disulfide equilibrium. Preliminary fluorescence spectroscopy measurements of roRFP2 indicate a highly reducing redox potential of −330mV indicating it may be a useful probe in reducing subcellular compartments such as mitochondria or in the cytoplasm. By employing vector recombination of shuttle vector PYX142, we successfully targeted roRFP2 in vivo to the mitochondria and cytoplasm of Saccharomyces cerevisiae. Expression of roRFP2 was visualized using fluorescence microscopy. Thus, through mutagenesis and residue substitution we successfully created a red version redox sensitive biosensor that tested effectively as a ratiometric indicator and expressed in the mitochondria and cytoplasm of S. cerevisiae. Moreover, the redox potential of roRFP2 is significantly more negative than the widely used roGFPs.
PMCID: PMC3186468
14.  Redox Regulation of Cell Survival 
Antioxidants & Redox Signaling  2008;10(8):1343-1374.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulation of cell survival. In general, moderate levels of ROS/RNS may function as signals to promote cell proliferation and survival, whereas severe increase of ROS/RNS can induce cell death. Under physiologic conditions, the balance between generation and elimination of ROS/RNS maintains the proper function of redox-sensitive signaling proteins. Normally, the redox homeostasis ensures that the cells respond properly to endogenous and exogenous stimuli. However, when the redox homeostasis is disturbed, oxidative stress may lead to aberrant cell death and contribute to disease development. This review focuses on the roles of key transcription factors, signal-transduction pathways, and cell-death regulators in affecting cell survival, and how the redox systems regulate the functions of these molecules. The current understanding of how disturbance in redox homeostasis may affect cell death and contribute to the development of diseases such as cancer and degenerative disorders is reviewed. We also discuss how the basic knowledge on redox regulation of cell survival can be used to develop strategies for the treatment or prevention of those diseases. Antioxid. Redox Signal. 10, 1343–1374.
Redox Biology and Regulatory Mechanisms
Redox homeostasis: ROS production and elimination
Oxidative stress and its consequences
Redox-mediated mechanisms in regulation of cellular processes
Transcriptional regulation
Direct oxidative modification
Regulation of redox-sensitive interacting proteins
Regulation of redox-sensitive modifying enzymes
Regulation of protein turnover
Redox Regulation of Signaling Proteins Affecting Cell Death and Survival
Redox regulation of cell survival at the transcription level
Role of NF-κB in cell survival
Redox regulation of NF-κB
Redox regulation of nuclear NF-κB
Cytoplasmic regulation of NF-κB
Role of AP-1 in cell survival
Redox regulation of AP-1
Role of Nrf2 in cell survival
Redox regulation of Nrf2
Role of HIF in cell survival
Redox regulation of HIF
Redox regulation of cell survival at the signal-transduction level
Mitogen-activated protein kinase
Role of MAPK for cell survival under oxidative stress
Redox regulation of MAPK
PI3K/Akt pathway
Role of PI3K/Akt in cell survival
Redox regulation of PI3K/Akt
Redox regulation of cell survival at the cell death–execution level
Role of caspases in cell death and survival
Redox regulation of caspases
Role of Bcl-2 in cell survival
Redox regulation of Bcl-2
Cytochrome c
Role of cytochrome c in cell survival
Redox regulation of cytochrome c
Integration of redox-sensitive signaling pathways in the regulation of cell survival
Crosstalk between signaling pathways
Role of p53
P53 serves as an antioxidant to maintain redox homeostasis and normal cell survival
Role of p53 in cell death
Redox regulation of p53
Role of Redox Regulation of Cell Survival in Pathogenesis of Diseases
Aberrant prolonged cell survival leads to cancer
Oncogene activation
Loss of functional p53
Aberrant expression of antioxidant enzymes
Superoxide dismutase (SOD)
Glutathione peroxidase (GPX) and peroxiredoxin (Prx)
Diseases with excessive cell death: aging and degenerative disorders
Therapeutic Strategies Based on Redox Regulation of Cell Survival
Manipulating redox homeostasis
Pro-oxidants as a therapeutic strategy for cancer
Antioxidants for prevention of degenerative diseases
Modulating redox-sensitive signaling molecules
Concluding Remarks
PMCID: PMC2932530  PMID: 18522489
15.  Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications 
Antioxidants & Redox Signaling  2012;17(11):1610-1655.
Several studies demonstrated that oxidative damage is a characteristic feature of many neurodegenerative diseases. The accumulation of oxidatively modified proteins may disrupt cellular functions by affecting protein expression, protein turnover, cell signaling, and induction of apoptosis and necrosis, suggesting that protein oxidation could have both physiological and pathological significance. For nearly two decades, our laboratory focused particular attention on studying oxidative damage of proteins and how their chemical modifications induced by reactive oxygen species/reactive nitrogen species correlate with pathology, biochemical alterations, and clinical presentations of Alzheimer's disease. This comprehensive article outlines basic knowledge of oxidative modification of proteins and lipids, followed by the principles of redox proteomics analysis, which also involve recent advances of mass spectrometry technology, and its application to selected age-related neurodegenerative diseases. Redox proteomics results obtained in different diseases and animal models thereof may provide new insights into the main mechanisms involved in the pathogenesis and progression of oxidative-stress-related neurodegenerative disorders. Redox proteomics can be considered a multifaceted approach that has the potential to provide insights into the molecular mechanisms of a disease, to find disease markers, as well as to identify potential targets for drug therapy. Considering the importance of a better understanding of the cause/effect of protein dysfunction in the pathogenesis and progression of neurodegenerative disorders, this article provides an overview of the intrinsic power of the redox proteomics approach together with the most significant results obtained by our laboratory and others during almost 10 years of research on neurodegenerative disorders since we initiated the field of redox proteomics. Antioxid. Redox Signal. 17, 1610–1655.
I. Introduction
II. Protein (/Lipid) Oxidation and Protein Dysfunction
A. Protein carbonyls
B. Protein nitration
1. Peroxynitrite (ONOO−)
2. Nitrogen dioxide (NO2)
C. HNE adduction to proteins
D. Importance of clearance and detoxification systems
1. The proteasome, parkin, ubiquitin carboxy-terminal hydrolase-L1, and HSPs
2. Superoxide dismutase
3. Catalase
4. Peroxiredoxins
5. Trx and Trx reductase
6. Glutathione reductase
7. Vitamins in neurodegeneration
8. Involvement of iron in neurodegeneration
E. Role of iron in neurodegeneration
1. Fe homeostasis in AD
2. Fe homeostasis in PD
3. Fe homeostasis in ALS
4. Fe homeostasis in HD
F. Some known consequences of protein oxidation
III. Overview of Redox Proteomics
A. Global, gel-based approaches
B. Targeted, gel-free approach
1. Enrichment of PCO modified proteins
2. Enrichment of HNE modified proteins
3. Enrichment of 3-NT modified proteins
IV. Application of Redox Proteomics to Selected Neurodegenerative Disorders
A. Alzheimer's disease
1. PCO in AD
2. Identification of carbonylated proteins in brain of subjects with AD
a. Sample: the brain
b. Energy dysfunction
c. Excitotoxicity
d. Proteosomal dysfunction
e. Neuritic abnormalities
f. APP regulation, tau hyperphosphorylation, and cell cycle regulation
g. Synaptic abnormalities and LTP
h. pH maintenance
i. Mitochondrial abnormalities
3. Carbonylated proteins in brain of subjects with amnestic MCI
4. EAD carbonylated proteins
5. PCAD vs. amnestic MCI protein carbonylation in brain
6. Protein-bound HNE in brain and progression of Alzheimer's disease
7. Protein-bound 3-NT in brain and progression of Alzheimer's disease
8. Nitrated brain proteins in MCI
9. Nitrated proteins in EAD
B. Parkinson disease
1. Redox proteomics in PD
C. Amyotrophic lateral sclerosis
1. Redox proteomics studies in ALS transgenic mice
D. Huntington disease
1. Redox proteomics-transgenic mouse model of HD
2. Proteomics of HD brain
E. Down syndrome
1. Redox proteomics in DS transgenic mice
V. Conclusions and Future Directions
PMCID: PMC3448942  PMID: 22115501
16.  Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise 
Frontiers in Genetics  2013;4:108.
Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive oxidants, which can be both upstream and downstream of ER stress. Accordingly, detrimental intracellular stress signals are amplified through establishment of a vicious cycle. An increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. Experimental monitoring of stress-induced, time-resolved changes in ER reduction-oxidation (redox) states is therefore important. Organelle-specific examination of redox changes has been facilitated by the advent of genetically encoded, fluorescent probes, which can be targeted to different subcellular locations by means of specific amino acid extensions. These probes include redox-sensitive green fluorescent proteins (roGFPs) and the yellow fluorescent protein-based redox biosensor HyPer. In the case of roGFPs, variants with known specificity toward defined redox couples are now available. Here, we review the experimental framework to measure ER redox changes using ER-targeted fluorescent biosensors. Advantages and drawbacks of plate-reader and microscopy-based measurements are discussed, and the power of these techniques demonstrated in the context of selected cell culture models for ER stress.
PMCID: PMC3680709  PMID: 23781233
endoplasmic reticulum; endoplasmic reticulum stress; glutathione; green fluorescent protein; hydrogen peroxide; unfolded protein response
17.  Organelle Redox of CF and CFTR-Corrected Airway Epithelia 
Free radical biology & medicine  2007;43(2):300-316.
In cystic fibrosis reduced CFTR function may alter redox properties of airway epithelial cells. Redox-sensitive GFP (roGFP1) and imaging microscopy were used to measure redox potentials of cytosol, ER, mitochondria and cell surface of cystic fibrosis nasal epithelial cells and CFTR-corrected cells. We also measured glutathione and cysteine thiol redox states in cell lysates and apical fluids to provide coverage over a range of redox potentials and environments that might be affected by CFTR. As measured with roGFP1, redox potentials at the cell surface (~ -207 ±8 mV) and in the ER (~ -217 ±1 mV) and rates of regulation of the apical fluid and ER lumen following DTT treatment were similar for CF and CFTR-corrected cells. CF and CFTR-corrected cells had similar redox potentials in mitochondria (-344 ±9 mV) and cytosol (-322 ±7 mV). Oxidation of carboxy-dichlorodihydrofluoresceindiacetate and of apical Amplex Red occurred at equal rates in CF and CFTR-corrected cells. Glutathione and cysteine redox couples in cell lysates and apical fluid were equal in CF and CFTR-corrected cells. These quantitative estimates of organelle redox potentials combined with apical and cell measurements using small molecule couples confirmed there were no differences in redox properties of CF and CFTR-corrected cells.
PMCID: PMC4085155  PMID: 17603939
redox-sensitive green fluorescent protein; cystic fibrosis; mitochondria; endoplasmic reticulum; cell surface; organelle redox potential
18.  Ratiometric biosensors that measure mitochondrial redox state and ATP in living yeast cells 
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. MitoroGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
PMCID: PMC3846110  PMID: 23912244
19.  Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin 
The Journal of General Physiology  2014;144(1):81-104.
A P2X4 receptor labeled with the pH-sensitive GFP superecliptic pHluorin represents a useful probe to investigate P2X4 receptor distribution, trafficking, and up-regulation.
P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.
PMCID: PMC4076521  PMID: 24935743
20.  Identification of Proteins Targeted by the Thioredoxin Superfamily in Plasmodium falciparum 
PLoS Pathogens  2009;5(4):e1000383.
The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites.
Author Summary
Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in various environments. As shown by glucose-6 phosphate dehydrogenase deficiency, a genetic variation protecting from malaria, the parasite–host cell unit is very susceptible to disturbances in redox equilibrium. This is the major reason why redox active proteins of Plasmodium currently belong to the most attractive antimalarial drug targets. The dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species, represent central players in the redox network of malarial parasites. To extend our knowledge of interacting partners and the functions of these proteins, we carried out pull-down assays with immobilized active site mutants of Trx, Grx, and Plrx and whole cell parasite lysate. After elution of bound proteins and mass spectrometric identification, about 20 interacting partners were identified for each of the redox proteins. Data was supported using BIAcore surface plasmon resonance. The identified interacting proteins, which are likely to be redox-regulated, are involved in important cellular processes including protein biosynthesis, energy metabolism, polyamine synthesis, and signal transduction. Our results contribute to our understanding of the redox interactome in malarial parasites.
PMCID: PMC2660430  PMID: 19360125
21.  Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes 
Free radical biology & medicine  2012;52(0):2254-2265.
Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast S. cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allow us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments in a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox system play distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol- rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.
PMCID: PMC3382975  PMID: 22561702
redox-sensitive sensor; redox; glutathione; yeast
22.  Transient light-induced intracellular oxidation revealed by redox biosensor 
Biochemical and biophysical research communications  2013;439(4):10.1016/j.bbrc.2013.09.011.
We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.
PMCID: PMC3830497  PMID: 24025674
Live cell imaging; redox-sensitive probe; green fluorescent protein (GFP); glutathione; light-induced oxidation
23.  Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis 
Free radical biology & medicine  2010;49(12):1925-1936.
Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.
PMCID: PMC3005834  PMID: 20937380
Reactive oxygen species; apoptosis; mitochondria; redox cycling agents; RoGFP
24.  Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk 
Molecular Biology of the Cell  2011;22(9):1440-1451.
Peroxisomes are capable of reactive oxygen species (ROS) generation, but their contribution to cellular redox balance is not well understood. This study demonstrates that peroxisomes and mitochondria functionally interact via ROS signaling, suggesting a potential broader role for the peroxisome in cellular aging and the initiation and progression of age-related diseases.
Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation–reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.
PMCID: PMC3084667  PMID: 21372177
25.  Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells☆ 
Redox Biology  2014;3:47-55.
Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn2+) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn2+ toxicity is not fully understood. H2O2 and Zn2+ have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn2+ to cause cellular H2O2 production. To determine the role of Zn2+-induced H2O2 production in the human airway epithelial cell response to Zn2+ exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50 µM Zn2+ for 5 min in the presence of 1 µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn2+ exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn2+-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn2+-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn2+ leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.
Graphical abstract
•We used targeted catalase expression to examine the role of H2O2 in Zn2+-induced effects.•Cytosolic or mitochondrial catalase ablated Zn2+-induced mitochondrial H2O2 production.•Catalase expression blunted Zn2+-induced cytosolic EGSH and HO-1 mRNA.•Independently, decreasing GSHtotal or increasing EGSH failed to induce HO-1 mRNA.•Zn2+ causes multiple oxidative effects by H2O2-dependent and independent mechanisms.
PMCID: PMC4297933  PMID: 25462065
Zinc; H2O2; Live cell imaging; Mitochondria; Catalase; Genetically encoded redox probes; PM, particulate matter; Zn2+, zinc; HAEC, human airway epithelial cells; PTP, protein tyrosine phosphatases; Cyto, cytosolic; Mito, mitochondria; PYRI, sodium pyrithione; HO-1, heme oxygenase 1; ZnP, zinc pyrithione

Results 1-25 (1187055)