Cellular Peroxidation Research Articles

Production of superoxide and hydrogen peroxide in the mitochondrial matrix is dominated by site IQ of complex I in diverse cell lines

Understanding how mitochondria contribute to cellular oxidative stress and drive signaling and disease is critical, but quantitative assessment is difficult. Our previous studies of cultured C2C12 cells used inhibitors of specific sites of superoxide and hydrogen peroxide production to show that mitochondria generate about half of the hydrogen peroxide released by the cells, and site IQ of respiratory complex I produces up to two thirds of the superoxide and hydrogen peroxide generated in the mitochondrial matrix. Here, we used the same approach to measure the engagement of these sites in seven diverse cell lines to determine whether this pattern is specific to C2C12 cells, or more general. These diverse cell lines covered primary, immortalized, and cancerous cells, from seven tissues (liver, cervix, lung, skin, neuron, heart, bone) of three species (human, rat, mouse). The rate of appearance of hydrogen peroxide in the extracellular medium spanned a 30-fold range from HeLa cancer cells (3 pmol/min/mg protein) to AML12 liver cells (84 pmol/min/mg protein). The mean contribution of identified mitochondrial sites to this extracellular hydrogen peroxide signal was 30 ± 7% SD; the mean contribution of NADPH oxidases was 60 ± 14%. The relative contributions of different sites in the mitochondrial electron transport chain were broadly similar in all seven cell types (and similar to published results for C2C12 cells). 70 ± 4% of identified superoxide/hydrogen peroxide generation in the mitochondrial matrix was from site IQ; 30 ± 4% was from site IIIQo. We conclude that although absolute rates vary considerably, the relative contributions of different sources of hydrogen peroxide production are similar in nine diverse cell types under unstressed conditions in vitro. Identified mitochondrial sites account for one third of total cellular hydrogen peroxide production (half each from sites IQ and IIIQo); in the mitochondrial matrix the majority (two thirds) of superoxide/hydrogen peroxide is from site IQ.

Biocatalysis of MnO2-Mediated Nanosystem for Enhanced Multimodal Therapy and Real-Time Tracking

Clearance of endogenous glutathione (GSH) to cytotoxic hydroxyl radicals (·OH) and insufficient cellular hydrogen peroxide (H2O2) limited the efficacy of chemodynamic therapy (CDT) to tumor. Herein...

Pad-printed Prussian blue doped carbon ink for real-time peroxide sensing in cell culture

Abstract Hydrogen peroxide has important roles within cellular functions, as a prevalent form of Reactive Oxygen Species, detection within mammalian cells is of metabolic importance; typically requiring cell lysis or fluorescence-based methods to quantify. Herein, we explore the novel use of Prussian blue mediated, pad printed carbon electrodes to allow the indirect detection of cellular peroxides in bulk culture media, which facilitates non-invasive, real-time detection. Electrodes demonstrated capacity to detect H2O2 with a linear range of 1-200 μM in CMEM (R2 = 0.9988), enabling detection of peroxides found in culture media and lysate. Developed electrodes had a Limit of Detection (LOD) of 0.41 μM H2O2 in Britton-Robinson Buffer (BRB), 0.38 μM in Eagle's Minimum Essential Medium (EMEM) and 9.19 μM in Dulbecco's Modified Eagles Medium (DMEM). Electrodes were tested in a conventional 5% serum supplemented EMEM (CMEM) and demonstrated an LOD of 0.5 μM and LOQ of 0.9 μM. The results demonstrate proof of concept for monitoring H2O2 in complex culture media with potential long-term use and reusability using simple, pad printed Prussian Blue / Carbon electrodes. The lack of further modification, and cost-effectiveness of these disposable electrodes could offer great advancement to monitoring of peroxides in complex media.

A porous hollow fiber sensor for detection of cellular hydrogen peroxide release based on cell-in-lumen configuration

The rapid and accurate detection of cellular hydrogen peroxide (H2O2) is of great significance in cell monitoring and drug screening. We have developed a porous hollow fiber sensor with a cell-in-lumen configuration to detect H2O2 released by cells in real-time. In contrast to the conventional two-dimensional electrode structure, the sensing unit was located at the outer surface of the hollow fiber, while cells grew in the lumen, and the released H2O2 can diffuse through the porous wall of the fiber. The sensing layer wrapped outside the porous hollow fiber can capture the released H2O2 in a flow-through mode, resulting in a high sensitivity to the nanomolar level. In short-term monitoring of cell respiratory burst, the current response of the cell-in-lumen sensing configuration was six times greater than that of a conventional cell-out-electrode configuration. The porous wall also separated the cells and electrodes, minimizing the impact of electrical field on the cells. The high sensitivity and stability of detection ensured the cell-in-lumen configuration could be used to monitor the cellular H2O2 gradual release process induced by anticancer drugs. Such in-situ monitoring of the cellular release of H2O2 provides a new in-vitro drug screening platform for personalized medicine and cancer therapy.

Clinical Validation Results of an Innovative Non-Invasive Device for Colorectal Cancer Preventive Screening through Fecal Exhalation Analysis.

Screening is recommended to reduce both incidence and mortality of colorectal cancer. Currently, many countries employ fecal occult blood test (FOBT). In Emilia-Romagna (Italy), since 2005, FOBT immunochemical version (FIT) is performed every two years on people aged between 50 and 69 years. A colonoscopy is then carried out on those who are FIT positive. However, FIT shows approximately 65% false positives (non-tumoral bleedings), leading to many negative colonoscopies. The use of an economic and easy-to-use method to check FOBT-positives will improve screening effectiveness, reducing costs to the national health service. This work illustrates the results of a three-year clinical validation protocol (started in 2016) of a patented device composed of a core of nanostructured gas sensors. This device was designed to identify CRC presence by fecal volatile compounds, with a non-invasive, in vitro and low-cost analysis. Feces are, in fact, affected by tumor-volatile biomarkers, produced by cellular peroxidation and metabolic alterations. The protocol consisted in the analysis of fecal samples of FIT-positive subjects, using colonoscopy as a gold standard. A total of 398 samples were analyzed with machine learning techniques, leading to a sensitivity and specificity of 84.1% and 82.4%, respectively, and a positive predictive value of 72% (25–35% for FIT).

Ultrathin polypyrrole films on self‐assembled monolayers as an efficient ultramicroelectrode assay

AbstractIn this work, we investigated the effect of electrodeposition of polypyrrole (PPy) films on two different self‐assembled monolayers (SAMs) modified gold electrode for the electrochemical construction of ultramicroelectrode (UME). In order to obtain SAM modified surfaces, 4‐mercapto‐1‐butanol and 11‐mercaptoundecanoic acid were used. The effect of these two chains on pyrrole electropolymerization was compared. Electropolymerization of pyrrole on SAM modified Au electrode was carried out by using cyclic voltammetry (CV) and constant potential electrolysis. To investigate the UME formation, the obtained surfaces were tested in Fe(CN)63−/4− redox system. UMEs were characterized using scanning electron microscopy energy‐dispersive X‐ray spectroscopy, attenuated total reflectance fourier transform infrared spectroscopy and electrochemical impedance spectroscopy. The designed UME was applied as an immobilization matrix to entrap a redox protein, Hemoglobin (Hb), as a model. Direct electron transfer between this protein and the fabricated thiol modified PPy based UME was achieved, which is very challenging on bare electrode surfaces. With this study, a simple, low‐cost and reproducible UME production way has been successfully accomplished and Hb modified UME is promising for different bioanalytical applications, for instance; cellular hydrogen peroxide or nitrite sensing.

Peroxisomal Dysfunction and Oxidative Stress in Neurodegenerative Disease: A Bidirectional Crosstalk.

Peroxisomes are multifunctional organelles best known for their role in cellular lipid and hydrogen peroxide metabolism. In this chapter, we review and discuss the diverse functions of this organelle in brain physiology and neurodegeneration, with a particular focus on oxidative stress. We first briefly summarize what is known about the various nexuses among peroxisomes, the central nervous system, oxidative stress, and neurodegenerative disease. Next, we provide a comprehensive overview of the complex interplay among peroxisomes, oxidative stress, and neurodegeneration in patients suffering from primary peroxisomal disorders. Particular examples that are discussed include the prototypic Zellweger spectrum disorders and X-linked adrenoleukodystrophy, the most prevalent peroxisomal disorder. Thereafter, we elaborate on secondary peroxisome dysfunction in more common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Finally, we highlight some issues and challenges that need to be addressed to progress towards therapies and prevention strategies preserving, normalizing, or improving peroxisome activity in patients suffering from neurodegenerative conditions.

The Soybean GmNAC019 Transcription Factor Mediates Drought Tolerance in Arabidopsis in an Abscisic Acid-Dependent Manner.

Being master regulators of gene expression, transcription factors (TFs) play important roles in determining plant growth, development and reproduction. To date, many TFs have been shown to positively mediate plant responses to environmental stresses. In the current study, the biological functions of a stress-responsive NAC [NAM (No Apical Meristem), ATAF1/2 (Arabidopsis Transcription Activation Factor1/2), CUC2 (Cup-shaped Cotyledon2)]-TF encoding gene isolated from soybean (GmNAC019) in relation to plant drought tolerance and abscisic acid (ABA) responses were investigated. By using a heterologous transgenic system, we revealed that transgenic Arabidopsis plants constitutively expressing the GmNAC019 gene exhibited higher survival rates in a soil-drying assay, which was associated with lower water loss rate in detached leaves, lower cellular hydrogen peroxide content and stronger antioxidant defense under water-stressed conditions. Additionally, the exogenous treatment of transgenic plants with ABA showed their hypersensitivity to this phytohormone, exhibiting lower rates of seed germination and green cotyledons. Taken together, these findings demonstrated that GmNAC019 functions as a positive regulator of ABA-mediated plant response to drought, and thus, it has potential utility for improving plant tolerance through molecular biotechnology.

Systemic Lupus Erythematosus: Pathogenesis at the Functional Limit of Redox Homeostasis

Systemic lupus erythematosus (SLE) is a disease characterized by the production of autoreactive antibodies and cytokines, which are thought to have a major role in disease activity and progression. Immune system exposure to excessive amounts of autoantigens that are not efficiently removed is reported to play a significant role in the generation of autoantibodies and the pathogenesis of SLE. While several mechanisms of cell death-based autoantigenic exposure and compromised autoantigen removal have been described in relation to disease onset, a significant association with the development of SLE can be attributed to increased apoptosis and impaired phagocytosis of apoptotic cells. Both apoptosis and impaired phagocytosis can be caused by hydrogen peroxide whose cellular production is enhanced by exposure to endogenous hormones or environmental chemicals, which have been implicated in the pathogenesis of SLE. Hydrogen peroxide can cause lymphocyte apoptosis and glutathione depletion, both of which are associated with the severity of SLE. The cellular accumulation of hydrogen peroxide is facilitated by the myriad of stimuli causing increased cellular bioenergetic activity that enhances metabolic production of this toxic oxidizing agent such as emotional stress and infection, which are recognized SLE exacerbating factors. When combined with impaired cellular hydrogen peroxide removal caused by xenobiotics and genetically compromised hydrogen peroxide elimination due to enzymatic polymorphic variation, a mechanism for cellular accumulation of hydrogen peroxide emerges, leading to hydrogen peroxide-induced apoptosis and impaired phagocytosis, enhanced autoantigen exposure, formation of autoantibodies, and development of SLE.

Biogenic capped selenium nano rods as naked eye and selective hydrogen peroxide spectrometric sensor

The Biogenic selenium(0) nano rods were synthesized and stabilized in citric acid and flavonoids from lemon juice by wet chemical route. As synthesized nanorods were characterized using physicochemical analysis techniques as UV–Vis, FITR, PXRD spectrometry, SEM and TEM surface morphology imaging techniques. These nano rods had exhibited trigonal Se packing lattice, 90 nm mean crystallite size and surface capping with flavonoid and citric acid functionality. The Se nanorods show selective sensing of peroxide by visible color change from reddish to faint pink and also through spectrometric sensing plots for concentrations from 60 ppm to 5 ppm with sensing limit up to 75 μM peroxide with interfering cellular cations as K, Na, Ca, Mg and Fe. These biogenic green sensor Se nano rods get converted to nano ovals after surface leaching with peroxide and show SPR based selective sensing mechanism in spectrometric plots. These developed nanorods finds suitable applications in biomedical cellular peroxide sensing with low limits through naked eye cost effective spectrometric sensing method.

Role of Catalase in Oxidative Stress- and Age-Associated Degenerative Diseases.

Reactive species produced in the cell during normal cellular metabolism can chemically react with cellular biomolecules such as nucleic acids, proteins, and lipids, thereby causing their oxidative modifications leading to alterations in their compositions and potential damage to their cellular activities. Fortunately, cells have evolved several antioxidant defense mechanisms (as metabolites, vitamins, and enzymes) to neutralize or mitigate the harmful effect of reactive species and/or their byproducts. Any perturbation in the balance in the level of antioxidants and the reactive species results in a physiological condition called “oxidative stress.” A catalase is one of the crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by destroying cellular hydrogen peroxide to produce water and oxygen. Deficiency or malfunction of catalase is postulated to be related to the pathogenesis of many age-associated degenerative diseases like diabetes mellitus, hypertension, anemia, vitiligo, Alzheimer's disease, Parkinson's disease, bipolar disorder, cancer, and schizophrenia. Therefore, efforts are being undertaken in many laboratories to explore its use as a potential drug for the treatment of such diseases. This paper describes the direct and indirect involvement of deficiency and/or modification of catalase in the pathogenesis of some important diseases such as diabetes mellitus, Alzheimer's disease, Parkinson's disease, vitiligo, and acatalasemia. Details on the efforts exploring the potential treatment of these diseases using a catalase as a protein therapeutic agent have also been described.

INDICES OF RESILIENCY IN CELLS FROM UM-HET3 MICE MAY CORRELATE WITH INDIVIDUAL FUTURE HEALTH OUTCOMES

The ability of an organism to respond to physical stresses and return to homeostasis (i.e. resilience) has been suggested to correlate with longevity. Here, we investigated whether this extends to resilience at a cellular level using primary fibroblasts isolated from tail skin of genetically heterogeneous young adult UM-HET3 mice. Cells isolated from each individual mouse (cell line) were tested in their response to concentrations of agents or conditions predicted to induce a cellular challenge, including paraquat, hydrogen peroxide, antimycin a, cadmium chloride, mdivi-1, thapsigargin, and nutrient starvation. Cell viability was monitored in real-time using an incucyte S3 live cell analysis system and we addressed the response following challenge as a marker of resilience. Cellular uptake of ethidium homodimer-1 was used to determine the loss of viability. Cellular bioenergetics were assessed using a seahorse XF24. We found that cell lines that were resistant to paraquat were also resistant to antimycin a, and hydrogen peroxide. Cell lines that were resistant to nutrient starvation were also resistant to mdivi-1. Indices of cellular bioenergetics status including ATP production rate and cell respiratory control ratio, revealed potential relationships with resiliency. Taken together, our data indicate that skin fibroblasts retain individual physiological programs that may in part explain the patterns of resiliency or sensitivity to a stressor at the organismal level. Since the cell lines tested in this study were obtained from living mice, future work will investigate whether these patterns of resiliency change with age and elucidate their utility in predicting future health outcome.

Polydopamine and ammonium bicarbonate coated and doxorubicin loaded hollow cerium oxide nanoparticles for synergistic tumor therapy

The development of effective nanoplatforms is extremely necessary for cancer therapy. Herein, we prepared polydopamine (PDA) and ammonium bicarbonate (NH4HCO3) coated and doxorubicin (Dox) loaded hollow cerium oxide (CeO2) NPs (PDAC NPs), which showed excellent synergistic effect for photothermal therapy, chemotherapy and chemodynamic therapy. Under near infrared laser irradiation, PDA shell could absorb the incident light and convert it into heat, which could not only kill tumor cells with hyperthermia, but also trigger the decomposition of NH4HCO3 into gaseous carbon dioxide and ammonia, leading to the destroy of PDA shell. The leakage of PDA further accelerated Dox release and exposed CeO2 surface, in which Dox could enter into cell nucleus to induce chemotherapy, and CeO2 could catalyze cellular hydrogen peroxide into hydroxyl radical to present chemodynamic therapy. In fact, PDAC NPs showed an excellent therapeutic efficacy both in vitro and in vivo. This design provides a new strategy for synergistic tumor therapy.

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12–18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

Deciphering the in vivo redox behavior of human peroxiredoxins I and II by expressing in budding yeast

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress. To understand this, we used oxidative-stress sensitive tsa1tsa2Δ yeast strain to express hPRXI or hPRXII. We found that hPrxI in yeast exists as a mixture of disulfide-linked dimer and reduced monomer but becomes hyperoxidized upon elevated oxidative stress as analyzed under denaturing conditions (SDS-PAGE). In contrast, hPrxII was present predominantly as the disulfide in unstressed cells and readily converted to its hyperoxidized, peroxidase-inactive form even with mild oxidative stress. Interestingly, we found that plant extracts containing polyphenol antioxidants provided further protection against the growth defects of the tsa1tsa2Δ strain expressing hPrx and preserved the peroxidase-active forms of the Prxs. The extracts also helped to protect against hyperoxidation of hPrxs in HeLa cells. Based on these findings we can conclude that resistance to oxidative stress of yeast cells expressing individual hPrxs requires the hPrx to be maintained in a redox state that permits redox cycling and peroxidase activity. Peroxidase activity decreases as the hPrx becomes hyperoxidized and the limited protection by hPrxII compared with hPrxI can be explained by its greater sensitivity to hyperoxidation.

Effect of antioxidants on BPA-induced stress on sperm function in a mouse model

In the past few years, bisphenol A, (BPA) an endocrine-disrupting chemical, has received increasing attention because of its detrimental health effects. There is ample evidence to support that BPA interferes with the reproductive health of humans and animals. In spermatozoa, BPA-induced adverse effects are mostly caused by increased oxidative stress. Using an in vitro experimental model, we examined whether antioxidants (glutathione, vitamin C, and vitamin E) have defensive effects against BPA-induced stress in spermatozoa. The results showed that antioxidants inhibit the overproduction of reactive oxygen species (basically cellular peroxides) and increase intracellular ATP levels, thereby preventing motility loss and abnormal acrosome reaction in BPA-exposed spermatozoa. In particular, glutathione and vitamin E reduced the protein kinase A-dependent tyrosine phosphorylation in spermatozoa and, thus, prevented the precocious acrosome reaction from occurring. Furthermore, we found that the compromised fertilisation and early embryo development mediated by BPA-exposed spermatozoa can be improved following their supplementation with glutathione and vitamin E. Based on these findings, we suggest that antioxidants reduce oxidative stress in BPA-exposed spermatozoa, thus preventing detrimental effects on their function and fertility.

Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction.

Molecular hydrogen (H2) was believed to be an inert and nonfunctional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H2 against oxidative stress. Subsequently, extensive studies revealed multiple functions of H2 by exhibiting the efficacies of H2 in various animal models and clinical studies. Here, we investigated the effect of H2 on free-radical-induced cytotoxicity using tert-butyl hydroperoxide in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase release assay and Hoechst 33342 and propidium iodide staining. Fatty acid peroxidation and mitochondrial viability were measured using 2 kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential was evaluated using tetramethylrhodamine methyl ester. As a result, H2 protected the cultured cells against the cytotoxic effects induced by tert-butyl hydroperoxide; H2 suppressed cellular fatty acid peroxidation and cell membrane permeability, mitigated the decline in mitochondrial oxidoreductase activity and mitochondrial membrane potential, and protected cells against cell death evaluated using propidium iodide staining. These results suggested that H2 suppresses free-radical-induced cell death through protection against fatty acid peroxidation and mitochondrial dysfunction.

Caution for the routine use of phenol red – It is more than just a pH indicator

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 μM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV–visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV–vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.

A Streptococcus aquaporin acts as peroxiporin for efflux of cellular hydrogen peroxide and alleviation of oxidative stress

Aquaporins (AQPs) are transmembrane proteins widely distributed in various organisms, and they facilitate bidirectional diffusion of water and uncharged solutes. The catalase-negative bacterium Streptococcus oligofermentans produces the highest H2O2 levels reported to date, which has to be exported to avoid oxidative stress. Here, we report that a S. oligofermentans aquaporin functions as a peroxiporin facilitating bidirectional transmembrane H2O2 transport. Knockout of this aquaporin homolog, So-AqpA, reduced H2O2 export by ∼50% and increased endogenous H2O2 retention, as indicated by the cellular H2O2 reporter HyPer. Heterologous expression of So-aqpA accelerated exogenous H2O2 influx into Saccharomyces cerevisiae and Escherichia coli cells, indicating that So-AqpA acts as an H2O2-transferring aquaporin. Alanine substitution revealed Phe-40 as a key residue for So-AqpA-mediated H2O2 transport. Northern blotting, qPCR, and luciferase reporter assays disclosed that H2O2 induces a >10-fold expression of So-aqpA Super-resolution imaging showed that H2O2 treatment increases So-AqpA protein molecules per cell by 1.6- to 3-fold. Inactivation of two redox-regulatory transcriptional repressors, PerR and MntR, reduced H2O2-induced So-aqpA expression to 1.8- and 4-fold, respectively. Electrophoretic mobility shift assays determined that MntR, but not PerR, binds to the So-aqpA promoter, indicating that MntR directly regulates H2O2-induced So-aqpA expression. Importantly, So-aqpA deletion decreased oxic growth and intraspecies competition and diminished the competitive advantages of S. oligofermentans over the caries pathogen Streptococcus mutans Of note, So-aqpA orthologs with the functionally important Phe-40 are present in all streptococci. Our work has uncovered an intrinsic, H2O2-inducible bacterial peroxiporin that has a key physiological role in H2O2 detoxification in S. oligofermentans.