2012 Articles

Dobrovolska O, Shumilina E, Gladyshev VN, Dikiy A.. (2012) Structural Analysis of Glutaredoxin Domain of Mus musculus Thioredoxin Glutathione Reductase. PLoS One 7, e52914.

AbstractThioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48±0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded β-sheet flanked by five α-helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol. More Information

Tsuji PA, Carlson BA, Naranjo-Suarez S, Yoo MH, Xu XM, Fomenko DE, Gladyshev VN, Hatfield DL, Davis CD. (2012) Knockout of the 15 kDa Selenoprotein Protects against Chemically-Induced Aberrant Crypt Formation in Mice. PLoS One 7, e50574.

AbstractEvidence suggests that selenium has cancer preventive properties that are largely mediated through selenoproteins. Our previous observations demonstrated that targeted down-regulation of the 15 kDa selenoprotein (Sep15) in murine colon cancer cells resulted in the reversal of the cancer phenotype. The present study investigated the effect of Sep15 knockout in mice using a chemically-induced colon cancer model. Homozygous Sep15 knockout mice, and wild type littermate controls were given four weekly subcutaneous injections of azoxymethane (10 mg/kg). Sep15 knockout mice developed significantly (p<0.001) fewer aberrant crypt foci than controls demonstrating that loss of Sep15 protects against aberrant crypt foci formation. Dietary selenium above adequate levels did not significantly affect aberrant crypt foci formation in Sep15 knockout mice. To investigate molecular targets affected by loss of Sep15, gene expression patterns in colonic mucosal cells of knockout and wild type mice were examined using microarray analysis. Subsequent analyses verified that guanylate binding protein-1 (GBP-1) mRNA and protein expression were strongly upregulated in Sep15 knockout mice. GBP-1, which is expressed in response to interferon-γ, is considered to be an activation marker during inflammatory diseases, and up-regulation of GBP-1 in humans has been associated with a highly significant, increased five-year survival rate in colorectal cancer patients. In agreement with these studies, we observed a higher level of interferon-γ in plasma of Sep15 knockout mice. Overall, our results demonstrate for the first time, that Sep15 knockout mice are protected against chemically-induced aberrant crypt foci formation and that Sep15 appears to have oncogenic properties in colon carcinogenesis in vivo. More Information

Tarrago L, Gladyshev VN. (2012) Recharging Oxidative Protein Repair: Catalysis by Methionine Sulfoxide Reductases towards Their Amino Acid, Protein, and Model Substrates. Biochemistry (Mosc) 77, 1097-1107.

AbstractThe sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins. More Information

Liang X, Kaya A, Zhang Y, Le DT, Hua D, Gladyshev VN. (2012) Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins. BMC Biochem. 13, 21.

AbstractBACKGROUND: Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.
RESULTS: We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.
CONCLUSION: Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
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Gerashchenko MV, Lobanov AV, Gladyshev VN. (2012) Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress. Proc. Natl. Acad. Sci. USA 109, 17394-17399.

AbstractInformation on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially the duration of the elongation step. We identified proteins whose synthesis was regulated rapidly by hydrogen peroxide posttranscriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. These data define the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggest that potentiation (coregulation of the transcript level and translation) is a feature of oxidative stress.
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Gladyshev VN. (2012) On the cause of aging and control of lifespan: Heterogeneity leads to inevitable damage accumulation, causing aging; Control of damage composition and rate of accumulation define lifespan. Bioessays 34, 925-928.

AbstractWhat the causes of aging are and which factors define lifespan are key questions in the understanding of aging. Here, it is argued that cellular life involves (i) inevitable accumulation of damage resulting from imperfectness and heterogeneity of every cellular process, and (ii) dilution of damage when cells divide. While severe damage is cleared by protective systems, milder damage can only be diluted. This is due to the high cost of accuracy, the greater number of damage forms compared to protective systems, and the constraints on cellular life inherited from the prokaryotic world. This strategy also applies to cancer cells, which are particularly dependent on damage dilution. Imposing restriction on cell division necessarily leads to aging. Interventions that extend lifespan act through metabolic reprogramming, thereby changing both damage composition and the rate of damage accumulation. Thus, heterogeneity leading to myriad mild damage forms represents the cause of aging, whereas the processes that affect the damage landscape and damage accumulation are lifespan regulators. More Information

Anisimov VN, Bartke A, Barzilai N, Batin MA, Blagosklonny MV, Brown-Borg H, Budovskaya Y, Campisi J, Friguet B, Fraifeld V, Franceschi C, Gems D, Gladyshev V, Gorbunova V, Gudkov AV, Kennedy B, Konovalenko M, Kraemer B, Moskalev A, Petropoulos I, Pasyukova E, Rattan S, Rogina B, Seluanov A, Shaposhnikov M, Shmookler Reis R, Tavernarakis N, Vijg J, Yashin A, Zimniak P. (2012) The second international conference “genetics of aging and longevity”. Aging (Albany NY) 4, 305-317.

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Carlson BA, Yoo MH, Tobe R, Mueller C, Naranjo-Suarez S, Hoffmann VJ, Gladyshev VN, Hatfield DL. (2012) Thioredoxin reductase 1 protects against chemically induced hepatocarcinogenesis via control of cellular redox homeostasis. Carcinogenesis 33, 1806-1813.

AbstractThioredoxin reductase 1 (TR1) controls the redox state of protein thiols in mammalian cells and has been shown to have roles in both preventing and promoting cancer. To define the role of this selenoenzyme in hepatocellular carcinoma development, we examined tumor incidence in the liver of mice with tissue-specific knockout of mouse TR1 subjected to the liver carcinogen, diethylnitrosamine (DEN). TR1-deficient livers manifested ~90% tumor incidence compared with ~16% in control livers. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. On the other hand, we observed upregulation of another selenoenzyme, glutathione peroxidase 2 (GPx2), and components of the glutathione (GSH) system, including those that generate reduced GSH. Overall, this study shows that TR1 protects against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal.
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Vinogradov DV, Tsoĭ OV, Zaika AV, Lobanov AV, Turanov AA, Gladyshev VN, Gel’fand MS. (2012) [Draft macronuclear genome of a ciliate Euplotes crassus]. Mol. Biol. (Mosk, Russia) 46, 361-6366.

AbstractBasic bioinformatical analysis of the draft Euplotes crassus macronuclear genome and transcriptome suggests that more than a quarter of E. crassus genes contain several exons. A large fraction of all introns is formed by “tiny” introns having length 20-30 bp. Analysis of the transcriptome revealed 63 possible cases of alternative splicing, and also 14 introns with non-standard splicing sites. About 2000 hypothetical genes do not have homologs in other ciliates, and since most of them have the closest homologs in bacterial genomes, they are likely an artifact of the sample preparation. Comparison of the E. crassus genome to the genomes of other ciliates showed an expansion of the same gene families, responsible for the free-living heterotrophic lifestyle.
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Tarrago L, Kaya A, Weerapana E, Marino SM, Gladyshev VN. (2012) Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding. J Biol Chem. 287, 24448-24459.

AbstractReduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work, we exploited an NADPH-dependent thioredoxin system and determined the kinetics parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the KM lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione-S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins, and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to a better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins. More Information

Tobe R, Yoo MH, Fradejas N, Carlson BA, Calvo S, Gladyshev VN, Hatfield DL. (2012) Thioredoxin reductase 1 deficiency enhances selenite toxicity in cancer cells via a thioredoxin-independent mechanism. Biochem J. 445, 423-430.

AbstractSelenium is an essential trace element in mammals, but is toxic at high levels. It is best known for its cancer prevention activity, but cancer cells are more sensitive to selenite toxicity than normal cells. Since selenite treatment leads to oxidative stress, and the thioredoxin system is a major antioxidative system, we examined the interplay between thioredoxin reductase 1 (TR1) and thioredoxin 1 (Trx1) deficiencies and selenite toxicity in DT cells, a malignant mouse cell line, and the corresponding parental NIH3T3 cells. TR1 deficient cells were far more sensitive to selenite toxicity than Trx1-deficient or control cells. In contrast, this effect was not seen in cells treated with hydrogen peroxide, suggesting that the increased sensitivity of TR1 deficiency to selenite was not due to oxidative stress caused by this compound. Further analyses revealed that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The data uncover a new role of TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The data also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy. More Information

Ross F, Hernández P, Porcal W, López GV, Cerecetto H, González M, Basika T, Carmona C, Fló M, Maggioli G, Bonilla M, Gladyshev VN, Boiani M, Salinas G. (2012) Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites. PLoS One 7, e35033.

AbstractParasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.
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Mariotti M, Ridge PG, Zhang Y, Lobanov AV, Pringle TH, Guigo R, Hatfield DL, Gladyshev VN. (2012) Composition and evolution of the vertebrate and Mammalian selenoproteomes. PLoS One 7, e33066.

AbstractBACKGROUND: Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24.
METHODOLOGY/PRINCIPAL FINDINGS: We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian (25 proteins) selenoproteomes, and describe how they evolved along lineages through gene duplication (20 events), gene loss (10 events) and replacement of Sec with cysteine (12 events). We show that an intronless selenophosphate synthetase 2 gene evolved in early mammals and replaced functionally the original multiexon gene in placental mammals, whereas both genes remain in marsupials. Mammalian thioredoxin reductase 1 and thioredoxin-glutathione reductase evolved from an ancestral glutaredoxin-domain containing enzyme, still present in fish. Selenoprotein V and GPx6 evolved specifically in placental mammals from duplications of SelW and GPx3, respectively, and GPx6 lost Sec several times independently. Bony fishes were characterized by duplications of several selenoprotein families (GPx1, GPx3, GPx4, Dio3, MsrB1, SelJ, SelO, SelT, SelU1, and SelW2). Finally, we report identification of new isoforms for several selenoproteins and describe unusually conserved selenoprotein pseudogenes.
CONCLUSIONS/SIGNIFICANCE: This analysis represents the first comprehensive survey of the vertebrate and mammal selenoproteomes, and depicts their evolution along lineages. It also provides a wealth of information on these selenoproteins and their forms.
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Shumilina E, Soldà A, Gerashchenko M, Gladyshev VN, Dikiy A. (2012) (1)H, (13)C, and (15)N NMR resonance assignments of reduced full length and shortened forms of the Grx domain of Mus musculus TGR. Biomol NMR Assign. 6,103-107.

AbstractTwo forms of the glutaredoxin (Grx) domain (full length Grx domain and short Grx lacking the N-terminal region) of Mus musculus thioredoxin glutathione reductase (TGR) were isotopically labelled with (15)N and (13)C isotopes, expressed and purified to homogeneity. We report here the (1)H, (13)C and (15)N NMR assignment for both Grx forms of this mouse TGR. This investigation represents the first NMR analysis of a mammalian TGR. More Information

Kasaikina MV, Hatfield DL, Gladyshev VN. (2012) Understanding selenoprotein function and regulation through the use of rodent models. Biochim Biophys Acta. 1823, 1633-1642.

AbstractSelenium (Se) is an essential micronutrient. Its biological functions are associated with selenoproteins, which contain this trace element in the form of the 21st amino acid, selenocysteine. Genetic defects in selenocysteine insertion into proteins are associated with severe health issues. The consequences of selenoprotein deficiency are more variable, with several selenoproteins being essential, and several showing no clear phenotypes. Much of these functional studies benefited from the use of rodent models and diets employing variable levels of Se. This review summarizes the data obtained with these models, focusing on mouse models with targeted expression of individual selenoproteins and removal of individual, subsets or all selenoproteins in a systemic or organ-specific manner. This article is part of a Special Issue entitled: Cell Biology of Metals. More Information

Marino SM, Gladyshev VN. (2012) Analysis and functional prediction of reactive cysteine residues. J Biol Chem. 287,4419-4425.

AbstractCys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pK(a), to algorithms for functional prediction of different types of Cys in proteins. More Information

Fomenko DE, Gladyshev VN. (2012) Comparative genomics of thiol oxidoreductases reveals widespread and essential functions of thiol-based redox control of cellular processes. Antioxid Redox Signal. 16,193-201.

AbstractAIMS: Redox regulation of cellular processes is an important mechanism that operates in organisms from bacteria to mammals. Much of the redox control is provided by thiol oxidoreductases: proteins that employ cysteine residues for redox catalysis. We wanted to identify thiol oxidoreductases on a genome-wide scale and use this information to obtain insights into the general principles of thiol-based redox control. RESULTS: Thiol oxidoreductases were identified by three independent methods that took advantage of the occurrence of selenocysteine homologs of these proteins and functional linkages among thiol oxidoreductases revealed by comparative genomics. Based on these searches, we describe thioredoxomes, which are sets of thiol oxidoreductases in organisms. Their analyses revealed that these proteins are present in all living organisms, generally account for 0.5%-1% of the proteome and that their use correlates with proteome size, distinguishing these proteins from those involved in core metabolic functions. We further describe thioredoxomes of Saccharomyces cerevisiae and humans, including proteins which have not been characterized previously. Thiol oxidoreductases occur in various cellular compartments and are enriched in the endoplasmic reticulum and cytosol. INNOVATION: We developed bioinformatics methods and used them to characterize thioredoxomes on a genome-wide scale, which in turn revealed properties of thioredoxomes. CONCLUSION: These data provide information about organization and properties of thiol-based redox control, whose use is increased with the increase in complexity of organisms. Our data also show an essential combined function of a set of thiol oxidoreductases, and of thiol-based redox regulation in general, in all living organisms.
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Malinouski M, Kehr S, Finney L, Vogt S, Carlson BA, Seravalli J, Jin R, Handy DE, Park TJ, Loscalzo J, Hatfield DL, Gladyshev VN. (2012) High-resolution imaging of selenium in kidneys: a localized selenium pool associated with glutathione peroxidase 3. Antioxid Redox Signal. 16, 185-192.

AbstractAIM: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution. More Information

Naranjo-Suarez S, Carlson BA, Tsuji PA, Yoo MH, Gladyshev VN, Hatfield DL. (2012) HIF-Independent Regulation of Thioredoxin Reductase 1 Contributes to the High Levels of Reactive Oxygen Species Induced by Hypoxia. PLoS One 7, e40370.

AbstractCellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS. More Information