A guide to genetically-encoded redox biosensors: State of the art and opportunities.

Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.

The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was found to be evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulfide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (the typical 2-Cys-Peroxiredoxin from Anabaena), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulfide bond between the two active site cysteines (Cys-38 and Cys-41). However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulfide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulfide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

ROS production and signalling in chloroplasts: cornerstones and evolving concepts.

Reactive oxygen species (ROS) such as singlet oxygen, superoxide (O2●- ) and hydrogen peroxide (H2 O2 ) are the markers of living cells. Oxygenic photosynthesis produces ROS in abundance, which act as a readout of a functional electron transport system and metabolism. The concept that photosynthetic ROS production is a major driving force in chloroplast to nucleus retrograde signalling is embedded in the literature, as is the role of chloroplasts as environmental sensors. The different complexes and components of the photosynthetic electron transport chain (PETC) regulate O2●- production in relation to light energy availability and the redox state of the stromal Cys-based redox systems. All of the ROS generated in chloroplasts have the potential to act as signals and there are many sulphhydryl-containing proteins and peptides in chloroplasts that have the potential to act as H2 O2 sensors and function in signal transduction. While ROS may directly move out of the chloroplasts to other cellular compartments, ROS signalling pathways can only be triggered if appropriate ROS-sensing proteins are present at or near the site of ROS production. Chloroplast antioxidant systems serve either to propagate these signals or to remove excess ROS that cannot effectively be harnessed in signalling. The key challenge is to understand how regulated ROS delivery from the PETC to the Cys-based redox machinery is organised to transmit redox signals from the environment to the nucleus. Redox changes associated with stromal carbohydrate metabolism also play a key role in chloroplast signalling pathways.

ThioredoxinA1 Controls the Oxidative Stress Response of Francisella tularensis Live Vaccine Strain (LVS).

Francisella tularensis is an intracellular, Gram-negative bacterium known for causing a disease known as tularemia in the Northern Hemisphere. F. tularensis is classified as a category A select agent by the CDC based on its possible use as a bioterror agent. F. tularensis overcomes oxidative stress encountered during its growth in the environment or host macrophages by encoding antioxidant enzymes such as superoxide dismutases, catalase, and alkylhydroperoxy reductase. These antioxidant enzymes are regulated by the oxidative stress response regulator, OxyR. In addition to these antioxidant enzymes, F. tularensis also encodes two thioredoxins, TrxA1 (FTL_0611) and TrxA2 (FTL_1224); however, their role in the oxidative stress response of F. tularensis is not known. This study investigated the role of thioredoxins of F. tularensis in the oxidative stress response and intracellular survival. Our results demonstrate that TrxA1 but not TrxA2 plays a major role in the oxidative stress response of F. tularensis. Most importantly, this study elucidates a novel mechanism through which the TrxA1 of F. tularensis controls the oxidative stress response by regulating the expression of the master regulator, oxyR. Further, TrxA1 is required for the intramacrophage survival and growth of Francisella. Overall, this study describes a novel role of thioredoxin, TrxA1, in regulating the oxidative stress response of F. tularensis. IMPORTANCE The role of thioredoxins in the oxidative stress response of F. tularensis is not known. This study demonstrates that of the two thioredoxins, TrxA1 is vital to counter the oxidative stress in F. tularensis live vaccine strain (LVS). Furthermore, this study shows differences in the well-studied thioredoxins of Escherichia coli. First, the expression of TrxA1 of F. tularensis is independent of the oxidative stress response regulator, OxyR. Second and most importantly, TrxA1 regulates the expression of oxyR and, therefore, the OxyR-dependent oxidative stress response of F. tularensis. Overall, this study reports a novel regulatory role of TrxA1 of F. tularensis in the oxidative stress response.

Thioredoxins m are major players in the multifaceted light-adaptive response in Arabidopsis thaliana.

Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modifications in target proteins. Chloroplast TRXs are divided into different types and have central roles in light energy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidopsis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism, we hypothesized that the impairment of the TRX m signalling would not only have harmful consequences on chloroplast metabolism but also at different levels of plant development. To uncover the physiological and developmental processes that depend on TRX m signalling, we carried out a comprehensive study of Arabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light and redox signalling are closely linked, we investigated the response to high light (HL) of the plants that are gradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX m and the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, and stomatal conductance. We also report new data indicating that the isoforms of TRX m fine-tune the response to HL, including the accumulation of the protective pigment anthocyanin. These results reveal novel signalling functions for the TRX m and underline their importance for plant growth and fulfilment of the acclimation/response to HL conditions.

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

Plants acclimate to Photosystem I photoinhibition by readjusting the photosynthetic machinery.

Photosynthetic light reactions require strict regulation under dynamic environmental conditions. Still, depending on environmental constraints, photoinhibition of Photosystem (PSII) or PSI occurs frequently. Repair of photodamaged PSI, in sharp contrast to that of PSII, is extremely slow and leads to a functional imbalance between the photosystems. Slow PSI recovery prompted us to take advantage of the PSI-specific photoinhibition treatment and investigate whether the imbalance between functional PSII and PSI leads to acclimation of photosynthesis to PSI-limited conditions, either by short-term or long-term acclimation mechanisms as tested immediately after the photoinhibition treatment or after 24 h recovery in growth conditions, respectively. Short-term acclimation mechanisms were induced directly upon inhibition, including thylakoid protein phosphorylation that redirects excitation energy to PSI as well as changes in the feedback regulation of photosynthesis, which relaxed photosynthetic control and excitation energy quenching. Longer-term acclimation comprised reprogramming of the stromal redox system and an increase in ATP synthase and Cytochrome b6 f abundance. Acclimation to PSI-limited conditions restored the CO2 assimilation capacity of plants without major PSI repair. Response to PSI inhibition demonstrates that plants efficiently acclimate to changes occurring in the photosynthetic apparatus, which is likely a crucial component in plant acclimation to adverse environmental conditions.

Characterization of the components of the thioredoxin system in Bacteroides fragilis and evaluation of its activity during oxidative stress.

OBJECTIVES: Bacteroides fragilis has a pronounced ability to survive prolonged exposure to atmospheric oxygen. The major objective of this study was to biochemically characterize the components of the thioredoxin system in B. fragilis. The nitroreductase activity of TrxR was also assayed. METHODS: Components of the thioredoxin system were expressed in E. coli and used in a disulfide reductase activity assay. Activity of TrxR was measured with purified recombinant enzyme or with cell extracts after or without exposure to oxygen or hydrogen peroxide, respectively. RESULTS: Of all six thioredoxins tested, only thioredoxins A, D, and F were reduced by recombinant TrxR and natural TrxR present in B. fragilis cell extracts. Exposure to oxygen and hydrogen peroxide increased the activity of TrxR. Further, B. fragilis TrxR acts as a nitroreductase with furazolidone or 1-Chloro-2,4-dinitrobenzene as substrates but cannot reduce metronidazole. CONCLUSION: TrxR shows an increase in activity under the conditions of oxidative stress and exerts nitroreductase activity.

Thiol-based redox-active proteins as cardioprotective therapeutic agents in cardiovascular diseases.

Thiol-based redox compounds, namely thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs), stand as a pivotal group of proteins involved in antioxidant processes and redox signaling. Glutaredoxins (Grxs) are considered as one of the major families of proteins involved in redox regulation by removal of S-glutathionylation and thereby reactivation of other enzymes with thiol-dependent activity. Grxs are also coupled to Trxs and Prxs recycling and thereby indirectly contribute to reactive oxygen species (ROS) detoxification. Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases, which play an essential role in the detoxification of hydrogen peroxide, aliphatic and aromatic hydroperoxides, and peroxynitrite. The Trxs, Grxs and Prxs systems, which reversibly induce thiol modifications, regulate redox signaling involved in various biological events in the cardiovascular system. This review focuses on the current knowledge of the role of Trxs, Grxs and Prxs on cardiovascular pathologies and especially in cardiac hypertrophy, ischemia/reperfusion (I/R) injury and heart failure as well as in the presence of cardiovascular risk factors, such as hypertension, hyperlipidemia, hyperglycemia and metabolic syndrome. Further studies on the roles of thiol-dependent redox systems in the cardiovascular system will support the development of novel protective and therapeutic strategies against cardiovascular diseases.

Thioredoxin-mediated regulation of (photo)respiration and central metabolism.

Thioredoxins (TRXs) are ubiquitous proteins engaged in the redox regulation of plant metabolism. Whilst the light-dependent TRX-mediated activation of Calvin-Benson cycle enzymes is well documented, the role of extraplastidial TRXs in the control of the mitochondrial (photo)respiratory metabolism has been revealed relatively recently. Mitochondrially located TRX o1 has been identified as a regulator of alternative oxidase, enzymes of, or associated with, the tricarboxylic acid (TCA) cycle, and the mitochondrial dihydrolipoamide dehydrogenase (mtLPD) involved in photorespiration, the TCA cycle, and the degradation of branched chain amino acids. TRXs are seemingly a major point of metabolic regulation responsible for activating photosynthesis and adjusting mitochondrial photorespiratory metabolism according to the prevailing cellular redox status. Furthermore, TRX-mediated (de)activation of TCA cycle enzymes contributes to explain the non-cyclic flux mode of operation of this cycle in illuminated leaves. Here we provide an overview on the decisive role of TRXs in the coordination of mitochondrial metabolism in the light and provide in silico evidence for other redox-regulated photorespiratory enzymes. We further discuss the consequences of mtLPD regulation beyond photorespiration and provide outstanding questions that should be addressed in future studies to improve our understanding of the role of TRXs in the regulation of central metabolism.

Imbalance in thioredoxin system activates NLRP3 inflammasome pathway in epicardial adipose tissue of patients with coronary artery disease.

Atherosclerosis is the leading cause of death worldwide and has in part an inflammatory basis. Since epicardial adipose tissue (EAT) is in close contact with coronary arteries we hypothesized that an imbalance in thioredoxin-1 (TRX-1) and thioredoxin interacting protein (TXNIP) in EAT, activates NLRP3 inflammasome and promotes production of IL-1ß, leading to the development of atherosclerosis. Thirty-eight patients with coronary artery disease (CAD) and thirty patients with no clinical signs of atherosclerosis who underwent open-heart surgery for valve replacement were classified as CAD and control groups, respectively. Biopsy samples from EAT were collected and expression of TXNIP, TRX-1, NLRP3 and IL-1ß genes were assessed using qRT-PCR. Tissue protein levels of TXNIP and TRX-1 were determined by Western blotting while ELISA was applied to measure IL-1ß. Haematoxylin and eosin staining was used for histological examination. mRNA and protein levels of TXNIP in EAT were significantly higher in patients with CAD compared with control group, whereas CAD patients showed lower TRX-1 gene and protein expression. In addition, in CAD patients the NLRP3 gene expression was almost doubled and IL-1ß significantly increased at the both mRNA and protein levels. Enhancment in NLRP3 gene expression and TXNIP protein levels were accompanied with the increase in IL-1ß protein level whereas TRX-1 protein content showed a negative correlation with IL-1ß level. Concurrent increase in TXNIP, NLRP3, and IL-1ß suggests possible involvement of thioredoxin system in the activation of NLRP3 inflammasome, production of IL-1ß, and the presence of inflammation in CAD patients.

YjbH Requires Its Thioredoxin Active Motif for the Nitrosative Stress Response, Cell-to-Cell Spread, and Protein-Protein Interactions in Listeria monocytogenes.

Listeria monocytogenes is a model facultative intracellular pathogen. Tight regulation of virulence proteins is essential for a successful infection, and the gene encoding the annotated thioredoxin YjbH was identified in two forward genetic screens as required for virulence factor production. Accordingly, an L. monocytogenes strain lacking yjbH is attenuated in a murine model of infection. However, the function of YjbH in L. monocytogenes has not been investigated. Here, we provide evidence that L. monocytogenes YjbH is involved in the nitrosative stress response, likely through its interaction with the redox-responsive transcriptional regulator SpxA1. YjbH physically interacted with SpxA1, and our data support a model in which YjbH is a protease adaptor that regulates SpxA1 protein abundance. Whole-cell proteomics identified eight additional proteins whose abundance was altered by YjbH, and we demonstrated that YjbH physically interacted with each in bacterial two-hybrid assays. Thioredoxin proteins canonically require active motif cysteines for function, but thioredoxin activity has not been tested for L. monocytogenes YjbH. We demonstrated that cysteine residues of the YjbH thioredoxin domain active motif are essential for L. monocytogenes sensitivity to nitrosative stress, cell-to-cell spread in a tissue culture model of infection, and several protein-protein interactions. Together, these results demonstrated that the function of YjbH in L. monocytogenes requires its thioredoxin active motif and that YjbH has a role in the posttranslational regulation of several proteins, including SpxA1.IMPORTANCE The annotated thioredoxin YjbH in Listeria monocytogenes has been implicated in virulence, but its function in the cell is unknown. In other bacterial species, YjbH is a protease adaptor that mediates degradation of the transcriptional regulator Spx. Here, we investigated the function of L. monocytogenes YjbH and demonstrated its role in the nitrosative stress response and posttranslational regulation of several proteins with which YjbH physically interacts, including SpxA1. Furthermore, we demonstrated that the cysteine residues of the YjbH thioredoxin active motif are required for the nitrosative stress response, cell-to-cell spread, and some protein-protein interactions. YjbH is widely conserved among Firmicutes, and this work reveals its unique requirement of the thioredoxin-active motif in L. monocytogenes.

Redox regulation by peroxiredoxins is linked to their thioredoxin-dependent oxidase function.

The chloroplast contains three types of peroxiredoxins (PRXs). Recently, 2-CysPRX was associated with thioredoxin (TRX) oxidation-dependent redox regulation. Here, this analysis was expanded to include PRXQ and PRXIIE. Oxidized PRXQ was able to inactivate NADPH malate dehydrogenase and fructose-1,6-bisphosphatase most efficiently in the presence of TRX-m1 and TRX-m4. The inactivation ability of TRXs did not entirely match their reductive activation efficiency. PRXIIE was unable to function as TRX oxidase in enzyme regulation. This conclusion was further supported by the observation that PRXQ adopts the oxidized form by about 50% in leaves, supporting a possible function as a TRX oxidase similar to 2-CysPRX. Results on the oxidation state of photosystem I (P700), plastocyanin, and ferredoxin in intact leaves indicate that each type of PRX has distinct regulatory functions, and that both 2-CysPRX and PRXQ conditionally assist in adjusting the redox state of target proteins for proper activity.

Photorespiration-how is it regulated and how does it regulate overall plant metabolism?

Under the current atmospheric conditions, oxygenic photosynthesis requires photorespiration to operate. In the presence of low CO2/O2 ratios, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs an oxygenase side reaction, leading to the formation of high amounts of 2-phosphoglycolate during illumination. Given that 2-phosphoglycolate is a potent inhibitor of photosynthetic carbon fixation, it must be immediately removed through photorespiration. The core photorespiratory cycle is orchestrated across three interacting subcellular compartments, namely chloroplasts, peroxisomes, and mitochondria, and thus cross-talks with a multitude of other cellular processes. Over the past years, the metabolic interaction of photorespiration and photosynthetic CO2 fixation has attracted major interest because research has demonstrated the enhancement of C3 photosynthesis and growth through the genetic manipulation of photorespiration. However, to optimize future engineering approaches, it is also essential to improve our current understanding of the regulatory mechanisms of photorespiration. Here, we summarize recent progress regarding the steps that control carbon flux in photorespiration, eventually involving regulatory proteins and metabolites. In this regard, both genetic engineering and the identification of various layers of regulation point to glycine decarboxylase as the key enzyme to regulate and adjust the photorespiratory carbon flow. Potential implications of the regulation of photorespiration for acclimation to environmental changes along with open questions are also discussed.

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues.

Cch1p, the yeast homolog of the pore-forming subunit α1 of the mammalian voltage-gated Ca2+ channel (VGCC), is located on the plasma membrane and mediates the redox-dependent influx of Ca2+ Cch1p is known to undergo both rapid activation (after oxidative stress and or a change to high pH) and slow activation (after ER stress and mating pheromone activation), but the mechanism of activation is not known. We demonstrate here that both the fast activation (exposure to pH 8-8.5 or treatment with H2O2) and the slow activation (treatment with tunicamycin or α-factor) are mediated through a common redox-dependent mechanism. Furthermore, through mutational analysis of all 18 exposed cysteine residues in the Cch1p protein, we show that the four mutants C587A, C606A, C636A and C642A, which are clustered together in a common cytoplasmic loop region, were functionally defective for both fast and slow activations, and also showed reduced glutathionylation. These four cysteine residues are also conserved across phyla, suggesting a conserved mechanism of activation. Investigations into the enzymes involved in the activation reveal that the yeast glutathione S-transferase Gtt1p is involved in the glutathionylation of Cch1p, while the thioredoxin Trx2p plays a role in the Cch1p deglutathionylation.

[The renal protective effect and mechanism of liraglutide in diabetic mice induced by high-fat diet].

Objective: To investigate the protective effect of liraglutide on kidney of diabetic mice induced by high-fat diet and its possible mechanisms. Methods: C57BL/6J male mice were randomly divided into normal chow diet (NC) group and high-fat diet (HFD) group, which were fed with normal chow diet and HFD for 12 weeks respectively. After diet challenge, the mice were randomly divided into normal control group, normal chow diet with liraglutide treatment (NC+Lira) group, HFD group and high-fat diet with liraglutide treatment (HFD+Lira) group. The mice in NC+Lira and HFD+Lira groups were given intraperitoneal injection of liraglutide (400 µg·kg(-1)·d(-1)) for 8 weeks, while mice in NC and HFD groups were given intraperitoneal injection of same amount of normal saline. Urinary albumin and creatinine levels were measured by enzyme-linked immunosorbent assay (ELISA). Renal morphology was observed by HE staining. The expression levels of silent mating type information regulation 2 homolog 1 (SIRT1) and thioredoxin-interacting protein (TXNIP) were determined by Western blot. Results: Compared with HFD group, liraglutide significantly lowered the body weight [(30.98±1.29) g vs (39.43±2.58) g], fasting blood glucose (FBG) [(7.21±0.15) mmol/L vs (9.55±0.29) mmol/L] and urinary albumin/creatinine ratio (ACR) [(205.48±17.14) µg/mg vs (319.86±34.14) µg/mg] in HFD+Lira group (all P<0.05). HE staining showed that glomerular hypertrophy of HFD group alleviated after liraglutide treatment. The expression level of TXNIP in the kidney of HFD mice significantly decreased after liraglutide treatment (0.41±0.10 vs 3.50±0.70), while expression level of SIRT1 significantly increased (0.75±0.15 vs 0.32±0.04) (both P<0.05). Conclusion: Liraglutide could improve diabetic nephropathy by up-regulation of SIRT1 expression and down-regulation of TXNIP expression in diabetic mice induced by HFD.

Human Plasma Thioredoxin-80 Increases With Age and in ApoE-/- Mice Induces Inflammation, Angiogenesis, and Atherosclerosis.

BACKGROUND: Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype. METHODS: TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1ß and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice. RESULTS: In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1ß and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE-/- mice have a significant increase of aortic atherosclerotic lesions. CONCLUSIONS: TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.

Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

Aberrant expression of redox regulatory proteins in patients with concomitant primary Sclerosing cholangitis/inflammatory bowel disease.

OBJECTIVE: Primary Sclerosing Cholangitis (PSC) is a severe cholestatic liver disease characterized by progressive peri-biliary tract inflammation, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%-80% of PSC patients (PSC/IBD). We previously reported dysregulation of key anti-oxidant pathways in PSC/IBD. The objective of this study was to expand previous data by examining the abundance of thioredoxins (Trx) in PSC/IBD. METHODS: Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, the protein abundance of thioredoxin, thioredoxin reductase (TrxR1), and their downstream substrates peroxiredoxins was assessed. RESULTS: Western blot analyses of thioredoxin and peroxiredoxin abundance revealed significant increases in abundance of Trx1 and TrxR1 whereas expression of thioredoxin-interacting protein was significantly decreased in PSC/IBD. Concurrently, abundance of cytosolic peroxiredoxins was not significantly impacted. The abundance of mitochondrial Trx2, along with peroxiredoxins 3, 5 and 6 were significantly decreased by concurrent PSC/IBD. Histological staining of Trx1/TrxR1 revealed elevated nuclear Trx1 and TrxR1 staining within cholangiocytes as well as an overall periportal increase in expression in PSC/IBD. An examination of additional anti-oxidant responses reveal suppression of gamma-glutamylcysteine synthetase and heme oxygenase (HO-1) whereas expression of the protein chaperone glucose regulated protein 78 increased suggesting elevated cellular stress in PSC/IBD. CONCLUSIONS: Results herein suggest that in addition to severe dysregulation of anti-oxidant responses, cholestasis impacts both cytosolic/nuclear (Trx1) as well as mitochondrial (Trx2) redox signaling and control pathways.