Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Evidence that peroxiredoxins are novel members of the thioredoxin fold superfamily.

Peroxiredoxins catalyze reduction of hydrogen peroxide or alkyl peroxide, to water or the corresponding alcohol. Detailed analysis of their sequences indicates that these enzymes possess a thioredoxin (Trx)-like fold and consequently are homologues of both thioredoxin and glutathione peroxidase (GPx). Sequence- and structure-based multiple sequence alignments indicate that the peroxiredoxin active site cysteine and GPx active site selenocysteine are structurally equivalent. Homologous peroxiredoxin and GPx enzymes are predicted to catalyze equivalent reactions via similar reaction intermediates.     

A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity

NADPH thioredoxin reductase C (NTRC) is an interesting NTR with a thioredoxin (Trx) domain at the C-terminus, able to conjugate both activities for 2-Cys peroxiredoxin (Prx) reduction. NTRC is dimeric in the presence of NADPH and interacted with dimeric 2-Cys Prx through the Trx module by a mixed disulfide between Cys377 of NTRC and Cys61 of the 2-Cys Prx. NTRC variants of both NTR and Trx active sites were inactive, but 1:1 mixtures of both variants allowed partial recovery of activity suggesting inter-subunit transfer of electrons during catalysis. Based on these results we propose a model for the reaction mechanism of NTRC.

Structured summary

MINT-7017333: 2cys Prx (uniprotkb:Q6ER94) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by molecular sieving (MI:0071)MINT-7017101, MINT-7017183: NTRC (uniprotkb:Q70G58) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by enzymatic studies (MI:0415)     

The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species‐scavenging enzyme that shows promiscuity in thiol redox control

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (‐SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H₂O₂ reduction, a sulfenic acid (‐SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S‐S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx‐MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H₂O₂ stress, and its gene expression is clearly induced upon H₂O₂ challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.     

Comparative structural analysis of oxidized and reduced thioredoxin from Drosophila melanogaster

Thioredoxins (Trx) participate in essential antioxidant and redox-regulatory processes via a pair of conserved cysteine residues. In dipteran insects like Drosophila and Anopheles, which lack a genuine glutathione reductase (GR), thioredoxins fuel the glutathione system with reducing equivalents. Thus, characterizing Trxs from these organisms contributes to our understanding of redox control in GR-free systems and provides information on novel targets for insect control. Cytosolic Trx of Drosophila melanogaster (DmTrx) is the first thioredoxin that was crystallized for X-ray diffraction analysis in the reduced and in the oxidized form. Comparison of the resulting structures shows rearrangements in the active-site regions. Formation of the C32-C35 disulfide bridge leads to a rotation of the side-chain of C32 away from C35 in the reduced form. This is similar to the situation in human Trx and Trx m from spinach chloroplasts but differs from Escherichia coli Trx, where it is C35 that moves upon change of the redox state. In all four crystal forms that were analysed, DmTrx molecules are engaged in a non-covalent dimer interaction. However, as demonstrated by gel-filtration analyses, DmTrx does not dimerize under quasi in vivo conditions and there is no redox control of a putative monomer/dimer equilibrium. The dimer dissociation constants K(d) were found to be 2.2mM for reduced DmTrx and above 10mM for oxidized DmTrx as well as for the protein in the presence of reduced glutathione. In human Trx, oxidative dimerization has been demonstrated in vitro. Therefore, this finding may indicate a difference in redox control of GR-free and GR-containing organisms.     

Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme

Insertion of selenocysteine (Sec) into protein scaffolds provides an opportunity for designing enzymes with improved and unusual catalytic properties. The use of a common thioredoxin fold with a high affinity for glutathione in glutaredoxin (Grx) and glutathione peroxidase (GPx) suggests a possibility of engineering Grx into GPx and vice versa. Here, we engineered a Grx domain of mouse thioredoxin/glutathione reductase (TGR) into a selenium-containing enzyme by substituting the active site cysteine (Cys) with selenocysteine (Sec) in a Cys auxotrophic system. The resulting selenoenzyme displayed an unusually high GPx catalytic activity rivaling that of several native GPxs. The engineered seleno-Grx was characterized by mass spectrometry and kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of naturally occurring GPxs. For example, its second rate constant (k(cat)/K(mH2O2)) was as high as 1.55x10(7) M(-1) min(-1). It appears that glutathione-dependent Grx, GPx and glutathione transferase (GST) evolved from a common thioredoxin-like ancestor to accommodate related glutathione-dependent functions and can be interconverted by targeted Sec insertion.     

Adrenomedullin protects against hypoxia/reoxygenation-induced cell death by suppression of reactive oxygen species via thiol redox systems

Adrenomedullin (ADM) functions as a survival factor against hypoxic cell death. However, molecular mechanisms underlying the cell survival pathway remain largely unknown. In this report, we showed that ADM suppressed reactive oxygen species (ROS) increase by inhibiting reduction of glutathione (GSH) level in hypoxia/reoxygenation (H/R) injury, and increased the activities of glutathione peroxidase and reductase. In addition, ADM maintained total and active reduced thioredoxin (Trx) levels against H/R. We also found that ADM blocked nuclear translocation of Trx induced by H/R. The results of the present study show that ADM regulates cellular ROS levels via the GSH and Trx system.     

NADPH依赖性硫氧还蛋白还原酶C在植物质体中的作用

硫氧还蛋白的氧化还原调节作用在生物界中普遍存在。它能够还原目标蛋白的二硫键,而自身的活性位点则被氧化。因此,对于新的催化循环,则需要由相应的还原酶将其再次还原成活性形式。硫氧还蛋白对维持高等植物的光合效率同样具有重要意义。叶绿体中的硫氧还蛋白分别由铁氧还蛋白依赖性硫氧还蛋白还原酶和NADPH依赖性硫氧还蛋白还原酶C(NTRC)两种酶还原。NTRC的本质是一种黄素蛋白,除了具有还原酶活性外,还整合了一个硫氧还蛋白结构域,在叶绿体和淀粉体的氧化还原调节中处于核心地位。这种特殊的双功能酶在卡尔文-本森循环、氧化戊糖磷酸途径、抗过氧化、四吡咯代谢、ATP和淀粉合成、生长素和光周期调控中扮演了多重角色。本综述总结了NTRC的生理功能,并讨论了该蛋白质对植物质体氧化还原稳态的调节机制。     

Catalytic activity of selenomethionine in removing amino acid, peptide, and protein hydroperoxides

Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products.     

Coupling of Domain Swapping to Kinetic Stability in a Thioredoxin Mutant

The thioredoxin (Trx) fold is a small monomeric domain that is ubiquitous in redox-active enzymes. Trxs are characterized by a typical WCGPC active-site sequence motif. A single active-site mutation of the tryptophan to an alanine in Staphylococcus aureus Trx converts the oxidized protein into a biologically inactive domain-swapped dimer. While the monomeric protein unfolds reversibly in a two-state manner, the oxidized dimeric form is kinetically stable and converts to the monomeric form upon refolding. After reduction, the half-life of the dimer decreases many orders of magnitude to ∼ 4.3 h, indicating that the active-site disulfide between Cys29 and Cys32 is an important determinant for the kinetics of unfolding. We propose kinetic stability as a possible evolutionary strategy in the evolution of multimeric proteins from their monomeric ancestors by domain swapping, which, for this biologically inactive Trx mutant, turned out to be an evolutionary dead end.     

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.     

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys129-His-Asn-Cys132-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be approximately 5.1, a relatively low pKa given its redox potential (approximately 265 mV) compared with most members of the thioredoxin family.     

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).     

The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function

The catalytic activity of selenocysteine-containing thioredoxin reductases can be mimicked by cysteine-variants if the local environment at the C-terminal redox center supports thiol activation. This concept of a linear catalytic site was challenged by structural data suggesting that the invariant residue His106 functions as a base catalyst for the dithiol-disulphide exchange reaction between enzyme and substrate. As reported here, we changed His106 to asparagine, glutamine, and phenylalanine in various C-terminal mutants of Drosophila melanogaster thioredoxin reductase. The catalytic activity dropped considerably, yet pH-profiles did not reveal differences, rendering a function for His106 as a base catalyst unlikely. Interestingly, the phenylalanine-mutants, designed as negative controls were the most active mutants which suggests rather a structural role of His106.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury

Background

Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.

Methods

Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl₄).

Results

TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.

Conclusions

Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.

General significance

The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.