The C-terminal selenenylsulfide of extracellular/non-reduced thioredoxin reductase endows this protein with selectivity to small-molecule electrophilic reagents under oxidative conditions.

Mammalian cytosolic thioredoxin reductase (TrxR1) serves as an antioxidant protein by transferring electrons from NADPH to various substrates. The action of TrxR1 is achieved via reversible changes between NADPH-reduced and non-reduced forms, which involves C-terminal selenolthiol/selenenylsulfide exchanges. TrxR1 may be released into extracellular environment, where TrxR1 is present mainly in the non-reduced form with active-site disulfide and selenenylsulfide bonds. The relationships between extracellular TrxR1 and tumor metastasis or cellular signaling have been discovered, but there are few reports on small-molecule compounds in targeted the non-reduced form of TrxR1. Using eight types of small-molecule thiol-reactive reagents as electrophilic models, we report that the selenenylsulfide bond in the non-reduced form of TrxR1 functions as a selector for the thiol-reactive reagents at pH 7.5. The non-reduced form of TrxR1 is resistant to hydrogen peroxide/oxidized glutathione, but is sensitive to certain electrophilic reagents in different ways. With 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and S-nitrosoglutathione (GSNO), the polarized selenenylsulfide bond breaks, and selenolate anion donates electron to the dynamic covalent bond in DTNB or GSNO, forming TNB-S-Se-TrxR1 complex or ON-Se-TrxR1 complex. The both complexes lose the ability to transfer electrons from NADPH to substrate. For diamide, the non-reduced TrxR1 actually prevents irreversible damage by this oxidant. This is consistent with the regained activity of TrxR1 through removal of diamide via dialysis. Diamide shows effective in the presence of human cytosolic thioredoxin (hTrx1), Cys residue(s) of which is/are preferentially affected by diamide to yield disulfide, hTrx1 dimer and the mixed disulfide between TrxR1-Cys497/Sec498 and hTrx1-Cys73. In human serum samples, the non-reduced form of TrxR1 exists as dithiothreitol-reducible polymer/complexes, which might protect the non-reduced TrxR1 from inactivation by certain electrophilic reagents under oxidative conditions, because cleavage of these disulfides can lead to regain the activity of TrxR1. The details of the selective response of the selenenylsulfide bond to electrophilic reagents may provide new information for designing novel small-molecule inhibitors (drugs) in targeted extracellular/non-reduced TrxR1.

Elevated serum 4HNE plus decreased serum thioredoxin: Unique feature and implications for acute exacerbation of chronic obstructive pulmonary disease.

Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is a global problem with high mortality. Its pathogenesis is not fully understood. To reveal new serum feature of AECOPD and their potential implications, we have analyzed 180 serum samples, and found that in the serum of AECOPD patients, 4-hydroxy-2-nonenal (4HNE)-protein adducts are dynamically increased as partial pressure of oxygen (PaO2) drops, which is accompanied by progressively decreasing thioredoxin reductase (TrxR1) and thioredoxin (Trx1), as compared with those of healthy people. This phenomenon is unique, because acute hypoxia patients have 1.1-fold or 1.7-fold higher serum TrxR1 or Trx1 activity, respectively, than healthy people, in keeping with low 4HNE level. Moreover, serum 4HNE-protein adducts may form disulfide-linked complexes with high-molecular-weight, the amount of which is significantly increased during AECOPD. Serum 4HNE-protein adducts include 4HNE-Trx1 adduct and 4HNE-TrxR1 adduct, but only the former is significantly increased during AECOPD. Through cell biology, biochemistry and proteomics methods, we have demonstrated that extracellular 4HNE and 4HNE-Trx1 adduct affect human bronchial epithelial cells via different mechanisms. 4HNE-Trx1 adduct may significantly alter the expression of proteins involved mainly in RNA metabolism, but it has no effect on TrxR1/Trx1 expression and cell viability. On the other hand, low levels of 4HNE promote TrxR1/Trx1 expression and cell viability, while high levels of 4HNE inhibit TrxR1/Trx1 expression and cell viability, during which Trx1, at least in part, mediate the 4HNE action. Our data suggest that increasing serum 4HNE and decreasing serum Trx1 in AECOPD patients are closely related to the pathological processes of the disease. This finding also provides a new basis for AECOPD patients to use antioxidant drugs.

New insights into serum/extracellular thioredoxin in regulating hepatic insulin receptor activation.

BACKGROUND: Serum thioredoxin of type-2 diabetic patients is significantly higher than that of healthy people. Pathophysiological significance is unclear. METHODS: Effects of serum/extracellular thioredoxin on phosphorylation (activation) of hepatic insulin receptor (IR) were investigated by using methods in biochemistry, cell/molecular biology and mass spectrometry. RESULTS: In human serum, thioredoxin and insulin may interact. Their mixture contains a mixed disulfide between insulin B-chain and thioredoxin-Cys73, which limits their activities. In contrast, free form of serum/extracellular thioredoxin is active, and can regulate phosphorylation of insulin receptor ß-subunits (IRß) via direct/indirect mechanisms. The direct mechanism associates with positive regulation. Serum/extracellular thioredoxin increases insulin binding to IR, facilitating insulin-induced phosphorylation of IRß and downstream AKT. The indirect mechanism is involved in negative regulation. Entry of extracellular thioredoxin into hepatic cells via IR enhances the expression and activity of cellular protein-tyrosine phosphatase 1B (PTP1B), which negatively regulates IRß phosphorylation. After coordination between these two mechanisms, the positive impact of serum/extracellular thioredoxin overwhelms its negative impact on IRß phosphorylation, which subsequently accelerates hepatic glucose uptake. In hepatic cells with thioredoxin deficiency, insulin-induced IRß phosphorylation is decreased, which could be restored by extracellular thioredoxin entry. Moreover, the results from assaying 475 serum samples demonstrate a discriminating value of serum thioredoxin activity in diagnosing type-2 diabetes. CONCLUSION: Serum/extracellular thioredoxin plays a critical role in regulating hepatic IRß phosphorylation. GENERAL SIGNIFICANCE: In case of insulin resistance/type-2 diabetes, hepatic IRß is at low phosphorylation level, thereby the improvement effect of serum/extracellular thioredoxin on insulin-induced IRß phosphorylation seems particularly important.

Role and mechanism of homocysteine in affecting hepatic protein-tyrosine phosphatase 1B.

BACKGROUND: Elevated homocysteine is epidemiologically related to insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling. However, the effect of homocysteine on PTP1B remains unclear. METHODS: S-homocysteinylated PTP1B was identified by LC-ESI-MS/MS. The ability of thioredoxin system to recover active PTP1B from S-homocysteinylated PTP1B was confirmed by RNA interference. To address the mechanism for homocysteine to affect PTP1B activity, we performed 5-IAF insertion, activity assays, Western blotting, co-immunoprecipitation and glucose uptake experiments. RESULTS: The thiol-containing form of homocysteine (HcySH) suppressed phosphorylation of insulin receptor-ß subunit, but enhanced PTP1B activity. This phenomenon was partially related to the fact that HcySH promoted PTP1B expression. Although the disulfide-bonded form of homocysteine (HSSH) modified PTP1B to form an inactive S-homocysteinylated PTP1B, HcySH-induced increase in the activities of cellular thioredoxin and thioredoxin reductase, components of thioredoxin system, could recover active PTP1B from S-homocysteinylated PTP1B. Thioredoxin system transferred electrons from NADPH to S-homocysteinylated PTP1B, regenerating active PTP1B in vitro and in hepatocytes. The actions of HcySH were also related with decrease in hepatic glucose uptake. CONCLUSIONS: The effect of HcySH/HSSH on PTP1B activity depends, at least partially, on the ratio of active PTP1B and S-homocysteinylated PTP1B. High HcySH-induced an increase in thioredoxin system activity is beneficial to de-S-homocysteinylation and is good for PTP1B activity. GENERAL SIGNIFICANCE: Our data provide a novel insight into post-translational regulation of PTP1B, and expand the biological functions of thioredoxin system.

A novel protein tyrosine phosphatase 1B inhibitor with therapeutic potential for insulin resistance.

BACKGROUND AND PURPOSE: Insulin-sensitizing drugs are currently limited, and identifying new candidates is a challenge. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signalling, and its inhibition is anticipated to improve insulin resistance. Here, the pharmacological properties of CX08005, a novel PTP1B inhibitor, were investigated. EXPERIMENTAL APPROACH: Recombinant hPTP1B protein was used to study enzyme activity and mode of inhibition. Docking simulation explored the interactions between CX08005 and PTP1B. Insulin sensitivity was evaluated by glucose tolerance test (GTT) in diet-induced obese (DIO) and KKAy mice; glucose-stimulated insulin secretion (GSIS), homeostasis model assessment of insulin resistance index (HOMA-IR) and whole-body insulin sensitivity (ISWB ) were also determined. A hyperinsulinaemic-euglycaemic clamp was performed to evaluate insulin-stimulated glucose disposal in both whole-body and insulin-sensitive tissues. Furthermore, CX08005's effects on muscle, fat and liver cells were determined in vitro. KEY RESULTS: CX08005 competitively inhibited PTP1B by binding to the catalytic P-loop through hydrogen bonds. In DIO mice, CX08005 ameliorated glucose intolerance dose-dependently (50-200 mg·kg(-1) ·day(-1) ) and decreased the HOMA-IR. In KKAy mice, CX08005 (50 mg·kg(-1) ·day(-1) ) improved glucose intolerance, GSIS, ISWB and hyperglycaemia. CX08005 also enhanced insulin-stimulated glucose disposal, increased glucose infusion rate and glucose uptake in muscle and fat in DIO mice (hyperinsulinaemic-euglycaemic clamp test). CX08005 enhanced insulin-induced glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and increased phosphorylation of IRß/IRS1 and downstream molecules in hepatocytes in a dose- and insulin-dependent manner respectively. CONCLUSIONS AND IMPLICATIONS: Our results strongly suggest that CX08005 directly enhances insulin action in vitro and in vivo through competitive inhibition of PTP1B.

Bacterial thioredoxin and thioredoxin reductase as mediators for epigallocatechin 3-gallate-induced antimicrobial action.

Epigallocatechin 3-gallate (EGCG) is the most abundant catechin in green tea and may combat bacteria with few side-effects. Its selectivity for different bacterial infections remains unclear, and hence the identification of the underlying mechanism is of practical importance. Both the thioredoxin (Trx) system and the glutathione/glutaredoxin (Grx) system support bacterial growth. Some pathogenic bacteria are naturally deficient in the Grx system. We analyzed the effect of green tea extract (GTE) and EGCG on wild-type and null mutants of Escherichia coli with either Trx or Grx system deficiency and found that GTE and EGCG selected the Trx system as a target and killed the mutant that is exclusively dependent on Trx/Trx reductase (TrxR). EGCG inhibited the activity of both Trx1 and TrxR of E. coli in a dose-dependent and time-dependent manner. The IC50 values of EGCG for the reduced forms of E. coli Trx1/TrxR were ~ 3-4-fold lower than those for their non-reduced forms. The IC50 value of EGCG for the E. coli Trx1 system was 56-fold lower than that for the mammalian Trx1 system. The inhibition by EGCG of both Trx1 and TrxR of E. coli was irreversible. EGCG-induced inactivation of E. coli Trx1 was a second-order process, and that of E. coli TrxR was an affinity-labeling process. The covalent binding sites for EGCG in E. coli Trx1 were Trp(28) , Trp(31) and Cys(32) , and in E. coli TrxR were Cys(135) and Cys(138) . Moreover, the sensitivity of Staphylococcus aureus to EGCG was similar to that of an E. coli mutant with Grx system deficiency. EGCG-induced inactivation of Trx/TrxR in S. aureus coincided with suppressed growth of this virulent pathogen. Our findings suggest a role for EGCG-dependent Trx/TrxR inactivation in potentiating antibacterial activity of EGCG.

New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation.

High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein-tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1.

Mechanistic insights into the inhibitory effects of palmitoylation on cytosolic thioredoxin reductase and thioredoxin.

Overnutrition can lead to oxidative stress, but its underlying mechanism remains unclear. In this study, we report that human liver-derived HepG2 cells utilize cytosolic thioredoxin reductase (TrxR1) and thioredoxin (hTrx1) to defend against the high glucose/palmitate-mediated increase in reactive oxygen species. However, enhanced TrxR1/hTrx1 palmitoylation occurs in parallel with a decrease in their activities under the conditions studied here. An autoacylation process appears to be the major mechanism for generating palmitoylated TrxR1/Trx1 in HepG2 cells. A novel feature of this post-translational modification is the covalent inhibition of TrxR1/hTrx1 by palmitoyl-CoA, an activated form of palmitate. The palmitoyl-CoA/TrxR1 reaction is NADPH-dependent and produces palmitoylated TrxR1 at an active site selenocysteine residue. Conversely, S-palmitoylation occurs at the structural Cys62/Cys69/Cys72 residues but not the active site Cys32/Cys35 residues of hTrx1. Palmitoyl-CoA concentration and the period of incubation with TrxR1/hTrx1 are important factors that influence the inhibitory efficacy of palmitoyl-CoA on TrxR1/hTrx1. Thus, an increase in TrxR1/hTrx1 palmitoylation could be a potential consequence of high glucose/palmitate. The time-dependent inactivation of the NADPH-TrxR1-Trx1 system by palmitoyl-CoA occurs in a biphasic manner - a fast phase followed by a slow phase. Kinetic analysis suggests that the fast phase is consistent with a fast and reversible association between TrxR1/hTrx1 and palmitoyl-CoA. The slow phase is correlated with a slow and irreversible inactivation, in which selenolate/thiolate groups nucleophilically attack the α-carbon of bound palmitoyl-CoA, leading to the formation of thioester/selenoester bonds. hTrx1 can enhance rate of fast phase but limits the rate of slow phase when it is present in a preincubation mixture containing NADPH, TrxR1 and palmitoyl-CoA. Therefore, hTrx1 may provide palmitoylation sites or partially protect the TrxR1 active site selenol/thiol group(s) from palmitoylation. Our data suggest that Se/S-palmitoylation acts as an important modulator of TrxR1/hTrx1 activities, representing a novel potential mechanism that underlies overnutrition-induced events.

High-fat diet-induced changes in liver thioredoxin and thioredoxin reductase as a novel feature of insulin resistance.

High-fat diet (HFD) can induce oxidative stress. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are critical antioxidant proteins but how they are affected by HFD remains unclear. Using HFD-induced insulin-resistant mouse model, we show here that liver Trx and TrxR are significantly decreased, but, remarkably, the degree of their S-acylation is increased after consuming HFD. These HFD-induced changes in Trx/TrxR may reflect abnormalities of lipid metabolism and insulin signaling transduction. HFD-driven accumulation of 4-hydroxynonenal is another potential mechanism behind inactivation and decreased expression of Trx/TrxR. Thus, we propose HFD-induced impairment of liver Trx/TrxR as major contributor to oxidative stress and as a novel feature of insulin resistance.

A thioredoxin reductase and/or thioredoxin system-based mechanism for antioxidant effects of ambroxol.

Long-term treatment with ambroxol (ABX), a bronchial expectorant, was found to prevent acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The underlying mechanism remains unclear. To address this, we have investigated the effect of ABX on critical antioxidant proteins thioredoxin (Trx) and thioredoxin reductase (TrxR) that are decreased in patients with AECOPD. Trx, TrxR and NADP(H) form Trx system, which is involved in regulating numerous oxidative stress-related events. In human bronchial epithelial cells, treatment with ABX from 0 to 200 µM gradually increased mRNA and protein levels of TrxR/Trx. At these ABX concentrations, TrxR activity was elevated progressively, whereas Trx activity exhibited a dose-dependent biphasic response, increasing at 50 and 75 µM, but decreasing at ABX over 150 µM. Pre-treatment with 75 µM ABX enhanced the capacity of the cells to eliminate reactive oxygen species, which was largely prevented by knockdown of cytosolic Trx (hTrx1). In a purified system, ABX shortened the initial lag phase during the reduction of insulin disulfide by Trx system. Pre-treatment of NADPH-reduced TrxR with ABX caused a dose- and time-dependent increase in thiolate/selenolate species, i.e. the catalytically active form of TrxR. Kinetic analysis demonstrated that the reduction of H2O2 by TrxR or Trx system were enhanced by 100 or 200 µM ABX. When hTrx1 was mixed with ABX in a molar ratio of 1:1 to 1:100 (which could occur in human plasma), changes in intrinsic Trp fluorescence occurred, and the response of reduced hTrx1 was especially remarkable. These data reveal an ABX-sensing mechanism of TrxR/Trx. We therefore conclude that the antioxidant actions of ABX at physiological concentrations are, at least partially, mediated by TrxR and/or Trx system.

Finite element analysis of pedestrian lower limb fractures by direct force: the result of being run over or impact?

The elucidation and prediction of the biomechanics of lower limb fractures could serve as a useful tool in forensic practices. Finite element (FE) analysis could potentially help in the understanding of the fracture mechanisms of lower limb fractures frequently caused by car-pedestrian accidents. Our aim was (1) to develop and validate a FE model of the human lower limb, (2) to assess the biomechanics of specific injuries concerning run-over and impact loading conditions, and (3) to reconstruct one real car-pedestrian collision case using the model created in this study. We developed a novel lower limb FE model and simulated three different loading scenarios. The geometry of the model was reconstructed using Mimics 13.0 based on computed tomography (CT) scans from an actual traffic accident. The material properties were based upon a synthesis of data found in published literature. The FE model validation and injury reconstruction were conducted using the LS-DYNA code. The FE model was validated by a comparison of the simulation results of three-point bending, overall lateral impact tests and published postmortem human surrogate (PMHS) results. Simulated loading scenarios of running-over the thigh with a wheel, the impact on the upper leg, and impact on the lower thigh were conducted with velocities of 10 m/s, 20 m/s, and 40 m/s, respectively. We compared the injuries resulting from one actual case with the simulated results in order to explore the possible fracture bio-mechanism. The peak fracture forces, maximum bending moments, and energy lost ratio exhibited no significant differences between the FE simulations and the literature data. Under simulated run-over conditions, the segmental fracture pattern was formed and the femur fracture patterns and mechanisms were consistent with the actual injury features of the case. Our study demonstrated that this simulation method could potentially be effective in identifying forensic cases and exploring of the injury mechanisms of lower limb fractures encountered due to inflicted lesions. This model can also help to distinguish between possible and impossible scenarios.

Blunt liver injury with intact ribs under impacts on the abdomen: a biomechanical investigation.

Abdominal trauma accounts for nearly 20% of all severe traffic injuries and can often result from intentional physical violence, from which blunt liver injury is regarded as the most common result and is associated with a high mortality rate. Liver injury may be caused by a direct impact with a certain velocity and energy on the abdomen, which may result in a lacerated liver by penetration of fractured ribs. However, liver ruptures without rib cage fractures were found in autopsies in a series of cases. All the victims sustained punches on the abdomen by fist. Many studies have been dedicated to determining the mechanism underlying hepatic injury following abdominal trauma, but most have been empirical. The actual process and biomechanism of liver injury induced by blunt impact on the abdomen, especially with intact ribs remained, are still inexhaustive. In order to investigate this, finite element methods and numerical simulation technology were used. A finite element human torso model was developed from high resolution CT data. The model consists of geometrically-detailed liver and rib cage models and simplified models of soft tissues, thoracic and abdominal organs. Then, the torso model was used in simulations in which the right hypochondrium was punched by a fist from the frontal, lateral, and rear directions, and in each direction with several impact velocities. Overall, the results showed that liver rupture was primarily caused by a direct strike of the ribs induced by blunt impact to the abdomen. Among three impact directions, a lateral impact was most likely to cause liver injury with a minimum punch speed of 5 m/s (the momentum was about 2.447 kg.m/s). Liver injuries could occur in isolation and were not accompanied by rib fractures due to different material characteristics and injury tolerance.

Thioredoxin and thioredoxin reductase control tissue factor activity by thiol redox-dependent mechanism.

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP(+) states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP(+), hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H(2)O(2) is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.

Molecular bases of thioredoxin and thioredoxin reductase-mediated prooxidant actions of (-)-epigallocatechin-3-gallate.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) function as antioxidant and anti-apoptotic proteins, which are often up-regulated in drug-resistant cancer cells. (-)-epigallocatechin-3-gallate (EGCG) is a naturally occurring antioxidant in green tea, but also exhibits prooxidant and apoptosis-inducing properties. We have previously showed a linkage between EGCG-induced inactivation of TrxR and decreased cell survival, revealing TrxR as a new target of EGCG. However, the molecular events underlying the importance of Trx/TrxR in EGCG-induced cytotoxicity remain unclear. Here, we show that the crosstalk between EGCG and Trx/TrxR occurred in a redox-dependent manner, and EGCG induced inactivation of Trx/TrxR in parallel with increased ROS levels in HeLa cells. Moreover, EGCG displayed great reactivity with Cys/Sec residues that have low pK(a) values. The structure of EGCG suggests that its quinone form would readily react with thiolate and selenolate nucleophiles. Using mass spectrometry, we have demonstrated the formation of EGCG-Trx1 (Cys(32)) and EGCG-TrxR (Cys/Sec) conjugates, confirming that EGCG quinone specifically conjugates with active-site Cys(32) in Trx or C-terminal Cys/Selenocysteine (Sec) couple in TrxR under conditions where Trx/TrxR are reduced. Non-reduced form of Trx/TrxR could escape from EGCG inhibition. These data reveal a potential mechanism for enhancing EGCG-induced cancer cell death by the NADPH-dependent reduction of Trx/TrxR.

Decreased serum levels of thioredoxin in patients with coronary artery disease plus hyperhomocysteinemia is strongly associated with the disease severity.

OBJECTIVE: Elevation of homocysteine and thioredoxin (Trx) levels was found in some patients with coronary artery diseases (CAD). However, their correlations with CAD were not clear. Dysfunction of thioredoxin/thioredoxin reductase (TrxR) may cause oxidative stress that is common to CAD. We seek to determine the association among homocysteine, Trx/TrxR and CAD. METHODS: Serum samples were collected from 150 CAD patients under statin treatment and 122 non-CAD controls. Risk factors for atherosclerosis including homocysteine, lipids and glucose levels were analyzed. Trx/TrxR activities and protein levels were determined using super-insulin assay and Western blot, respectively. One-way ANOVA, Tukey's post hoc test and Spearman's rank correlation coefficient were used for statistical analysis. CAD severity was evaluated by angiographic Gensini score. RESULTS: Compared with non-CAD group, CAD group had significantly increased TrxR activity (P<0.05) and homocysteine levels (P<0.01), but not Trx activity. After further dividing CAD group using homocysteine below 15 microM as reference, Trx activity decreased significantly in CAD group with high homocysteine, and was inversely associated with homocysteine levels (r=-0.199, P<0.05) that was, however, weakly positively associated with TrxR activity. Neither lipids nor glucose significantly affected Trx/TrxR activity. Association of CAD severity with low Trx plus high homocysteine was strong (r=-0.458, P<0.001), but with high homocysteine alone was rather weak (r=0.125, P=0.225). CONCLUSION: In CAD patients, high homocysteine levels may cause low Trx activity, which is closely correlated to the extent and severity of CAD.

Opposing regulation of histamine-induced calcium signaling by sodium selenite and ebselen via alterations of thiol redox status.

Elevated blood histamine plays a role in the pathogenesis of atherosclerosis. Calcium signaling mediates histamine action in endothelial cells. Selenium (Se) is a dietary essential trace element for humans. Se compounds in different oxidation states were found to exhibit an opposing effect on the histamine-induced calcium signaling in the ECV304 cell line. When Se in the form of sodium selenite was added in the cell culture, the reactivity of the histamine H(1)-receptor was increased as reported in our previous paper. We here show that as a culture supplement, sodium selenite enhanced the activity of selenoprotein thioredoxin reductase (TrxR) and the calcium response to histamine stimulation, which were reversed by treating the cells with gold thioglucose, a nucleophilic drug that selectively modifies thiolate/selenolate groups. Sodium selenite most likely caused a reductive shift in the thiol/disulfide redox balance through increasing TrxR activity. In contrast, when the cells were treated with Se in the form of ebselen, a thiol oxidant with peroxidase-like activity, histamine-induced calcium release and calcium entry were significantly suppressed. This effect appeared related to the thiol-directed modification rather than the peroxidase-like activity of ebselen, because this inhibitory effect was not replicated by increasing cellular peroxidase activity. Thus, the opposing effects of sodium selenite and ebselen on histamine-induced calcium signaling are achieved, at least in part, through their opposite actions in modulating the thiol/disulfide redox state.

Penultimate selenocysteine residue replaced by cysteine in thioredoxin reductase from selenium-deficient rat liver.

Selenium is an essential micronutrient for humans and animals, and its deficiency can predispose to the development of pathological conditions. This study evaluates the effect of selenium deficiency on the thioredoxin system, consisting of NADPH, selenoprotein thioredoxin reductase (TrxR), and thioredoxin (Trx); and the glutathione system, including NADPH, glutathione reductase, glutathione, and glutaredoxin coupled with selenoprotein glutathione peroxidase (GPx). We particularly investigate whether inactive truncated TrxR is present under selenium-starvation conditions due to reading of the UGA codon as stop. Feeding rats a selenium-deficient diet resulted in a large decrease in activity of TrxR and GPx in rat liver but not in the levels of Trx1 and Grx1. However, selenium deficiency induced mitochondrial Grx2 10-fold and markedly changed the expression of some flavoproteins that are involved in the cellular folate, glucose, and lipid metabolism. Liver TrxR mRNA was nearly unchanged, but no truncated enzyme was found. Instead, a low-activity form of TrxR with a cysteine substituted for the penultimate selenocysteine in the C-terminal active site was identified in selenium-deficient rat liver. These results show a novel mechanism for decoding the UGA stop codon, inserting cysteine to make a full-length enzyme that may be required for selenium assimilation.

Systematic characterization of the covalent interactions between (-)-epigallocatechin gallate and peptides under physiological conditions by mass spectrometry.

(-)-Epigallocatechin gallate (EGCG) is a major bioactive component in leaves of green tea, and has been widely investigated for its anti-tumor activity. The interaction between EGCG and the key peptides or proteins (e.g. glutathione, enzymes) in vivo is thought to be involved in the toxicity and anti-cancer mechanism of EGCG. However, the true anti-tumor mechanism of EGCG is not clear, and few studies have focused on the reactivity of EGCG toward peptides or proteins under physiological conditions (pH 7.4, 37 degrees C). In this work, the covalent interactions between EGCG and model peptides containing one or more nucleophilic residues (i.e. Arg, Cys, Met, and alpha-NH(2) of the N-terminus of peptides) under physiological condition were fully characterized using mass spectrometry. It was found that EGCG can react with the thiol groups of peptides to form adducts under physiological conditions (pH 7.4, 37 degrees C), even in the absence of the peroxidase/hydrogen peroxide system. Besides the thiol groups of peptides, it is firstly reported that EGCG also reacts with alpha-NH(2) of the N-terminus or arginine residues of model peptides to form Schiff base adducts, and the methionine residues of model peptides can be easily oxidized by hydrogen peroxide (H(2)O(2)) generated during the process of EGCG auto-oxidation to form methionine sulfoxide products. The preference for the reaction of nucleophlic residues of peptides with EGCG was determined to have the following order: Cys > alpha-NH(2) of the N-terminus > Arg. The neutral loss ions of [M+H-170](+) and [M+H-138](+) were detected in all tandem mass spectra of the EGCG adducts of peptides, which indicates that these two neutral loss ions can be considered as the characteristic neutral loss ions of peptides modified by EGCG. Results of the present research provide insights into the toxicology and anti-tumor mechanism of EGCG in vivo.