Arne Holmgren

[21] Thioredoxin and thioredoxin reductase

Publisher Summary This chapter summarizes current methods to determine Trx and thioredoxin reductase (TR). Thioredoxin (Trx) is a small (Mr 12,000) multifunctional and ubiquitous protein characterized by having a redox-active disulfide/dithiol within the conserved active site sequence: -Trp-Cys-Gly-Pro-Cys-. Oxidized thioredoxin (Trx-S 2 ) has a disulfide, and reduced thioredoxin [Trx-(SH) 2 ] has a dithiol. Thioredoxin reductase specifically reduces Trx-S 2 to Trx-(SH) 2 using NADPH. The Trx-(SH) 2 form is a powerful protein disulfide reductase. Thus, Trx, TR, and NADPH, collectively called the thioredoxin system, operate as a powerful NADPH-dependent protein disulfide reductase system. Thioredoxin has been isolated and characterized from a wide variety of prokaryotic and eukaryotic species. Mammalian thioredoxins show about 25% sequence identity to the well-characterized E. coli protein with 108 residues. One classic function of the thioredoxin system is to act as a hydrogen donor for ribonucleotide reductase, which is essential for DNA synthesis. Redox control processes involve changes in the activity of an enzyme, a receptor, or a transcription factor via dithiol/disulfide interchange reactions. Mammalian thioredoxin reductase, with its broader substrate specificity, is likely to be involved in multiple signaling systems for redox control of cellular processes.

THE ROLE OF THE THIOREDOXIN AND GLUTAREDOXIN PATHWAYS IN REDUCING PROTEIN DISULFIDE BONDS IN THE ESCHERICHIA COLI CYTOPLASM

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

The thioredoxin antioxidant system

The thioredoxin (Trx) system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin, is a key antioxidant system in defense against oxidative stress through its disulfide reductase activity regulating protein dithiol/disulfide balance. The Trx system provides the electrons to thiol-dependent peroxidases (peroxiredoxins) to remove reactive oxygen and nitrogen species with a fast reaction rate. Trx antioxidant functions are also shown by involvement in DNA and protein repair by reducing ribonucleotide reductase, methionine sulfoxide reductases, and regulating the activity of many redox-sensitive transcription factors. Moreover, Trx systems play critical roles in the immune response, virus infection, and cell death via interaction with thioredoxin-interacting protein. In mammalian cells, the cytosolic and mitochondrial Trx systems, in which TrxRs are high molecular weight selenoenzymes, together with the glutathione-glutaredoxin (Grx) system (NADPH, glutathione reductase, GSH, and Grx) control the cellular redox environment. Recently mammalian thioredoxin and glutathione systems have been found to be able to provide the electrons crossly and to serve as a backup system for each other. In contrast, bacteria TrxRs are low molecular weight enzymes with a structure and reaction mechanism distinct from mammalian TrxR. Many bacterial species possess specific thiol-dependent antioxidant systems, and the significance of the Trx system in the defense against oxidative stress is different. Particularly, the absence of a GSH-Grx system in some pathogenic bacteria such as Helicobacter pylori, Mycobacterium tuberculosis, and Staphylococcus aureus makes the bacterial Trx system essential for survival under oxidative stress. This provides an opportunity to kill these bacteria by targeting the TrxR-Trx system.

Unraveling the Biological Roles of Reactive Oxygen Species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide

The recent high-resolution solution structures of human and Escherichia coli thioredoxin in their oxidized and reduced states support a catalytic model of protein disulfide reduction involving binding of a target protein and nucleophilic attack by the active-site Cys32 thiolate to form a transition state mixed disulfide.

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2C). Standard state redox potential (E°′) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E°′ resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E°′ values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coliglutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E°′ = −233 and −198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculatedE°′ values from a number of proteins ranging from −270 mV for E. coli Trx to −124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as −240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide

Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia and has potential anticancer activity against a wide range of solid tumors. ATO exerts its effect mainly through elevated oxidative stress, but the exact molecular mechanism remains elusive. The thioredoxin (Trx) system comprising NADPH, thioredoxin reductase (TrxR), and Trx and the glutathione (GSH) system composed of NADPH, glutathione reductase, and GSH supported by glutaredoxin are the two electron donor systems that control cellular proliferation, viability, and apoptosis. Recently, the selenocysteine-dependent TrxR enzyme has emerged as an important molecular target for anticancer drug development. Here, we have discovered that ATO irreversibly inhibits mammalian TrxR with an IC50 of 0.25 μM. Both the N-terminal redox-active dithiol and the C-terminal selenothiol-active site of reduced TrxR may participate in the reaction with ATO. The inhibition of MCF-7 cell growth by ATO was correlated with irreversible inactivation of TrxR, which subsequently led to Trx oxidation. Furthermore, the inhibition of TrxR by ATO was attenuated by GSH, and GSH depletion by buthionine sulfoximine enhanced ATO-induced cell death. These results strongly suggest that the ATO anticancer activity is by means of a Trx system-mediated apoptosis. Blocking cancer cell DNA replication and repair and induction of oxidative stress by the inhibition of both Trx and GSH systems are suggested as cancer chemotherapeutic strategies.

S-Nitrosoglutathione Is Cleaved by the Thioredoxin System with Liberation of Glutathione and Redox Regulating Nitric Oxide

In activated human neutrophils a burst of nitric oxide (NO) converts intracellular GSH to S-nitrosoglutathione (GSNO) which is subsequently cleaved to restore GSH by an unknown mechanism. We discovered that GSNO is an NADPH oxidizing substrate for human or calf thymus thioredoxin reductase (TR) with an apparent Km value of 60 μM and a Kcat of 0.6 × s−1. Addition of human thioredoxin (Trx) stimulated the initial NADPH oxidation rate severalfold but was accompanied by progressive inactivation of TR. Escherichia coli TR lacked activity with GSNO, but with E. coli Trx present, GSNO was reduced without inhibition of the enzyme. Chemically reduced E. coli Trx-(SH)2 was oxidized to Trx-S2 by GSNO with a rate constant of 760 M−1s−1 (7-fold faster than by GSSG) as measured by tryptophan fluorescence. Analysis of this reaction in the presence of oxymyoglobin revealed quantitative formation of metmyoglobin indicative of NO· release. Analysis of GSNO reduction demonstrated that oxidation of NADPH produced a stoichiometric amount of free GSH. These results demonstrate a homolytic cleavage mechanism of GSNO, giving rise to GSH and NO·. GSNO efficiently inhibited the protein disulfide reductase activity of the complete human or calf thymus thioredoxin systems. Our results demonstrate enzymatic cleavage of GSNO by TR or Trx and suggest novel mechanisms for redox signaling.

Thiol redox control via thioredoxin and glutaredoxin systems

The Trx (thioredoxin) and Grx (glutaredoxin) systems control cellular redox potential, keeping a reducing thiol-rich intracellular state, which on generation of reactive oxygen species signals through thiol redox control mechanisms. Here, we give a brief overview of the human Trx and Grx systems. The main part focuses on our current knowledge about mitochondrial Grx2, which facilitates mitochondrial redox homoeostasis during oxidative stress-induced apoptosis.

Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to de-glutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.