Advertisement

Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2–R1-dependent ribonucleotide reductase

  • Author Footnotes
    1 Present address: Amity Institute of Biotechnology, Amity University, Major Arterial Road, Rajarhat, New Town, Kolkata 700135, West Bengal, India.
    Rajib Sengupta
    Footnotes
    1 Present address: Amity Institute of Biotechnology, Amity University, Major Arterial Road, Rajarhat, New Town, Kolkata 700135, West Bengal, India.
    Affiliations
    Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institute, Stockholm SE-17177, Sweden
    Search for articles by this author
  • Lucia Coppo
    Affiliations
    Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institute, Stockholm SE-17177, Sweden
    Search for articles by this author
  • Pradeep Mishra
    Affiliations
    Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institute, Stockholm SE-17177, Sweden
    Search for articles by this author
  • Arne Holmgren
    Correspondence
    To whom correspondence should be addressed: Karolinska Institute, SE 17177 Stockholm, Sweden. Tel.:46-8-52487686; Fax:46-8-7284716
    Affiliations
    Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institute, Stockholm SE-17177, Sweden
    Search for articles by this author
  • Author Footnotes
    1 Present address: Amity Institute of Biotechnology, Amity University, Major Arterial Road, Rajarhat, New Town, Kolkata 700135, West Bengal, India.
    3 The abbreviations used are: RNRribonucleotide reductase[3H]CDPtritium-labeled cytidine 5′-diphosphate[3H]dCDPtritium-labeled deoxycytidine 5′-diphosphate[3H]CMPtritium-labeled cytidine 5′-monophosphateDi-E-GSSGdi-eosin–labeled oxidised glutathioneGrxglutaredoxinGSH-Grx systemglutathione-glutaredoxin systemR1-SG-ER1 subunit glutathionylated by eosin-labeled glutathioneTrxthioredoxinTrxRthioredoxin reductaseGRGSH reductase.
Open AccessPublished:July 02, 2019DOI:https://doi.org/10.1074/jbc.RA119.008752
      Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1–R2 and R1–p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4–6 times higher than that of the Trx1 system. For both complexes, the Vmax values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1–p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.

      Introduction

      Ribonucleotide reductases (RNR)
      The abbreviations used are: RNR
      ribonucleotide reductase
      [3H]CDP
      tritium-labeled cytidine 5′-diphosphate
      [3H]dCDP
      tritium-labeled deoxycytidine 5′-diphosphate
      [3H]CMP
      tritium-labeled cytidine 5′-monophosphate
      Di-E-GSSG
      di-eosin–labeled oxidised glutathione
      Grx
      glutaredoxin
      GSH-Grx system
      glutathione-glutaredoxin system
      R1-SG-E
      R1 subunit glutathionylated by eosin-labeled glutathione
      Trx
      thioredoxin
      TrxR
      thioredoxin reductase
      GR
      GSH reductase.
      catalyze the rate-limiting step in deoxyribonucleotides synthesis required for DNA replication and repair (
      • Aberg A.
      • Hahne S.
      • Karlsson M.
      • Larsson A.
      • Ormö M.
      • Ahgren A.
      • Sjöberg B.M.
      Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli.
      ,
      • Stubbe J.
      Ribonucleotide reductases: the link between an RNA and a DNA world?.
      ) (Fig. 1). The two main antioxidant systems in mammalian cells, thioredoxin and GSH-glutaredoxin, support RNR catalysis. The mammalian class Ia RNR enzyme is composed of two homodimeric subunits: R1 (87 kDa) and R2 (40 kDa), which together form a heterotetrameric active enzyme (
      • Nordlund P.
      • Reichard P.
      Ribonucleotide reductases.
      ). The R1 subunit carries the substrate-binding site, the active site composed of three redox-active cysteine residues, allosteric effector binding sites, and two redox-active thiols in the C-terminal (
      • Holmgren A.
      • Sengupta R.
      The use of thiols by ribonucleotide reductase.
      ) (Fig. 1). The R2 subunit contains a diferric-tyrosyl radical cofactor required for enzyme catalysis. The p53R2, an additional mammalian RNR protein, functions as a catalytic partner of the R1 subunit (
      • Håkansson P.
      • Hofer A.
      • Thelander L.
      Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.
      ,
      • Tanaka H.
      • Arakawa H.
      • Yamaguchi T.
      • Shiraishi K.
      • Fukuda S.
      • Matsui K.
      • Takei Y.
      • Nakamura Y.
      A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage.
      • Bourdon A.
      • Minai L.
      • Serre V.
      • Jais J.P.
      • Sarzi E.
      • Aubert S.
      • Chrétien D.
      • de Lonlay P.
      • Paquis-Flucklinger V.
      • Arakawa H.
      • Nakamura Y.
      • Munnich A.
      • Rötig A.
      Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion.
      ). The mouse p53R2 (40 kDa) showed 81% sequence identity to the mouse R2 subunit at the amino acid level (
      • Pontarin G.
      • Ferraro P.
      • Håkansson P.
      • Thelander L.
      • Reichard P.
      • Bianchi V.
      p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage.
      ). Like R2, the p53R2 subunit also contains a diferric-tyrosyl radical cofactor. In contrast to the S-phase–specific R2 subunit, the p53R2 is induced by DNA damage and the expression is mediated by the tumor suppressor protein p53 (
      • Thelander L.
      Ribonucleotide reductase and mitochondrial DNA synthesis.
      ).
      Figure thumbnail gr1
      Figure 1Thioredoxin and GSH-glutaredoxin systems as electron donors to the RNR. Thioredoxin and glutaredoxin are known electron donors to the RNR that maintains the dNTP pool and thus DNA synthesis. RNR catalyzes the de novo synthesis of 2′-deoxyribonucleotides (dNDPs) from the corresponding ribonucleotides (NDPs). Each cycle of RNR catalysis generates a disulfide in the active site of the R1 subunit, which is reduced by C-terminal dithiols leaving a disulfide to be reduced. Electrons for reduction come ultimately from NADPH via thioredoxin reductase (TR) and Trx, or GR, GSH, and Grx.
      The requirement of dNTPs is widely different during the cell cycle. The RNR concentration and composition change following the requirement of the cell cycle with the R2 subunit being degraded after S-phase. The concentration of R1 subunit instead remains constant also in resting cells. In fact, the R1–p53R2 complex has a crucial role in dNTP supply for basal DNA repair and also for mitochondrial DNA synthesis outside the S-phase (
      • Håkansson P.
      • Hofer A.
      • Thelander L.
      Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.
      ,
      • Pontarin G.
      • Fijolek A.
      • Pizzo P.
      • Ferraro P.
      • Rampazzo C.
      • Pozzan T.
      • Thelander L.
      • Reichard P.A.
      • Bianchi V.
      Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.
      ).
      Inefficient RNR activity may lead to fork retardation, reduction of DNA replication, and blockage of cell cycle. Thus, RNR inhibitors, such as hydroxyurea, act as inducer of replication stress.
      The RNR reaction cycle involves formation of a disulfide in the active site, with the reduction of a substrate ribonucleoside diphosphate (
      • Holmgren A.
      • Sengupta R.
      The use of thiols by ribonucleotide reductase.
      ,
      • Sengupta R.
      • Holmgren A.
      Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase.
      ). In the R1 subunit, the narrow entrance to the catalytic site sterically prohibits a direct reduction of the active-site disulfide by thioredoxin (Trx) or glutaredoxin (Grx). The active-site thiols are regenerated by a pair of thiols in the swinging arm of R1 by a thiol-disulfide exchange reaction (
      • Aberg A.
      • Hahne S.
      • Karlsson M.
      • Larsson A.
      • Ormö M.
      • Ahgren A.
      • Sjöberg B.M.
      Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli.
      ). Then, Trx and GSH-Grx systems are required to reduce the resulting C-terminal disulfide for the next cycle of RNR reaction (
      • Laurent T.C.
      • Moore E.C.
      • Reichard P.
      Enzymatic synthesis of deoxyribonucleotides: IV. isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B.
      ,
      • Holmgren A.
      Glutathione-dependent synthesis of deoxyribonucleotides: characterization of the enzymatic mechanism of Escherichia coli glutaredoxin.
      ). Trx is reduced by thioredoxin reductase (TrxR), whereas Grx is reduced by glutathione (GSH) and GSH reductase (GR) (
      • Lillig C.H.
      • Holmgren A.
      Thioredoxin and related molecules: from biology to health And Disease.
      ,
      • Lillig C.H.
      • Berndt C.
      • Holmgren A.
      Glutaredoxin systems.
      ). Several reports have been published describing the catalytic mechanism of RNR (
      • Gon S.
      • Faulkner M.J.
      • Beckwith J.
      In vivo requirement for glutaredoxins and thioredoxins in the reduction of the ribonucleotide reductases of Escherichia coli.
      • Guittet O.
      • Håkansson P.
      • Voevodskaya N.
      • Fridd S.
      • Gräslund A.
      • Arakawa H.
      • Nakamura Y.
      • Thelander L.
      Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating Cells.
      ,
      • Kashlan O.B.
      • Scott C.P.
      • Lear J.D.
      • Cooperman B.S.
      A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase: functional consequences of ATP- and dATP-induced oligomerization of the large subunit.
      • Wang J.
      • Lohman G.J.
      • Stubbe J.
      Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases.
      ), although the electron transfer flow is still not completely characterized.
      Our results show that both Trx and the GSH-Grx system are able to sustain the RNR activity for both complexes. However, at the physiological concentration of GSH, the Grx system seems more efficient. The role of GSH as a key mediator of cell proliferation has been shown by several groups of researchers. Co-localization of GSH with nuclear DNA was observed in proliferating cells (
      • Thelander L.
      • Reichard P.
      Reduction of ribonucleotides.
      ). In highly purified normal human T cells, GSH modulated proliferation and regulated DNA synthesis (
      • Suthanthiran M.
      • Anderson M.E.
      • Sharma V.K.
      • Meister A.
      Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens.
      ). Depletion of GSH blocks DNA synthesis in mammary carcinoma cells (
      • Dethlefsen L.A.
      • Lehman C.M.
      • Biaglow J.E.
      • Peck V.M.
      Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells.
      ). In a similar study done in 3T3 fibroblast cells, the relationship between the distribution of nuclear GSH and cell cycle progression was observed (
      • Markovic J.
      • Mora N.J.
      • Broseta A.M.
      • Gimeno A.
      • de-la-Concepción N.
      • Viña J.
      • Pallardó F.V.
      The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.
      ). Thus, these data emphasize the role of the GSH-Grx system in RNR activity. Considering also that the R1–p53R2 complex has higher affinity for the GSH-Grx system, these data may be used to better understand replication stress, tumor cell growth, and treatment.

      Results

      Characterization of mammalian R1–p53R2 compared with the R1–R2 complex

      A common way to get active RNR is to use DTT as electron donor (
      • Aberg A.
      • Hahne S.
      • Karlsson M.
      • Larsson A.
      • Ormö M.
      • Ahgren A.
      • Sjöberg B.M.
      Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli.
      ,
      • Stubbe J.
      Ribonucleotide reductases: the link between an RNA and a DNA world?.
      ). To investigate the effect of DTT for mammalian RNR catalysis, experiments were carried out with R1–R2 and R1–p53R2 complexes (Fig. 2, A and B). The active RNR heterotetramer was formed by mixing 200 μg/ml of R1 with 100 μg/ml of R2 (Fig. 2A) or p53R2 (Fig. 2B) in the presence of Mg2+ and ATP as cofactors. RNR reduced [3H]CDP to [3H]dCDP with maximum activity at 10 mm DTT for the R1–R2 complex as previously described (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ) and 2.5 mm DTT for the R1–p53R2 complex. Inhibition by higher concentrations of DTT was observed for both the RNR complexes but was much more pronounced for R1–p53R2; in fact this complex lost half of the activity at 15 mm, whereas the corresponding R1–R2 complex has 50% of maximal activity at 40 mm DTT.
      Figure thumbnail gr2
      Figure 2A and B, effect of DTT on RNR activity. Enzyme activity of 200 μg/ml of R1 with 100 μg/ml of R2 (A) or 100 μg/ml of p53R2 (B) were measured in the presence of increasing amounts of DTT with standard assay conditions. C and D, effect of physiological reductants on RNR activity. Enzyme activity of 200 μg/ml of R1 with different concentrations of R2 (C) and p53R2 (D) were measured in the presence of 5 μm Trx1, 1 mm NADPH, and 0.1 μm TR as the Trx system (closed circles) or with 1 μm Grx1, 1 mm NADPH, 5 mm GSH, and 0.1 μm GR as the Grx system (open circles). Results are representative of three independent experiments. The y axis represents the nanomoles of dCDP formed after 30 min of incubation or enzymatic reaction.
      We titrated the concentrations of R2 and p53R2 using the physiological electron donors for RNR, such as Trx and Grx-GSH systems to check if increasing concentrations of p53R2 were leading to a similar activity as the complex of R1–R2. In Fig. 2, C and D, it is possible to see that both R2 and p53R2 reached saturation at 200 μg/ml concentrations (1:2 molar ratio R1:R2; R1:p53R2) but the R1–p53R2 complex had a lower enzymatic catalytic efficiency.

      Thioredoxin and GSH-glutaredoxin systems as electron donors for RNR complexes

      Different concentrations of Trx (0.01–10 μm) (Fig. 3, A and B) and Grx1 (0.1–1 μm) (Fig. 3, C and D) were used to measure RNR activity. The result of the kinetic parameters are summarized in Table 2. The Trx system as electron donor showed a five times higher value of Vmax for the R1–R2 complex and a typical apparent Km value of 1.4 μm (Fig. 3A), whereas the R1–p53R2 complex had a Km equal to 0.7 μm (Fig. 3B).
      Figure thumbnail gr3
      Figure 3A and B, RNR activity with the Trx system as electron donor. Trx1, 1 mm NADPH, and 0.1 μm TR were mixed with 200 μg/ml of R1 and 100 μg/ml of R2. The y axis represents the nanomoles of dCDP formed after 30 min of incubation (A) or 100 μg/ml of p53R2 (B). C and D, RNR activity with the GSH-Grx system as electron donor. Grx1, 1 mm NADPH, 0.1 μm GR, and different concentrations of GSH were mixed with 200 μg/ml of R1 and 100 μg/ml of R2 (C) or 100 μg/ml of p53R2 (D). Results are representative of five independent experiments.
      Table 2Kinetic constants of RNR complexes with Trx and Grx systems
      R1–R2kcat/KmR1–p53R2kcat/Km
      VmaxKmkcatVmaxKmkcat
      nm/sμms−1μm−1 s−1nm/sμms−1μm−1 s−1
      Trx199.331.40.0450.03219.220.700.00870.012
      Grx143.330.150.0200.13113.330.080.00610.076
      Grx235.110.30.0160.05313.660.210.00620.030
      Grx2C40S23.660.360.0110.03012.770.380.00580.015
      The Km value for the R1–R2 complex with varying concentrations of human Grx1 in combination with 5 or 10 mm GSH, NADPH, and excess GR (Fig. 3C) was equal to 0.15 μm, in agreement with previously reported data (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ). However, we obtained a 1.9-fold higher value of Vmax compared with the previous report (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ). The same experiment was performed with the R1–p53R2 complex (Fig. 3D) and it displayed an apparent Km value of 0.08 μm for Grx1.
      The specific activities, apparent Vmax, and corresponding kcat were found to be lower for the GSH-Grx1 system compared with the Trx1 system for both of the RNR complexes (Table 1, Table 2). However, the catalytic efficiency (kcat/Km) was higher for GSH-Grx1, having lower Km values for both RNR complexes (Table 2).
      Table 1Specific activities of RNR complexes
      Specific activity
      R1–R2R1–p53R2
      nmol [dCDP]/min/mg of R1
      DTT66.6618.00
      Trx system59.6011.53
      Grx1 system (5 mm GSH)26.336.66
      Grx1 system (10 mm GSH)39.8614.46
      Mammalian cells also express a cytosolic isoform of Grx1, named Grx2, with similar enzymatic properties but a different active-site motif (
      • Lundberg M.
      • Johansson C.
      • Chandra J.
      • Enoksson M.
      • Jacobsson G.
      • Ljung J.
      • Johansson M.
      • Holmgren A.
      Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.
      ,
      • Gladyshev V.N.
      • Liu A.
      • Novoselov S.V.
      • Krysan K.
      • Sun Q.A.
      • Kryukov V.M.
      • Kryukov G.V.
      • Lou M.F.
      Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2.
      ). We wanted to test if R1–p53R2 is a substrate also of this other Grx isoform. We performed experiments with different concentrations of Grx2 and also with the active-site mutant Grx2C40S that has been shown to operate via a monothiol mechanism with the R1–R2 complex (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ) (Fig. 4). Grx2 was able to support the activity of both mammalian RNR complexes. The Km values (Table 2) were higher for Grx2 in the R1–R2 complex, 0.3 μm, compared with R1–p53R2 complex, 0.21 μm. The C40S mutant of Grx2 was found to act with similar efficiency for both RNR complexes (Km values of 0.36 and 0.38 μm, respectively).
      Figure thumbnail gr4
      Figure 4RNR activity using different reducing systems. Samples with 200 μg/ml of R1 and 100 μg/ml of R2 (A) or 100 μg/ml of p53R2 (B) were used for the assays with different reductants as indicated in each column. The GSH-Grx system contained 1 μm Grx1 or Grx2 or Grx2C40S, 5 mm GSH, 1 mm NADPH, and 0.1 μm GR. The Trx system contained 3.6 μm Trx1 with 1 mm NADPH and 0.1 μm TrxR. The results are expressed as the mean ± S.E. of two independent experiments performed in triplicates.

      Importance of GSH concentration in mammalian RNR catalysis

      From the results of Fig. 3, C and D, we saw that increased concentrations of GSH lead to an increase of RNR activity. The importance of reduced GSH was confirmed by running the RNR assay with 5 mm GSH but in the absence of GR (Table 3). Moreover, we titrated GSH (0.5–20 mm) in the presence of 1 μm Grx1 plus excess GR and 1 mm NADPH (Fig. 5, A and B). The Km value for GSH was found to be higher than previously reported for the R1–R2 complex (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ). The R1–p53R2 complex showed again a lower Km value compared with R1–R2 complex (Fig. 5B).
      Table 3Specific activities of human R1–p53R2 complex in presence and absence glutathione reductase
      Specific activity
      nmol [dCDP]/min/mg of R1
      R1-p53R2 + GSH-Grx system without human GR0.71 ± 0.44
      R1-p53R2 + GSH-Grx system with1 μm human GR5.7 ± 0.56
      Figure thumbnail gr5
      Figure 5A–C, effect of GSH on mouse RNR activity. Enzyme activity of 200 μg/ml of R1 with 100 μg/ml of R2 (A) or 100 μg/ml of p53R2 (B) were measured in the presence of increasing amounts of GSH, 1 μm Grx1, 1 mm NADPH, and 0.1 μm GR. Each point represents the mean value of two independent experiments with duplicate samples. Enzyme activities of 200 μg/ml of R1 with 100 μg/ml of R2 were measured with 5 μm Trx1 (open circles) and 10 μm Trx1 (closed circles) in the presence of freshly prepared GSH, 1 mm NADPH, 0.1 μm GR (n = 2, mean ± S.E.) (C). D and E, glutathionylation of R1. 1 μm pre-reduced R1 was treated with 20 μm Di-E-GSSG and the release of E-GSH was monitored with an emission at 545 nm after excitation at 520 nm (black circles). Fluorescent background intensity from Di-E-GSSG was used as control (white circles) (E). R1 subunit was treated with Di-E-GSSG. After incubation, samples were run on a nonreducing SDS-PAGE. After gel electrophoresis, R1-SG-E were visualized on a UV transilluminator. The gel was subsequently stained with Coomassie Blue staining solution. Lane 1, untreated R1; lane 2, R1-SG-E; lane 3, DTT-treated R1-SG-E; lane 4, Grx1 system-treated R1-SGE. The Grx1 system contained 1 μm Grx1, 1 mm NADPH, 5 mm GSH, and 0.1 μm GR. Results are representative of three independent experiments (insets in D and ). Similarly, 2.5 (triangle) and 5 μm (black circle) of C-terminal mutant peptide R1C787S was treated with 20 μm Di-E-GSSG and the release of E-GSH was measured at 545 nm after excitation at 520 nm. The fluorescence increase of Di-E-GSSG (white circle) was used as a background.
      To further verify the role of GSH for RNR catalysis, the R1–R2 complex was incubated with Trx and GSH in the presence of GR and NADPH (Fig. 5C). RNR activity increased with increasing concentrations of GSH. The result suggests that GSH could maintain the level of reduced Trx to support the RNR catalysis. Altogether, the results clearly show that GSH is necessary for the reduction of both mammalian RNR complexes.
      To study glutathionylation of the R1 subunit, pre-reduced R1 was incubated with eosin-labeled GSSG called Di-E-GSSG and the release of E-GSH in the reaction mixture was monitored by measuring fluorescence at 545 nm after excitation at 520 nm (Fig. 5D). Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. The fluorescence values increased over time, suggesting a reaction between R1 and Di-E-GSSG with release of the highly fluorescent E-GSH molecule.
      We were able to visualize the glutathionylated R1 in SDS-PAGE because the eosin group can be detected when exposed to UV light (
      • Coppo L.
      • Montano S.J.
      • Padilla A.C.
      • Holmgren A.
      Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.
      ). The glutathionylated R1 (R1-SG-E) was detected only in the Di-E-GSSG–treated samples and a subsequent incubation with either DTT or Grx1 systems was able to deglutathionylate R1-SG-E (Fig. 5D, inset). Also addition of the Grx2 system showed a similar effect (data not shown).
      Considering that the R1 subunit has Cys residues exposed not only in the swinging arm, we wanted to test if the thiols in the C-terminal part, Cys-787 and Cys-790, which are active to reduce the R1 active site, can be glutathionylated. We used the peptide corresponding to the C-terminal part of R1, which had mutations of Cys-787 or Cys-790 to Ser. The C-terminal C787S peptide was treated with Di-E-GSSG,and the release of E-GSH in the reaction mixture was monitored by measuring an increase in fluorescence (Fig. 5E). The increase in fluorescence over time suggests the glutathionylation of Cys-790 with the concomitant release of free E-GSH. Similar results were obtained when the same reaction was performed with the C790S peptide (data not shown) suggesting the glutathionylation of Cys-787.

      Discussion

      A balanced reduced cellular redox environment is essential to maintain normal physiological functions. Within cells, enzymes containing redox-active cysteines play a crucial role in regulating different signaling events. The regulatory and catalytic subunit (R1) of RNR complexes has two different classes of redox-active cysteines that control the enzyme activity. Trx and Grx systems play crucial roles in keeping the swinging arm cysteines of R1 reduced to maintain a balanced supply of the dNTPs for proper DNA replication and repair. Unbalanced dNTP pools result in replication stress, misincorporation of nucleotides into DNA, mutations, and cell death. As cells enter S-phase of the cell cycle, the dNTP pools increase severalfold. This event is possible because of increased synthesis of R1 and induction of R2 (
      • Nordlund P.
      • Reichard P.
      Ribonucleotide reductases.
      ). In contrast, the p53R2 protein is present constantly at low levels in both nonproliferating and proliferating cells (
      • Håkansson P.
      • Hofer A.
      • Thelander L.
      Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.
      ,
      • Guittet O.
      • Håkansson P.
      • Voevodskaya N.
      • Fridd S.
      • Gräslund A.
      • Arakawa H.
      • Nakamura Y.
      • Thelander L.
      Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating Cells.
      ).
      The active-site cleft in R1 between the barrel and N-terminal domain does not allow Trx or Grx to enter in the active-site pocket to reduce the disulfide bond between Cys-218 and Cys-444, which is formed after substrate reduction (Fig. 6). Two thiols located at the C-terminal tail (Cys-787 and Cys-790 in mouse R1) reduce that disulfide bond formed in the active site (Fig. 6). The critical role for the C-terminal cysteine pair (in a CXXXXC or CXXC motif) of R1 subunit in regeneration of its active site have been suggested by in vitro mutagenesis and kinetic studies (
      • Aberg A.
      • Hahne S.
      • Karlsson M.
      • Larsson A.
      • Ormö M.
      • Ahgren A.
      • Sjöberg B.M.
      Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli.
      ,
      • Lin A.N.
      • Ashley G.W.
      • Stubbe J.
      Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.
      ,
      • Mao S.S.
      • Holler T.P.
      • Yu G.X.
      • Bollinger Jr., J.M.
      • Booker S.
      • Johnston M.I.
      • Stubbe J.
      A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.
      ). Moreover, interallelic complementation studies with R1 from Saccharomyces cerevisiae demonstrated that the C terminus of one monomer acts in trans to regenerate the active site of its neighboring monomeric subunit (
      • Zhang Z.
      • Yang K.
      • Chen C.C.
      • Feser J.
      • Huang M.
      Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.
      ). The last 24 residues at the C-terminal tail, including the shuttle dithiols, are not visible in the electron density map. For the Escherichia coli R1 subunit, the distance from the last visible residue, 737 to Cys-225 is about 30 Å. This unique arrangement of the flexible C-terminal tail allows the enzyme to retain a buried active site for the substrate reduction.
      Figure thumbnail gr6
      Figure 6Structure of human R1 dimer. A, each R1 subunit contains one catalytic site and two allosteric regulatory sites. The allosteric specificity site regulates the substrate preference and the allosteric activity site regulates the ribonucleotide reductase activity. Adapted and modified from Ref.
      • Fairman J.W.
      • Wijerathna S.R.
      • Ahmad M.F.
      • Xu H.
      • Nakano R.
      • Jha S.
      • Prendergast J.
      • Welin R.M.
      • Flodin S.
      • Roos A.
      • Nordlund P.
      • Li Z.
      • Walz T.
      • Dealwis C.G.
      Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization.
      . B, model of reduction of RNR by the Trx and GSH-Grx system: for simplicity, interaction of only one active site and one CXXC motif are shown. The active-site pocket of each R1 subunit is shown in a circle. The cysteine residue (−SH) in the active site of R1 is converted to a transient thiyl radical through electron transfer from the tyrosyl radical located in R2. At the completion of each turnover cycle, a disulfide bond is formed between the conserved cysteine pair at the active site (1). Reduction of the R1 active site is mediated by the C-terminal CXXC motif of the neighboring subunit (2). The resulting disulfide bond is reduced by Trx and Grx systems through disulfide-exchange resulting in an active R1 (3 → 4). The reaction leads the RNR reaction cycle to continue (4 → 1). The role of the Grx system for a monothiol mechanism involving glutathionylation is also shown (3 → 5 and 5 → 4). Adapted from Ref.
      • Sengupta R.
      • Holmgren A.
      Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase.
      .
      The efficiency of the Trx1 and Grx1 system as electron donor for RNR activity is summarized (Table 1, Table 2) for both of the mammalian RNR complexes. In mammalian cells, the R1–R2 complex supports the S-phase–specific DNA replication, whereas the R1–p53R2 complex performs the RNR activity in G0/G1 phase cells. Our data shows that the R1–p53R2 complex has 3–5 times lower activity compared with R1–R2 complex (Table 1). Thus, low constitutive levels of p53R2 with low levels of R1 subunit in mammalian cells provide the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis during the G0/G1 phase. Our study suggests that the Trx1 system, having a higher kcat, has the advantage to act as an electron donor for the RNR complexes. However, Grx1 has a higher catalytic efficiency (kcat/Km) compared with Trx1 with both RNR complexes. Interestingly, the two main cytosolic Grx isoforms showed activity with both RNR complexes. The C40S mutant of Grx2 that act with a monothiol mechanism was found to act as an efficient mediator also for R1–p53R2 activity (Fig. 4B). Here, it is noteworthy to mention that the bacterial monothiol mutant of Grx1 showed no activity with E. coli RNR. Recently, it has been shown that human monothiol Grx5 is active as a GSH-dependent electron donor for mammalian ribonucleotide reductase (
      • Johansson C.
      • Roos A.K.
      • Montano S.J.
      • Sengupta R.
      • Filippakopoulos P.
      • Guo K.
      • von Delft F.
      • Holmgren A.
      • Oppermann U.
      • Kavanagh K.L.
      The crystal structure of human GLRX5: iron–sulfur cluster co-ordination, tetrameric assembly and monomer activity.
      ). However, the apparent Vmax and specific activity with the RNR complexes were found to be much lower for Grx5 compared with Grx1 or Grx2. Our results confirm the glutathionylation mechanism for Grx catalysis in contrast to the dithiol mechanism for Trx (Fig. 6). The treatment with Di-E-GSSG resulted in glutathionylation of the R1 subunit, which could be detected by the increase in fluorescence values (Fig. 5, D and E). Furthermore, the glutathionylated R1 (R1-SG-E) was also detected in SDS-PAGE with the help of a UV transilluminator and the R1-SG-E was found to be deglutathionylated in the presence of either DTT or the complete Grx1 system (Fig. 5D, insets). Moreover, the C787S peptide of the R1 subunit was found to be glutathionylated by Di-E-GSSG, which suggests the glutathionylation of Cys-790 (Fig. 5E). In a similar way, the C790S peptide of the R1 subunit was also found to be glutathionylated (data not shown), suggesting the glutathionylation of Cys-787. Thus, both C-terminal dithiols of the R1 subunit can be glutathionylated, which could be regulated by the Grx-GSH system.
      Our study also shows that GSH can reduce Trx1 directly to support RNR catalysis and the RNR activity was very much dependent on the concentrations of GSH (Fig. 5C). This is in agreement with a recent study where it has been shown that both GSH and Grx1 directly and effectively reduced Trx1 (
      • Du Y.
      • Zhang H.
      • Lu J.
      • Holmgren A.
      Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose.
      ). Interestingly, it was found that normal mice and mice having TrxR1-deficient hepatocytes showed similar liver growth rates during development and regeneration (
      • Rollins M.F.
      • van der Heide D.M.
      • Weisend C.M.
      • Kundert J.A.
      • Comstock K.M.
      • Suvorova E.S.
      • Capecchi M.R.
      • Merrill G.F.
      • Schmidt E.E.
      Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration.
      ). Thus, in mouse hepatocytes, a TrxR1-independent electron transfer pathway to RNR is most likely dependent on GSH, and can support DNA replication. Similarly, it was shown that the TrxR1-lacking lymphomas can initiate and progress normally in mice (
      • Mandal P.K.
      • Schneider M.
      • Kölle P.
      • Kuhlencordt P.
      • Förster H.
      • Beck H.
      • Bornkamm G.W.
      • Conrad M.
      Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation.
      ). A critical experiment demonstrating that the thioredoxin system is not essential for DNA replication has recently been published by Prigge et al. (
      • Prigge J.R.
      • Coppo L.
      • Martin S.S.
      • Ogata F.
      • Miller C.G.
      • Bruschwein M.D.
      • Orlicky D.J.
      • Shearn C.T.
      • Kundert J.A.
      • Lytchier J.
      • Herr A.E.
      • Mattsson Å.
      • Taylor M.P.
      • Gustafsson T.N.
      • Arnér E.S.J.
      • Holmgren A.
      • Schmidt E.E.
      Hepatocyte hyperproliferation upon liver-specific co-disruption of thioredoxin-1, thioredoxin reductase-1, and glutathione reductase.
      ). The authors used a conditional knockout of Trx, TrxR, and GR in hepatocytes and the resulting mice with co-disruption of all three proteins surprisingly showed a 2–3–fold larger liver per body mass with hyper-proliferation. When GSH synthesis was inhibited by addition of BSO the mice died from liver necrosis. These results demonstrate the importance of the glutaredoxin system for DNA synthesis. Another important result particularly regarding RNR in tumor tissues comes from results presented in an article by Hammond and co-workers (
      • Foskolou I.P.
      • Jorgensen C.
      • Leszczynska K.B.
      • Olcina M.M.
      • Tarhonskaya H.
      • Haisma B.
      • D'Angiolella V.
      • Myers W.K.
      • Domene C.
      • Flashman E.
      • Hammond E.M.
      Ribonucleotide reductase requires subunit switching in hypoxia to maintain DNA replication.
      ). These authors showed that in hypoxic conditions the R1–p53R2 is the predominant subunit during DNA replication because p53R2 is better suited to retain activity during hypoxia. A further insight in the role of Grx is shown by results that Grx-1 silencing induces cell senescence via the p53/p21/p16b signaling axis (
      • Yang F.
      • Yi M.
      • Liu Y.
      • Wang Q.
      • Hu Y.
      • Deng H.
      Glutaredoxin-1 silencing induces cell senescence via p53/p21/p16 signaling axis.
      ). In conclusion our results show that the p53R2 has better activity with the GSH-Grx system and that makes p53R2 and the GSH-Grx system an important new anticancer therapeutic target to be further investigated.

      Experimental procedures

      Materials

      [3H]CMP and [3H]CDP were obtained from Moravek Biochemicals and Radiochemicals, respectively. The cation exchanger resin AG-50W® was purchased from Bio-Rad. Yeast GSH reductase, GSH, DTT, NADPH, and insulin were from Sigma. Di-Eosin-GSSG was purchased form IMCO, Sweden. Human Trx1, Grx1, Grx2, and Grx2C40S were prepared as described previously (
      • Lundberg M.
      • Johansson C.
      • Chandra J.
      • Enoksson M.
      • Jacobsson G.
      • Ljung J.
      • Johansson M.
      • Holmgren A.
      Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.
      ,
      • Johansson C.
      • Lillig C.H.
      • Holmgren A.
      Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase.
      ,
      • Ren X.
      • Bjöernstedt M.
      • Shen B.
      • Ericson M.L.
      • Holmgren A.
      Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione.
      ). Rat recombinant TrxR was a kind gift from Dr. Elias Arner (
      • Rengby O.
      • Johansson L.
      • Carlson L.A.
      • Serini E.
      • Vlamis-Gardikas A.
      • Kårsnäs P.
      • Arnér E.S.
      Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.
      ).

      Conversion of [3H]CMP to [3H]CDP

      E. coli CMP kinase plasmid was a kind gift from Prof. Anne-Marie Gilles (Laboratoire d'Enzymologie et de Biochimie Structurales, France). E. coli CMP kinase was expressed, purified, and used to convert [3H]CMP to [3H]CDP as described by Bucurenci et al. (
      • Bucurenci N.
      • Sakamoto H.
      • Briozzo P.
      • Palibroda N.
      • Serina L.
      • Sarfati R.S.
      • Labesse G.
      • Briand G.
      • Danchin A.
      • Barzu O.
      • Gilles A.M.
      CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.
      ). The [3H]CDP was separated from the reaction mixture by a C18 HPLC column using a mobile phase comprising 20 mm ammonium acetate buffer, pH 5, and 5% acetonitrile (data not shown).

      Expression and purification of mouse glutaredoxin

      Expression and purification of Grx1, Grx2, and Grx2C40S were done as previously described (
      • Lundberg M.
      • Johansson C.
      • Chandra J.
      • Enoksson M.
      • Jacobsson G.
      • Ljung J.
      • Johansson M.
      • Holmgren A.
      Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.
      ).

      Expression and purification of RNR subunits

      The E. coli strains expressing mouse R1, R2, and p53R2 were kindly provided by Dr. Lars Thelander (Umeå University, Umeå, Sweden). Recombinant proteins were expressed and purified as previously described (
      • Guittet O.
      • Håkansson P.
      • Voevodskaya N.
      • Fridd S.
      • Gräslund A.
      • Arakawa H.
      • Nakamura Y.
      • Thelander L.
      Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating Cells.
      ,
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ,
      • Davis R.
      • Thelander M.
      • Mann G.J.
      • Behravan G.
      • Soucy F.
      • Beaulieu P.
      • Lavallée P.
      • Gräslund A.
      • Thelander L.
      Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.
      ,
      • Mann G.J.
      • Gräslund A.
      • Ochiai E.
      • Ingemarson R.
      • Thelander L.
      Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.
      ).

      C-terminal peptides of R1

      C-terminal peptides of R1 subunit were purchased from Caslo Laboratory, Denmark. The peptide represents the last 10 amino acids (mutation at Cys-787 or Cys-790) of the C-terminal tail of mouse R1 subunit. The two monothiol mutant peptides (C790S and C787S) have sequences of NREECLMSGS (1125.61 Da) and NREESLMCGS (1124.70 Da), respectively.

      Ribonucleotide reductase activity assay

      Determination of RNR activities were carried out as described previously (
      • Zahedi Avval F.
      • Holmgren A.
      Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
      ). Briefly, the RNR system was reconstituted by mixing recombinant R1 and R2 or p53R2 proteins. Activity was assayed following the conversion of [3H]CDP into [3H]dCDP. R1 and R2 or p53R2 proteins were pre-incubated with 2 mm ATP and 10 mm MgCl2 at 37 °C for 15 min and the reaction was initiated by adding a reaction mixture containing 40 mm Tris-HCl buffer, pH 7.6, 2 mm ATP, 10 mm MgCl2, 200 mm KCl, 20 μm FeCl3, and 0.5 mm [3H]CDP (10,000 cpm/nmol) in a final volume of 50 μl. Incubation was carried out at 37 °C for 30 min. The reaction was stopped by the addition of 1 m perchloric acid. 100 μl of 10 mm dCMP was added to determine the recovery of the product. To hydrolyze CDP and dCDP into CMP and dCMP, respectively, the samples were incubated in boiling water for 10 min. The amount of radioactivity derived from [3H]dCMP was quantified by liquid scintillation counting after ion exchange chromatography on Dowex-50 columns (
      • Nordlund P.
      • Reichard P.
      Ribonucleotide reductases.
      ). As reductant, 5 mm DTT or 5 μm Trx1 and 0.2 μm TrxR plus 1 mm NADPH were used in separate experiments. When the Grx1 system was used together with RNR, the samples contained 1 μm Grx1 (or Grx2 or Grx2C40S), 0.15 μm GR, 1 mm NADPH, and 5 mm GSH. To avoid oxidation of GSH to GSSG, each time a freshly prepared stock of GSH was titrated to pH 7.0. The enzyme activity was measured as nanomole of dCDP produced after 30 min of reaction. The activity data were analyzed with Prism 8 (GraphPad Software, Inc.) and fitted with Michaelis-Menten kinetics for calculation of catalytically efficiency of the RNR complex with various reducing system.

      Glutathionylation of R1 subunit and peptides

      To study the glutathionylation of R1, Di-E-GSSG was used to glutathionylate R1 protein and C-terminal peptides of R1 (
      • Coppo L.
      • Montano S.J.
      • Padilla A.C.
      • Holmgren A.
      Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.
      ,
      • Montano S.J.
      • Lu J.
      • Gustafsson T.N.
      • Holmgren A.
      Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.
      ). Di-E-GSSG has quenched fluorescence in the disulfide form that increases up to 20-fold on reduction of its disulfide bond and formation of E-GSH. Black 96-well-plates were used in a PerkinElmer Victor3 multilabel counter containing a final well volume of 200 μl in 0.1 m potassium phosphate buffer (pH 7.5), 1 mm EDTA. The reaction was started by addition of 20 μm Di-E-GSSG to R1 or C-terminal peptide solutions, followed by recording the fluorescence emission at 545 nm after excitation at 520 nm. Controls where no R1 protein or peptide was added were used as fluorescent background.
      To confirm the glutathionylation of R1 with a different method, R1 subunit was incubated with Di-E-GSSG. Aliquots of S-glutathionylated R1 (R1-SG-E) were further treated with 10 mm DTT or with the complete GSH-Grx system. All samples were run on a nonreducing SDS-PAGE 4–12% acrylamide. The gel was exposed to UV transilluminator to visualize eosin-tagged glutathionylated protein. The same gels were later stained with Coomassie Blue staining.

      Author contributions

      R. S. and A. H. conceptualization; R. S. data curation; R. S., L. C., and P. M. software; R. S. formal analysis; R. S. and A. H. methodology; R. S., L. C., P. M., and A. H. writing-review and editing; L. C. and P. M. visualization; A. H. supervision; A. H. funding acquisition; A. H. investigation; A. H. writing-original draft.

      Acknowledgments

      We gratefully thank Rolf Eliasson for excellent technical assistance and Jacek Andrzejewski for large-scale cultivation of bacteria. We thank Prof. Lars Thelander for providing the plasmids encoding the RNR subunits and Prof. Pär Nordlund for human R1. The gift of TrxR by Dr. Elias Arnér is also acknowledged.

      Supplementary Material

      References

        • Aberg A.
        • Hahne S.
        • Karlsson M.
        • Larsson A.
        • Ormö M.
        • Ahgren A.
        • Sjöberg B.M.
        Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli.
        J. Biol. Chem. 1989; 264 (2663852): 12249-12252
        • Stubbe J.
        Ribonucleotide reductases: the link between an RNA and a DNA world?.
        Curr. Opin. Struct. Biol. 2000; 10 (11114511): 731-736
        • Nordlund P.
        • Reichard P.
        Ribonucleotide reductases.
        Annu. Rev. Biochem. 2006; 75 (16756507): 681-706
        • Holmgren A.
        • Sengupta R.
        The use of thiols by ribonucleotide reductase.
        Free Radic. Biol. Med. 2010; 49 (20851762): 1617-1628
        • Håkansson P.
        • Hofer A.
        • Thelander L.
        Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.
        J. Biol. Chem. 2006; 281 (16436374): 7834-7841
        • Tanaka H.
        • Arakawa H.
        • Yamaguchi T.
        • Shiraishi K.
        • Fukuda S.
        • Matsui K.
        • Takei Y.
        • Nakamura Y.
        A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage.
        Nature. 2000; 404 (10716435): 42-49
        • Bourdon A.
        • Minai L.
        • Serre V.
        • Jais J.P.
        • Sarzi E.
        • Aubert S.
        • Chrétien D.
        • de Lonlay P.
        • Paquis-Flucklinger V.
        • Arakawa H.
        • Nakamura Y.
        • Munnich A.
        • Rötig A.
        Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion.
        Nat. Genet. 2007; 39 (17486094): 776-780
        • Pontarin G.
        • Ferraro P.
        • Håkansson P.
        • Thelander L.
        • Reichard P.
        • Bianchi V.
        p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage.
        J. Biol. Chem. 2007; 282 (17416930): 16820-16828
        • Thelander L.
        Ribonucleotide reductase and mitochondrial DNA synthesis.
        Nat. Genet. 2007; 39 (17534360): 703-704
        • Pontarin G.
        • Fijolek A.
        • Pizzo P.
        • Ferraro P.
        • Rampazzo C.
        • Pozzan T.
        • Thelander L.
        • Reichard P.A.
        • Bianchi V.
        Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18997010): 17801-17806
        • Sengupta R.
        • Holmgren A.
        Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase.
        World J. Biol. Chem. 2014; 5 (24600515): 68-74
        • Laurent T.C.
        • Moore E.C.
        • Reichard P.
        Enzymatic synthesis of deoxyribonucleotides: IV. isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B.
        J. Biol. Chem. 1964; 239 (14245400): 3436-3444
        • Holmgren A.
        Glutathione-dependent synthesis of deoxyribonucleotides: characterization of the enzymatic mechanism of Escherichia coli glutaredoxin.
        J. Biol. Chem. 1979; 254 (34620): 3672-3678
        • Lillig C.H.
        • Holmgren A.
        Thioredoxin and related molecules: from biology to health And Disease.
        Antioxid. Redox Signal. 2007; 9 (17115886): 25-47
        • Lillig C.H.
        • Berndt C.
        • Holmgren A.
        Glutaredoxin systems.
        Biochim. Biophys. Acta. 2008; 1780: 1304-1317
        • Gon S.
        • Faulkner M.J.
        • Beckwith J.
        In vivo requirement for glutaredoxins and thioredoxins in the reduction of the ribonucleotide reductases of Escherichia coli.
        Antioxid. Redox Signal. 2006; 8 (16771665): 735-742
        • Guittet O.
        • Håkansson P.
        • Voevodskaya N.
        • Fridd S.
        • Gräslund A.
        • Arakawa H.
        • Nakamura Y.
        • Thelander L.
        Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating Cells.
        J. Biol. Chem. 2001; 276 (11517226): 40647-40651
        • Kashlan O.B.
        • Scott C.P.
        • Lear J.D.
        • Cooperman B.S.
        A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase: functional consequences of ATP- and dATP-induced oligomerization of the large subunit.
        Biochemistry. 2002; 41 (11781084): 462-474
        • Wang J.
        • Lohman G.J.
        • Stubbe J.
        Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17726094): 14324-14329
        • Thelander L.
        • Reichard P.
        Reduction of ribonucleotides.
        Annu. Rev. Biochem. 1979; 48 (382982): 133-158
        • Suthanthiran M.
        • Anderson M.E.
        • Sharma V.K.
        • Meister A.
        Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens.
        Proc. Natl. Acad. Sci. U.S.A. 1990; 87 (1970635): 3343-3347
        • Dethlefsen L.A.
        • Lehman C.M.
        • Biaglow J.E.
        • Peck V.M.
        Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells.
        Radiat. Res. 1988; 114 (3375425): 215-224
        • Markovic J.
        • Mora N.J.
        • Broseta A.M.
        • Gimeno A.
        • de-la-Concepción N.
        • Viña J.
        • Pallardó F.V.
        The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.
        PLoS ONE. 2009; 4 (19641610): e6413
        • Zahedi Avval F.
        • Holmgren A.
        Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S phase ribonucleotide reductase.
        J. Biol. Chem. 2009; 284 (19176520): 8233-8240
        • Lundberg M.
        • Johansson C.
        • Chandra J.
        • Enoksson M.
        • Jacobsson G.
        • Ljung J.
        • Johansson M.
        • Holmgren A.
        Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.
        J. Biol. Chem. 2001; 276 (11297543): 26269-26275
        • Gladyshev V.N.
        • Liu A.
        • Novoselov S.V.
        • Krysan K.
        • Sun Q.A.
        • Kryukov V.M.
        • Kryukov G.V.
        • Lou M.F.
        Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2.
        J. Biol. Chem. 2001; 276 (11397793): 30374-30380
        • Coppo L.
        • Montano S.J.
        • Padilla A.C.
        • Holmgren A.
        Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.
        Anal. Biochem. 2016; 499 (26836485): 24-33
        • Lin A.N.
        • Ashley G.W.
        • Stubbe J.
        Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.
        Biochemistry. 1987; 26 (3322391): 6905-6909
        • Mao S.S.
        • Holler T.P.
        • Yu G.X.
        • Bollinger Jr., J.M.
        • Booker S.
        • Johnston M.I.
        • Stubbe J.
        A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.
        Biochemistry. 1992; 31 (1382592): 9733-9743
        • Zhang Z.
        • Yang K.
        • Chen C.C.
        • Feser J.
        • Huang M.
        Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17277086): 2217-2222
        • Johansson C.
        • Roos A.K.
        • Montano S.J.
        • Sengupta R.
        • Filippakopoulos P.
        • Guo K.
        • von Delft F.
        • Holmgren A.
        • Oppermann U.
        • Kavanagh K.L.
        The crystal structure of human GLRX5: iron–sulfur cluster co-ordination, tetrameric assembly and monomer activity.
        Biochem. J. 2011; 433 (21029046): 303-311
        • Du Y.
        • Zhang H.
        • Lu J.
        • Holmgren A.
        Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose.
        J. Biol. Chem. 2012; 287 (22977247): 38210-38219
        • Rollins M.F.
        • van der Heide D.M.
        • Weisend C.M.
        • Kundert J.A.
        • Comstock K.M.
        • Suvorova E.S.
        • Capecchi M.R.
        • Merrill G.F.
        • Schmidt E.E.
        Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration.
        J. Cell Sci. 2010; 123 (20571049): 2402-2412
        • Mandal P.K.
        • Schneider M.
        • Kölle P.
        • Kuhlencordt P.
        • Förster H.
        • Beck H.
        • Bornkamm G.W.
        • Conrad M.
        Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation.
        Cancer Res. 2010; 70 (21045148): 9505-9514
        • Prigge J.R.
        • Coppo L.
        • Martin S.S.
        • Ogata F.
        • Miller C.G.
        • Bruschwein M.D.
        • Orlicky D.J.
        • Shearn C.T.
        • Kundert J.A.
        • Lytchier J.
        • Herr A.E.
        • Mattsson Å.
        • Taylor M.P.
        • Gustafsson T.N.
        • Arnér E.S.J.
        • Holmgren A.
        • Schmidt E.E.
        Hepatocyte hyperproliferation upon liver-specific co-disruption of thioredoxin-1, thioredoxin reductase-1, and glutathione reductase.
        Cell Rep. 2017; 19 (5730093): 2771-2781
        • Foskolou I.P.
        • Jorgensen C.
        • Leszczynska K.B.
        • Olcina M.M.
        • Tarhonskaya H.
        • Haisma B.
        • D'Angiolella V.
        • Myers W.K.
        • Domene C.
        • Flashman E.
        • Hammond E.M.
        Ribonucleotide reductase requires subunit switching in hypoxia to maintain DNA replication.
        Mol. Cell. 2017; 66 (28416140): 206-220
        • Yang F.
        • Yi M.
        • Liu Y.
        • Wang Q.
        • Hu Y.
        • Deng H.
        Glutaredoxin-1 silencing induces cell senescence via p53/p21/p16 signaling axis.
        J. Proteome Res. 2018; 17 (29356545): 1091-1100
        • Johansson C.
        • Lillig C.H.
        • Holmgren A.
        Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase.
        J. Biol. Chem. 2004; 279 (14676218): 7537-7543
        • Fairman J.W.
        • Wijerathna S.R.
        • Ahmad M.F.
        • Xu H.
        • Nakano R.
        • Jha S.
        • Prendergast J.
        • Welin R.M.
        • Flodin S.
        • Roos A.
        • Nordlund P.
        • Li Z.
        • Walz T.
        • Dealwis C.G.
        Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization.
        Nat. Struct. Mol. Biol. 2011; 18 (21336276): 316-322
        • Ren X.
        • Bjöernstedt M.
        • Shen B.
        • Ericson M.L.
        • Holmgren A.
        Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione.
        Biochemistry. 1993; 32 (8373774): 9701-9708
        • Rengby O.
        • Johansson L.
        • Carlson L.A.
        • Serini E.
        • Vlamis-Gardikas A.
        • Kårsnäs P.
        • Arnér E.S.
        Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.
        Appl. Environ. Microbiol. 2004; 70 (15345395): 5159-5167
        • Bucurenci N.
        • Sakamoto H.
        • Briozzo P.
        • Palibroda N.
        • Serina L.
        • Sarfati R.S.
        • Labesse G.
        • Briand G.
        • Danchin A.
        • Barzu O.
        • Gilles A.M.
        CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.
        J. Biol. Chem. 1996; 271 (8576266): 2856-2862
        • Davis R.
        • Thelander M.
        • Mann G.J.
        • Behravan G.
        • Soucy F.
        • Beaulieu P.
        • Lavallée P.
        • Gräslund A.
        • Thelander L.
        Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.
        J. Biol. Chem. 1994; 269 (8083221): 23171-23176
        • Mann G.J.
        • Gräslund A.
        • Ochiai E.
        • Ingemarson R.
        • Thelander L.
        Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.
        Biochemistry. 1991; 30 (1847079): 1939-1947
        • Montano S.J.
        • Lu J.
        • Gustafsson T.N.
        • Holmgren A.
        Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.
        Anal. Biochem. 2014; 449 (24374250): 139-146