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Arne Holmgren

Publications and source records attributed to Arne Holmgren.

At least 19 recordsLinked to original sources
PubMed2007

How does iron-sulfur cluster coordination regulate the activity of human glutaredoxin 2?

Human mitochondrial glutaredoxin (Grx2) was described as the first iron-sulfur protein from the thioredoxin superfamily of proteins. The [2Fe-2S] cluster was proposed to serve as redox sensor for the activation of Grx2 during oxidative stress. The authors have demonstrated that the iron-sulfur cluster is complexed by the two N-terminal active site thiols of two Grx2 monomers and two molecules of glutathione that are bound noncovalently to the proteins and in equilibrium with glutathione in solution. When reduced glutathione becomes the limiting factor for cluster coordination, the holo-Grx2 complex dissociates, yielding enzymatically active Grx2.

Gene Expression Regulation↗
PubMed2007

Thioredoxin and related molecules--from biology to health and disease.

Thioredoxin and glutaredoxin systems in mammalian cells utilize thiol and selenol groups to maintain a reducing intracellular redox state acting as antioxidants and reducing agents in redox signaling with oxidizing reactive oxygen species. During the last decade, the functional roles of thioredoxin in particular have continued to expand, also including novel functions such as a secreted growth factor or a chemokine for immune cells. The role of thioredoxin and glutaredoxin in antioxidant defense and the role of thioredoxin in controlling recruitment of inflammatory cells offer potential use in clinical therapy. The fundamental differences between bacterial and mammalian thioredoxin reductases offer new principles for treatment of infections. Clinical drugs already in use target the active site selenol in thioredoxin reductases, inducing cell death in tumor cells. Thioredoxin and binding proteins (ASK1 and TBP2) appear to control apoptosis or metabolic states such as carbohydrate and lipid metabolism related to diseases such as diabetes and atherosclerosis.

Amino Acid Sequence↗
PubMed2006

Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system.

Reactive oxygen species (ROS) and the cellular thiol redox state are crucial mediators of multiple cell processes like growth, differentiation, and apoptosis. Excessive ROS production or oxidative stress is associated with several diseases, including cardiovascular disorders like ischemia-reperfusion. To prevent ROS-induced disorders, the heart is equipped with effective antioxidant systems. Key players in defense against oxidative stress are members of the thioredoxin-fold family of proteins. Of these, thioredoxins and glutaredoxins maintain a reduced intracellular redox state in mammalian cells by the reduction of protein thiols. The reversible oxidation of Cys-Gly-Pro-Cys or Cys-Pro(Ser)-Tyr-Cys active site cysteine residues is used in reversible electron transport. Thioredoxins and glutaredoxins belong to corresponding systems consisting of NADPH, thioredoxin reductase, and thioredoxin or NADPH, glutathione reductase, glutathione, and glutaredoxin, respectively. Thioredoxin as well as glutaredoxin activities appear to be very important for the progression and severity of several cardiovascular disorders. These proteins function not only as antioxidants, they inhibit or activate apoptotic signaling molecules like apoptosis signal-regulating kinase 1 and Ras or transcription factors like NF-kappaB. Thioredoxin activity is regulated by the endogenous inhibitor thioredoxin-binding protein 2 (TBP-2), indicating an important role of the balance between thioredoxin and TBP-2 levels in cardiovascular diseases. In this review, we will summarize cardioprotective effects of endogenous thioredoxin and glutaredoxin systems as well as the high potential in clinical applications of exogenously applied thioredoxin or glutaredoxin or the induction of endogenous thioredoxin and glutaredoxin systems.

Animals↗
PubMed2006

The thioredoxin system in cancer.

Thioredoxin (Trx), NADPH and thioredoxin reductase (TrxR) comprise a thioredoxin system which exists in nearly all living cells. It functions in thiol-dependent thiol-disulfide exchange reactions crucial to control of the reduced intracellular redox environment, cellular growth, defense against oxidative stress or control of apoptosis and has multi-facetted roles in mammalian cells including implications in cancer. Eg reduced Trx activates DNA binding of transcription factors and is involved in antioxidant defense through repair of oxidatively damaged proteins or as an electron donor to peroxiredoxins. The Trx system functions in synthesis of deoxyribonucleotides for DNA synthesis, both replication and repair, by ribonucleotide reductase. Trx and truncated Trx (Trx80) act in modulation of immune cell function. TrxR isoforms in the cytosol and the mitochondria are essential selenoenzymes with a selenocysteine in the active site. These enzymes display a remarkably broad substrate specificity but are also targets for existing chemotherapeutic drugs. Mammalian TrxR enzymes are linked to selenium metabolism as a result of being selenoproteins, but can also directly reduce low molecular selenium compounds like selenite and have been implicated in the chemoprevention effects of selenium against cancer. Numerous scientific reports describe higher expression of Trx and TrxR in some, but not all tumors. Some data suggest that high Trx could be linked to resistance to chemotherapies while others suggest that high Trx and TrxR may induce apoptosis and reduce the mitotic index of certain tumors linked to the p53 dependent cell death. Recent data suggest that TrxR is essential for the carcinogenic process and invasive phenotype of cancer. Both Trx and TrxR have been regarded as interesting targets for chemotherapy.

Humans↗
PubMed2006

Inhibition of Mammalian thioredoxin reductase by some flavonoids: implications for myricetin and quercetin anticancer activity.

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, exerts a wide range of activities in cellular redox control, antioxidant function, cell viability, and proliferation. Recently, the selenocysteine (Sec)-containing mammalian TrxR has emerged as a new target for anticancer drug development because TrxR and Trx are overexpressed in many aggressive tumors and the tumor cells seem to be more dependent on Trx system than normal cells. Here we have investigated the inhibition of mammalian TrxR by flavonoids which have been presumed to be cancer chemoprevention agents because of their antioxidant activities. Myricetin and quercetin were found to have strong inhibitory effects on mammalian TrxRs with IC50 values of 0.62 and 0.97 micromol/L, respectively. The inhibition was shown to be concentration, NADPH, and time dependent and involved an attack on the reduced COOH-terminal -Cys-Sec-Gly active site of TrxR. Oxygen-derived superoxide anions enhanced the inhibitory effect whereas anaerobic conditions attenuated inhibition. Spectral analysis suggested that the flavonols might perform their inhibitory effects via semiquinone radicals. Additionally, the flavonols had the potential to inhibit the growth of A549 cells with the same potency as inhibition of TrxR. TrxR activity in the cell lysates was reduced on treatment with myricetin >50 micromol/L, which coincided with the oxidization of Trx. The cell cycle was arrested in S phase by quercetin and an accumulation of cells in sub-G1 was observed in response to myricetin. Thus, the anticancer activity of quercetin and myricetin may be due to inhibition of TrxR, consequently inducing cell death.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2006

Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo.

Lipid peroxidation is a cellular process that takes place under physiological conditions and particularly after oxidative stress. 4-Hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, is known to exert a multitude of biological effects and has high reactivity to various cellular components, including DNA and protein. The thioredoxin system, composed of the selenoenzyme thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, plays a key role in redox regulation and is involved in many signaling pathways. The selenocysteine (Sec) and cysteine (Cys) residues (Cys-496/Sec-497) in the active site of TrxR and a pair of Cys residues (Cys-32/Cys-35) in Trx are sensitive to various alkylating reagents. Herein, we report a mechanistic study on the inhibition of rat TrxR by HNE. The inhibition occurs with TrxR only in its reduced form and persists after removal of HNE. Inhibition of TrxR by HNE added to cultured HeLa cells is also observed. In addition, HNE inactivates reduced Escherichia coli Trx irreversibly. We proved that the redox residues (Cys-496/Sec-497 in TrxR and Cys-32/Cys-35 in Trx) were primary targets for HNE modification. The covalent adducts formed between HNE and Trx were also confirmed by mass spectrum. Because the thioredoxin system is one of the core regulation enzymes of cells' function, inhibition of both TrxR and Trx by HNE provides a possibly novel mechanism for explanation of its cytotoxic effect and signaling activity, as well as the further damage indirectly caused under oxidative stress conditions.

Aldehydes↗
PubMed2006

Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase.

Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 microM (kcat/Km of 4.86 x 10(4) M(-1) s(-1)). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 microM) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 x 10(5) M(-1) s(-1)) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 microM reduced human Trx (IC50 2 microM) or 4 mM dithiothreitol (IC50 6 microM) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair.

Animals↗
PubMed2006

The redox state of SECIS binding protein 2 controls its localization and selenocysteine incorporation function.

Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins.

Amino Acid Motifs↗
PubMed2005

Ebselen: a thioredoxin reductase-dependent catalyst for alpha-tocopherol quinone reduction.

The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if alpha-tocopherol quinone (TQ), a product of alpha-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity, while the product of reduction of TQ, alpha-tocopherolhydroquinone (TQH(2)), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo.

Animals↗
PubMed2005

Human glutaredoxin 2 affinity tag for recombinant peptide and protein purification.

Recombinant proteins are commonly expressed in fusion with an affinity tag to facilitate purification. We have in the present study evaluated the possible use of the human glutaredoxin 2 (Grx2) as an affinity tag for purification of heterologous proteins. Grx2 is a glutathione binding protein and we have shown in the present study that the protein can be purified from crude bacterial extracts by a one-step affinity chromatography on glutathione-Sepharose. We further showed that short peptides could be fused to either the N- or C-terminus of Grx2 without affecting its ability to bind to the glutathione column. However, when Grx2 was fused to either the 27 kDa green fluorescent protein or the 116 kDa beta-galactosidase, the fusion proteins lost their ability to bind glutathione-Sepharose. Insertion of linker sequences between the Grx2 and the fusion protein did not restore binding to the column. In summary, our findings suggest that Grx2 may be used as an affinity tag for purification of short peptides and possibly also certain proteins that do not interfere with the binding to glutathione-Sepharose. However, the failure of purifying either green fluorescent protein or beta-galactosidase fused to Grx2 suggests that the use of Grx2 as an affinity tag for recombinant protein purification is limited.

Chromatography, Affinity↗
PubMed2005

Characterization of human glutaredoxin 2 as iron-sulfur protein: a possible role as redox sensor.

Human mitochondrial glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase (active site: Cys-Ser-Tyr-Cys) that facilitates the maintenance of mitochondrial redox homeostasis upon induction of apoptosis by oxidative stress. Here, we have characterized Grx2 as an iron-sulfur center-containing member of the thioredoxin fold protein family. Mossbauer spectroscopy revealed the presence of a four cysteine-coordinated nonoxidizable [2Fe-2S]2+ cluster that bridges two Grx2 molecules via two structural Cys residues to form dimeric holo Grx2. Coimmunoprecipitation of radiolabeled iron with Grx2 from human cell lines indicated the presence of the cluster in vivo. The [2Fe-2S]-bridged dimer was enzymatically inactive, but degradation of the cluster and the resulting monomerization of Grx2 activated the protein. Slow degradation under aerobic conditions was prevented by the presence of glutathione, whereas glutathione disulfide as well as one-electron oxidants or reductants promoted monomerization of Grx2. We propose that the iron-sulfur cluster serves as a redox sensor for the activation of Grx2 during conditions of oxidative stress when free radicals are formed and the glutathione pool becomes oxidized.

Apoproteins↗
PubMed2005

Thioredoxin reductase is irreversibly modified by curcumin: a novel molecular mechanism for its anticancer activity.

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a -Gly-Cys-Sec-Gly active site. TrxRs are the only enzymes catalyzing the NADPH-dependent reduction of the active site disulfide in thioredoxins (Trxs), which play essential roles in substrate reductions, defense against oxidative stress, and redox regulation by thiol redox control. TrxRs have been found to be overexpressed by a number of human tumors. Curcumin, which is consumed daily by millions of people, is a polyphenol derived from the plant Curcuma longa. This phytochemical has well known anticancer and antiangiogenic properties. In this study we report that rat TrxR1 activity in Trx-dependent disulfide reduction was inhibited by curcumin. The IC(50) value for the enzyme was 3.6 microM after incubation at room temperature for 2 h in vitro. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of curcumin. By using mass spectrometry and blotting analysis, we proved that this irreversible inhibition by curcumin was caused by alkylation of both residues in the catalytically active site (Cys(496)/Sec(497)) of the enzyme. However, the curcumin-modified enzyme showed a strongly induced NADPH oxidase activity to produce reactive oxygen species. Inhibition of TrxR by curcumin added to cultured HeLa cells was also observed with an IC(50) of around 15 microM. Modification of TrxR by curcumin provides a possible mechanistic explanation for its cancer preventive activity, shifting the enzyme from an antioxidant to a prooxidant.

Animals↗
PubMed2005

Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli.

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.

Amino Acid Motifs↗
PubMed2005

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase.

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

Amino Acid Sequence↗
PubMed2005

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities.

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.

Animals↗
PubMed2005

Overexpression of glutaredoxin 2 attenuates apoptosis by preventing cytochrome c release.

Human mitochondrial glutaredoxin 2 (Grx2) catalyzes glutathione-dependent dithiol reaction mechanisms, reducing protein disulfides, and monothiol reactions, reducing mixed disulfides between proteins and GSH (de-/glutathionylation). Here, we have overexpressed Grx2 in HeLa cells in its mitochondrial form (mGrx2-HeLa) as well as a truncated cytosolic form, lacking the mitochondrial translocation signal (tGrx2-HeLa). The resulting clones were less susceptible to apoptosis induced by 2-deoxy-d-glucose (2-DG) or doxorubicin (Dox). Overexpression of Grx2 inhibited cytochrome c release and caspase activation induced by both agents. In addition, Grx2 prevented 2-DG- and Dox-induced loss of cardiolipin, the phospholipid anchoring cytochrome c to the inner mitochondrial membrane. Overexpression of mGrx2 provided better protection than tGrx2 overexpression, especially after treatment with 2-DG. We propose that Grx2 facilitates the maintenance of cellular redox homeostasis upon treatment with apoptotic agents, thereby preventing cardiolipin oxidation and cytochrome c release.

Apoptosis↗