Binding Of Thioredoxin Research Articles

Parallel TXNIP‐ and TLR2‐ Dependent Pathways Synergistically Mediate Saturated Fatty Acid‐Induced Apoptosis in Cardiomyocytes

BACKGROUNDSaturated fatty acids (SFA) induce apoptosis in many cell types in vitro, and high SFA diet increases apoptotic cardiomyocytes, but mechanism remains unclear. We had previously demonstrated that p38α mitogen activated protein kinase (MAPK)–dependent signaling mediates cardiomyocyte apoptosis induced by palmitic acid (PA), a SFA. More recently, direct binding of thioredoxin interacting protein (TXNIP) and p38 MAPK has been demonstrated. SFA has been shown to bind to and activate Toll‐like receptor‐2 (TLR2), as well as the TLR4 accessory protein, MD2, triggering inflammation, oxidative stress, and cell death. We hypothesized that parallel TXNIP‐, and TLR‐ dependent pathways, both triggered by SFA, synergistically mediate apoptosis in cardiomyocytes.METHODS AND RESULTSA human cardiomyocyte line, AC16, was used in the study. TXNIP was inhibited by pre‐treating (24hours) cells with SBI‐477, a chemical inhibitor of MondoA/ChREBP. TXNIP knockdown was achieved with siRNA. TXNIP was overexpressed by transfecting with TXNIP plasmid, then 48 h later treated with TLR2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS), for an additional 2–3 days. TXNIP expression was determined by Western blotting of whole cell lysates. Apoptosis was measured by flow cytometry using Annexin V/PI staining.PA increased TXNIP protein levels dose‐dependently (1.5‐ and 2.3‐fold increase at 150 uM and 300 uM PA respectively) (ANOVA p<0.01, n=3). PA did not affect MondoA and ChREBP protein expression but increased nuclear translocation 2‐fold. TXNIP inhibition reduced PA‐induced apoptosis by 79% (p<0.05; n=3). TXNIP knockdown reduced PA‐induced cardiomyocyte apoptosis by 61% (p<0.01; n=7).TXNIP overexpression for 24 h increased apoptosis 1.8‐fold compared to control plasmid (PCDNA), but this was attenuated by siRNA knockdown of p38α or c‐fos, 59% and 84% respectively. TXNIP overexpression for 4–5 days markedly increased apoptosis rate to 4.0‐fold. The combined stressors of transfection with control plasmid and TLR ligand increased cell death rate 3.2 and 4.6‐fold for PGN and LPS respectively, compared to PCDNA + Vehicle. TXNIP overexpression followed by PGN increased apoptosis rate 8‐fold, whereas LPS reduced it to 2.7‐fold.CONCLUSIONSThis study in a human cardiomyocyte line demonstrated that (1) PA increased TXNIP levels, (2) TXNIP inhibition or knockdown attenuated PA‐induced apoptosis, (3) TXNIP overexpression increased apoptosis, (4) TXNIP overexpression combined with a TLR2 ligand, PGN, synergistically amplified the apoptosis rate, whereas LPS, a TLR4 ligand did not. These findings suggest that TXNIP‐dependent pathway synergistically combine with TLR2‐dependent signaling, but not TLR4‐, to amplify apoptosis, and suggest a mechanism by which elevated plasma SFA levels may lead to development of lipotoxic cardiomyopathy.Support or Funding InformationThis work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&D 5I01CX000598 grant (to P.R.), and VA Merit Grant BLRD I01BX007080 (to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. K.T. is supported by the M. Lowell Edwards Endowment.

Cysteine residues mediate high-affinity binding of thioredoxin to ASK1.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.

Apoptosis of cholangiocytes modulated by thioredoxin of carcinogenic liver fluke

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini, frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis.

Somatic Mutations in MAP3K5 Attenuate Its Proapoptotic Function in Melanoma through Increased Binding to Thioredoxin

Patients with advanced metastatic melanoma have poor prognosis and the genetics underlying its pathogenesis are poorly understood. High throughput sequencing has allowed comprehensive discovery of somatic mutations in cancer samples. Here, upon analysis of our whole-genome and whole-exome sequencing data of 29 melanoma samples we identified several genes that harbor recurrent non-synonymous mutations. These included MAP3K5, which in a prevalence screen of 288 melanomas was found to harbor a R256C substitution in 5 cases. All MAP3K5 mutated samples were wild-type for BRAF, suggesting a mutual exclusivity for these mutations. Functional analysis of the MAP3K5 R256C mutation revealed attenuation of MKK4 activation through increased binding of the inhibitory protein thioredoxin (TXN/TRX-1/Trx); resulting in increased proliferation and anchorage-independent growth of melanoma cells. This mutation represents a potential target for the design of new therapies to treat melanoma.

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.

A rice lipid transfer protein binds to plasma membrane proteinaceous sites

Nonspecific lipid transfer protein (nsLTP) is usually basic and secreted low-molecular-mass protein in plants. The 3-D structure of nsLTP1 resembles that of elicitin produced by the plant pathogen Phytophthora cryptogea, which can bind to the plant plasma membrane putative receptor and activate the downstream responses. It is inferred that nsLTP1 may have similar binding sites on the plasma membranes. In this work, rice recombinant protein TRX-nsLTP110 labeled with (125)I was shown to bind to rice plasma membrane preparations in a saturable curve, with an apparent K(d) of 13.6 nM and B(max) of 150 fmol/mg proteins. Competition experiments revealed that the binding of TRX-nsLTP110 was specific, in contrast to the nonspecific binding of the fusion tag thioredoxin. Protease treatment assay showed that the binding sites were proteinaceous. Our results suggest that the binding sites of nsLTPs on plasma membranes may be ubiquitous in the plant kingdom. They may be competed out from the binding sites under pathogen attack, supporting a role for nsLTP1 in host defense response to pathogens.

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin.

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.

Structural and Functional Organization of the DNA Polymerase of Bacteriophage T7

The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.

29] Modified T7 DNA polymerase for DNA sequencing

Publisher Summary This chapter discusses various modified T7 DNA polymerase for DNA sequencing. When bacteriophage T7 infects its Escherichia coli host, it directs the production of a phage-specific DNA polymerase. This enzyme is a two subunit protein. This enzyme has two known catalytic activities, DNA-dependent DNA polymerase and a 3' → 5'-exonuclease activity that is active on both single-stranded and double-stranded DNA. Both of these activities are also present in purified gene 5 protein alone, although the DNA polymerase activity and the double-stranded exonuclease are both greatly increased by the binding of thioredoxin. This increase has been attributed to the high processivity conferred by the thioredoxin subunit. Modified T7 DNA polymerase is able to incorporate the nucleotide analogs used for DNA sequencing. The most commonly used method for sequencing with modified T7 DNA polymerase was introduced by Tabor and Richardson to take advantage of some of its useful properties. The results of a sequencing experiment run on a high-resolution denaturing electrophoresis gel are also discussed in this chapter.

Isolation and characterization of a protease-nicked thioredoxin.

An altered form of thioredoxin, composed of two peptides with molecular weights of 7 and 5 X 10(3) has been isolated from Escherichia coli B after chromatography on Ultrogel AcA54. The position of the clip site, determined by amino acid sequencing to lie between Pro(64) and Gly(65), is consistent with a cleavage within the hinge region connecting the two prominent folding domains of thioredoxin. The NH2-terminal domain containing the active site, and the carboxyl-terminal domain correspond to the 7 and 5 X 10(3) fragments, respectively. The two peptides combine to form a tight complex, T'12. At concentrations below 10(-6) M, the clipped species was not efficiently reduced by thioredoxin reductase, showing only half the activity of native thioredoxin at a protein concentration of 9 X 10(-8) M. The Km of the clipped species for thioredoxin reductase (2.9 X 10(-6) M) was nearly equivalent to that of native thioredoxin (2.2 X 10(-6) M), and the Vmax for both native and clipped thioredoxins were nearly identical. These data are taken to imply a reversible association of the fragments to produce a thioredoxin equivalent to the intact species in its interaction with thioredoxin reductase. Some evidence exists for an unfolding of the 7 X 10(3) fragment upon dissociation of the complex. A functional role for the two-domain structure of thioredoxin is proposed in which the carboxyl domain serves to stabilize the active site within the amino domain and confer the species specificity in the binding of thioredoxin to thioredoxin reductase.

The Function of Thioredoxin and Glutathione in Deoxyribonucleic Acid Synthesis

Ribonucleotide reductases (EC 1 .I 7.4.1 and EC 1.17.4.2) catalyse the reduction of ribonucleoside 5’-diphosphates or triphosphates to the corresponding 2’-deoxyribonucleotides and are key enzymes for the onset of DNA replication during the cell cycle (Reichard, 1968; Follmann, 1974). The reduction of the 2’-hydroxyl group of the ribose moiety in a ribonucleotide is dependent on NADPH, but this coenzyme will not function directly with ribonucleotide reductase. In vitro, purified ribonucleotide reductase requires certain dithiols like lipoic acid or dithiothreitol for activity (Reichard, 1968). The thioredoxin system of Escherichia coli was discovered in 1964 by Reichard and coworkers (Laurent et al., 1964) as a physiological NADPH-dependent hydrogen-donor systemforribonucleotidereductase. Thioredoxin is asmall acidic dithiol protein (mol.wt. 12000) which can occur in an oxidized (thioredoxin-S,) or a reduced [thioredoxin-(SH),] form. Thioredoxin reductase is a flavoprotein (mol.wt. 66000) which will reduce thioredoxin-S, by NADPH (Reichard, 1968). Ribonucleotide reduction as studied in vitro thus appears as a complex multienzyme system. Further, evidence exists that the organization in vivo of the ribonucleotide reductase system may involve interactions between the proteins to form a highly effective multienzyme complex of unknown properties. This is supported by the low activity of ribonucleotide reductase obtained by reconstitution in vitro as compared with the activity in cells made permeable to nucleotides by diethyl ether treatment or the activity in ‘high-speed’ supernatants from gently lysed E. coli cells (Eriksson, 1975). Steady-state kinetics of ribonucleotide reductase indicated that the enzyme acted by a Ping Pong mechanism, in the presence of thioredoxin-(SH)z, that is, it akernated between two stable forms during catalysis (Thelander, 1974). The electron acceptor in ribonucleotide reductase was suggested to be oxidation-reduction-active disulphides of protein B1 (Thelander, 1974). The Ping Pong mechanism might explain the difliculties in observing any binding between Escherichia coli thioredoxin and ribonucleotide reductase in vitro (A. Holmgren, unpublished work). From an X-ray-crystallographic study of E. coli thioredoxin-S, to 0.28 nm (2.8A) resolution, the oxidation-reductionactive disulphide bond has been located in a protrusion of the molecule (Holmgren et al., 1975). Reduction of thioredoxin-Sz is suggested to be accompanied by a local conformational change within this active-centre protrusion (Holmgren et al., 1975). From such a model the simultaneous binding of thioredoxin to thioredoxin reductase and ribonucleotide reductase seems unlikely. Both these enzymes have oxidationreduction disulphide bonds at their active sites (Thelander, 1974) that should interact with the S-S bond protrusion in thioredoxin for transfer of electrons via thiol-disulphide interchange. The structural data for thioredoxin thus also support Ping Pong mechanisms for the interaction of thioredoxin with both ribonucleotide and thioredoxin reductase. A completely new and unexpected role for thioredoxin in DNA synthesis was discovered in the work of Richardson and co-workers (Mark & Richardson, 1976). E. coli thioredoxin was found to be a subunit of a phage TFinduced DNA polymerase, detected after virus infection, which is essential for the phage life cycle. Phage T7 DNA polymerase is composed of one 84000-dalton phage-coded subunit and 1 molecule of thioredoxin (Mark & Richardson, 1976). The specific function of thioredoxin in the phage T7 DNA polymerase is presently unknown. However, since the enzyme can be reconstituted in vitro from purified gene-5 protein of phage T7 by addition of E. coli thioredoxin, the function of the thioredoxin subunit can be studied in some detail. The discovery of the new function for E. coli thioredoxin as subunit of phage T7 DNA polymerase was based on the isolation of E. coli mutants (tsnC) which could not