Biomedical subjects

A Holmgren

Publications and source records attributed to A Holmgren.

At least 91 records · Page 5Linked to original sources

PubMed1997

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.

Amino Acid Sequence↗

PubMed1997

Human IgG is substrate for the thioredoxin system: differential cleavage pattern of interchain disulfide bridges in IgG subclasses.

Thioredoxin, a 12,000 mol. wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity. Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH)2. It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherichia coli thioredoxin systems. The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH)2, which directly reduced IgG disulfides in a time and dose-dependent manner. Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy light chain and inter-heavy chain disulfides, and SDS PAGE corroborated that the buried intrachain disulfides were left intact. The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4. In sharp contrast, IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times. A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide. Consistent results were obtained for several IgG myeloma proteins within each subclass. The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.

Disulfides↗

PubMed1997

The expression of thioredoxin mRNA is increased in the human cervix during pregnancy.

The classic function for thioredoxin is to act as a hydrogen donor for the enzyme ribonucleotide reductase, which is essential for DNA synthesis. In addition, thioredoxin participates in the regulation of different metabolic processes via thiol redox control. These kind of processes involve changes in the activity of different enzymes, receptors or transcription factors via dithiol/disulphide interchange reactions. Thioredoxin is present in the human decidua and trophoblasts. This study was performed to investigate whether thioredoxin mRNA is present in the human cervix, and differently expressed during pregnancy as compared with the non-pregnant state. Cervical biopsies and serum samples were obtained from 28 late pregnant, 41 post-partum and 15 non-pregnant menstruating women. The tissues were analysed for thioredoxin mRNA content using a solution hybridization technique. The thioredoxin mRNA level increased 3-fold at late pregnancy in comparison with the non-pregnant state. No further increase was seen immediately after parturition, either after spontaneous delivery or after pharmacological induction. There was a positive correlation between the cervical thioredoxin mRNA level and the serum oestradiol concentration in the non-pregnant group. We suggest that thioredoxin mRNA in the human cervix is regulated, at least partly, by oestradiol.

Adult↗

PubMed1997

Thioredoxin messenger ribonucleic acid is regulated by estradiol in the rat uterus.

Thioredoxin is a major cellular dithiol reductant with a large number of functions in electron transport and thiol redox control of enzymes and transcription factors. To investigate the expression and regulation of thioredoxin in the uterus, 35 rats were ovariectomized (OVX) and treated 14 days after surgery with either growth hormone (GH), dexamethasone (DEX), or estradiol (E2), or combinations of these, for 24 h. Thioredoxin mRNA levels were determined by solution hybridization. The animals receiving E2 or a combination of E2 and DEX showed significantly increased thioredoxin mRNA levels, by 4-fold and 5-fold, respectively, as compared to the OVX control group. The GH or GH+DEX-treated groups did not display any difference in thioredoxin mRNA levels. Thioredoxin mRNA was also measured at different time points in uteri from OVX rats treated with daily E2 injections and was found to be transiently increased, with a maximum 48 h after the initiation of the E2 treatment. In contrast, the thioredoxin mRNA level in the liver of OVX rats was about 10-fold higher than in the uterus but remained unaffected by the different hormone treatments. We conclude that thioredoxin mRNA is expressed in the rat uterus, and up-regulated by E2 in a tissue-specific manner, with a maximum at 48 h after the initiation of hormone treatment.

Animals↗

PubMed1997

Selenium and the thioredoxin and glutaredoxin systems.

Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FAD-containing enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a K(m)-value (6 mumol.L-1) and a high turnover number (kappa cat 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH-dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the human plasma glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furthermore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SECIS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of selenium for cell proliferation.

Amino Acid Sequence↗

PubMed1996

Selenite and selenate inhibit human lymphocyte growth via different mechanisms.

Selenium compounds like selenite and selenate have strong inhibitory effects, particularly on mammalian tumor cell growth by unknown mechanisms. We found that the addition of sodium selenite and sodium selenate inhibited the growth of human 3B6 and BL41 lymphocytes. Selenite was more potent because 10 microM selenite produced a growth inhibitory effect similar to that of 250 microM selenate. The mechanism of action of selenite and selenate appears to be different. 3B6 and BL41 cells treated with selenite accumulated in the S-phase; however, selenate caused an accumulation of cells in G2. Selenite-mediated growth inhibition was irreversible, although the effects of selenate could be reversed. Selenite, in contrast to selenate, is efficiently reduced by the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). At concentrations required to observe a similar effect on cell growth, the activity of thioredoxin reductase, recently shown to be a selenoprotein, increased in selenite-treated cells and decreased in selenate-treated cells. Ribonucleotide reductase activity was inhibited in an in vitro assay by selenite and selenodiglutathione but not by selenate. These results show that selenite and selenate use different mechanisms to inhibit cell growth.

B-Lymphocytes↗

PubMed1996

The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12.

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

Bacterial Proteins↗

PubMed1996

S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide.

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

Animals↗

PubMed1996

Efficient reduction of lipoamide and lipoic acid by mammalian thioredoxin reductase.

Reduction of the antioxidant lipoic acid has been proposed to be catalyzed in vivo by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR). We have found that thioredoxin reductase (TR) from calf thymus, calf liver, human placenta, and rat liver efficiently reduced both lipoic acid and lipoamide with Michaelis-Menten type kinetics in NADPH-dependent reactions. In contrast to LipDH, lipoic acid was reduced almost as efficiently as lipoamide. Under equivalent conditions at 20 degrees C, pH 8.0, mammalian TR reduced lipoic acid by NADPH 15 times more efficiently than the corresponding NADH dependent reduction catalyzed by LipDH (297 min-1 for TR vs. 20.3 min-1 for LipDH). Moreover, TR was 2.5 times faster in reducing lipoic acid with NADPH than in catalyzing the reverse reaction (oxidation of dihydrolipoic acid with NADP+). In contrast, LipDH was only 0.048 times as efficient in the forward reaction as compared to the reverse reaction (using NADH and NAD+). We conclude that all or part of the previously described NADPH-dependent lipoamide dehydrogenase (diaphorase) activities in mammalian systems should be attributed to TR. Our results suggest that in mammalian cells a significant part of the therapeutically important reduction of lipoic acid is catalyzed by thioredoxin reductase.

Animals↗

PubMed1996

Selenite incubated with NADPH and mammalian thioredoxin reductase yields selenide, which inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron.

Selenite and selenodiglutathione (GS-Se-SG) efficiently inhibited 5-lipoxygenase activity in sonicates of human monoclonal B-lymphocytes. The apparent IC50 of GS-Se-SG was 0.5 microM. The inhibitory effect of these compounds was observed within 10 min of incubation. In order to elucidate if the mechanism of inhibition by these compounds was result of direct interference with lipoxygenase or indirectly mediated by cellular factors, pure 15-lipoxygenase from soybeans was used as a model system for enzyme assays and electron spin resonance (ESR) measurements. Incubation of 15-lipoxygenase with a mixture of human placenta thioredoxin reductase (TR) or calf-thymus TR, selenite, and NADPH blocked the activity of the enzyme. Neither TR and NADPH nor selenite inhibited soybean lipoxygenase when incubated separately. These results suggest that selenite must be reduced to selenide in order to inhibit 5- and 15-lipoxygenase activities. Preincubation anaerobically of 15-lipoxygenase with chemically generated selenide (6 microM) resulted in a strong inhibition of activity, in assays with arachidonic acid in the presence of oxygen. In contrast, selenide exposed to air prior to preincubation did not inhibit the enzyme. Since selenide is known to be efficiently oxidized by oxygen and to form elemental selenium the results evidence that selenide was the inhibitor of lipoxygenase activity in the anaerobic preincubations. After incubation with TR, NADPH, and selenite or with chemically generated selenide, the ESR spectrum of 15-lipoxygenase changed: the dominant axial component with a peak at g = 6.1 decreased, and a rhombic form with a feature at g = 4.28 grew. The results suggest that selenide produced by the reduction of selenite reduces the active site iron to the ESR invisible state and changes the ligation geometry of the oxidized form.

Arachidonate 15-Lipoxygenase↗

PubMed1996

NK-lysin, a disulfide-containing effector peptide of T-lymphocytes, is reduced and inactivated by human thioredoxin reductase. Implication for a protective mechanism against NK-lysin cytotoxicity.

The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK-lysin was treated with dithiothreitol prior to incubation with the cells. NK-lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, e. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated.

Cells, Cultured↗

PubMed1996

Glutaredoxin-3 from Escherichia coli. Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis.

The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.

Amino Acid Sequence↗

PubMed1996

The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14.

Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe.

Animals↗

PubMed1996

Deoxyribonucleoside triphosphate pools and growth of glutathione-depleted 3T6 mouse fibroblasts.

Buthionine sulfoximine (BSO) selectively blocks g-glutamylcysteine synthetase and thereby depletes cells of glutathione (GSH). In cultures of exponentially growing 3T6 mouse fibroblasts, 0.1 mM BSO rapidly stopped GSH synthesis after treatment for 12 hours. The GSH-depleted cells grew as well as control 3T6 cells with no decrease in DNA synthesis. Furthermore, the pools of deoxyribonucleoside triphosphates (dNTPs), typically tightly regulated in cultured cells, did not change in size. Ribonucleotide reductase catalyzes the reduction of all four ribonucleotides and occupies a key position in dNTP regulation. Our data suggest that the GSH-glutaredoxin (a GSH-dependent disulfide-oxidoreductase) system is not the sole/major hydrogen carrier from NADPH for the reduction of ribonucleoside diphosphates by ribonucleotide reductase.

Amino Acid Sequence↗

PubMed1996

Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function.

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.

Amino Acid Sequence↗

PubMed1996

High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.

Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.

Amino Acid Sequence↗

PubMed1996

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3: use of 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC.

Virtually complete sequence specific 1H and 15N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F1-decoupled ROESY-15N-HSQC and 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC using a single uniformly 15N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-15N-HSQC and a homonuclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without 13C/15N double labelling. Chemical shifts, 3J(alpha H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.

Amino Acid Sequence↗

PubMed1996

Elevation of plasma thioredoxin levels in HIV-infected individuals.

Thioredoxin (Trx), a ubiquitous protein intimately involved in redox and protein disulfide reductions, has been shown to be released from cells and to have cytokine-like activities. In addition, Trx has been implicated in the redox regulation of immunological responses and shown to be deficient in tissues from AIDS patients. In studies presented here, plasma Trx levels were measured by ELISA in plasma samples from HIV-infected individuals (n = 136) and HIV-negative controls (n = 47). To account for the release of Trx into plasma due to hemolysis, the Trx measurements were corrected according to the level of hemoglobin in the plasma sample. Data presented show that, in contrast to tissue Trx levels, corrected plasma Trx levels are significantly higher in HIV-infected individuals than in controls (P < 0.0001). Furthermore, approximately 25% of the HIV-infected individuals studied have plasma Trx levels greater than the highest levels found in controls (37 ng/ml). Detailed multiparameter FACS analysis of peripheral blood mononuclear cells (PBMC) from the infected individuals demonstrates that those with higher plasma Trx levels (37 ng/ml or greater) tend to have lower overall CD4 counts. In addition, increases in plasma Trx levels correlate with decreases in monochlorobimane staining (indicative of lower intracellular glutathione levels in PBMC) and with changes in surface antigen expression (CD62L, CD38 and CD20) that occur in the later stages of HIV infection. These correlations suggest that elevation of plasma Trx levels may be an important component of advanced HIV disease, perhaps related to the oxidative stress that often occurs at this stage.

Antigens, CD↗