Protein Disulfide Oxidoreductase Research Articles

Temperature-, SDS-, and pH-Induced Conformational Changes in Protein Disulfide Oxidoreductase from the Archaeon Pyrococcus furiosus: A Dynamic Simulation and Fourier Transform Infrared Spectroscopic Study

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.

Requirements for the Different Cysteines in the Chemotactic and Desensitizing Activity of Human Thioredoxin

Thioredoxin (Trx) is a protein disulfide oxidoreductase that can be secreted and act as a chemoattractant for leukocytes. Like chemokines, it causes desensitization of monocytes against its chemotactic activity and that of monocyte chemoattractant protein-1 (MCP-1). To investigate the role of the redox properties of Trx, and particularly of some of its five cysteines, in its chemotactic and desensitizing action, we tested different mutants, including Trx80, a truncated form, and various mutants lacking specific cysteines: Trx C62S/C73S and the redox-inactive mutant Trx C32S/C35S. Of the mutants, only Trx80 maintained the chemotactic activity of wild-type Trx toward both monocytes and polymorphonuclear neutrophils, all of them desensitized monocytes against wild-type Trx or MCP-1, but not chemotactic peptide formyl-methionyl-leucil peptide. These data indicate that different redox-active cysteines are important for Trx chemotactic action, whereas its desensitizing action does not have these requirements, suggesting a redox-independent mechanism.

The Genomics of Disulfide Bonding and Protein Stabilization in Thermophiles

Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.

Crystal structure of a protein disulfide oxidoreductase fromAquifex aeolicus

Crystal structure of a protein disulfide oxidoreductase from<i>Aquifex aeolicus</i>

Characteristics of Thiol:Protein Disulfide Oxidoreductase from Wheat (Triticum aestivum L.) Grain

Biochemical properties of a homogenous preparation of thiol:protein disulfide oxidoreductase (TPDO, EC 1.8.4.2) isolated for the first time from mature wheat (Triticum aestivum L.) grain were studied. According to polyacrylamide gel electrophoresis data, the molecular weight of TPDO is around 167 kD, the enzyme consisting of two subunits of 77 and 73 kD, which differentiates TPDO from known enzymes of SH/SS-metabolism of wheat caryopses. In substrate specificity and enzymatic characteristics (pH and temperature optima) TPDO is similar to analogous enzymes of animal tissues. Inhibition of disulfide reductase activity by alkylating agents and heavy metal ions suggests the participation of active center SH-groups in the catalytic act and classes the enzyme as a member of the thioredoxin superfamily. The SS-reductase reduces aggregating capacity of acetic acid-soluble fraction of wheat storage proteins. The proposed physiological role of TPDO is participation in creation and regulation of SH/SS-status of wheat endosperm proteins and formation of the rheological properties of gluten.

Diabetic changes in the redox status of the microsomal protein folding machinery

Changes in assisted protein folding are largely unexplored in diabetes. In the present studies, we have identified a reductive shift in the redox status of rat liver microsomes after 4 weeks of streptozotocin-induced diabetes. This change was reflected by a significant increase in the total- and protein-sulfhydryl content, as well as in the free sulfhydryl groups of the major protein disulfide isomerases (PDIs), the 58 kDa PDI and the 57 kDa ERp57 but not other chaperones. A parallel decrease of the protein-disulfide oxidoreductase activity was detected in the microsomal fraction of diabetic livers. The oxidant of PDI, Ero1-Lα showed a more oxidized status in diabetic rats. Our results reveal major changes in the redox status of the endoplasmic reticulum and its redox chaperones in diabetic rats, which may contribute to the defective protein secretion of the diabetic liver.

Crystallization and preliminary X-ray diffraction studies of a protein disulfide oxidoreductase fromAeropyrum pernixK1

A protein disulfide oxidoreductase from the archaeon Aeropyrum pernix K1 has been overexpressed in Escherichia coli and crystallized at 298 K using the hanging-drop vapour-diffusion method. Crystals belong to the space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 90.59, b = 102.43, c = 128.96 A. A complete data set has been collected at the Elettra synchrotron source in Trieste to 1.93 A resolution using a single frozen crystal.

Crystallization and preliminary X-ray diffraction studies of a protein disulfide oxidoreductase fromAquifex aeolicus

A protein disulfide oxidoreductase from the thermophilic bacterium Aquifex aeolicus has been overexpressed in Escherichia coli and crystallized at 298 K using the hanging-drop vapour-diffusion method. Crystals belong to space group R32, with unit-cell parameters a = b = 161.1, c = 153.1 A. A complete data set has been collected to 2.4 A using synchrotron radiation. Packing-density considerations agree with the presence of 2-4 monomers in the asymmetric unit, with a corresponding solvent content of 66-32%.

Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel protein family related to protein disulfide-isomerase.

Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 degrees C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family.

Mitochondrial Thioredoxin System: EFFECTS OF TrxR2 OVEREXPRESSION ON REDOX BALANCE, CELL GROWTH, AND APOPTOSIS

Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (DeltaPsi(m)), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of DeltaPsi(m) dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against beta-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells.

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1 h at 100 °C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.

γ-Glutamyl cysteine synthetase is up-regulated during recovery of brain mitochondrial complex I following neurotoxic insult in mice

β- N-Oxalyl amino- l-alanine ( l-BOAA), a naturally occurring excitatory amino acid inhibits mitochondrial complex I activity in motor cortex and lumbar spinal cord of mice through oxidation of critical thiol groups. Glutaredoxin, a protein disulfide oxido-reductase mediates recovery of complex I by regenerating protein thiols utilizing reducing equivalents of glutathione. We have examined the status of γ-glutamyl cysteine synthetase (γ-GCS), the rate limiting enzyme in glutathione synthesis during recovery of complex I function following l-BOAA toxicity. Sustained and maximal up-regulation of γ-GCS was seen in motor cortex which was associated with regeneration of complex I activity. In lumbosacral cord, however, the up-regulation was transient and complex I function did not recover. These studies demonstrate the important role of γ-GCS in mediating the recovery of mitochondrial function following excitotoxic insult and its differential regulation in central nervous system regions.

Thioltransferase (Glutaredoxin) Mediates Recovery of Motor Neurons from Excitotoxic Mitochondrial Injury

Mitochondrial dysfunction involving electron transport components is implicated in the pathogenesis of several neurodegenerative disorders and is a critical event in excitotoxicity. Excitatory amino acid L-beta-N-oxalylamino-L-alanine (L-BOAA), causes progressive corticospinal neurodegeneration in humans. In mice, L-BOAA triggers glutathione loss and protein thiol oxidation that disrupts mitochondrial complex I selectively in motor cortex and lumbosacral cord, the regions affected in humans. We examined the factors regulating postinjury recovery of complex I in CNS regions after a single dose of L-BOAA. The expression of thioltransferase (glutaredoxin), a protein disulfide oxidoreductase regulated through AP1 transcription factor was upregulated within 30 min of L-BOAA administration, providing the first evidence for functional regulation of thioltransferase during restoration of mitochondrial function. Regeneration of complex I activity in motor cortex was concurrent with increase in thioltransferase protein and activity, 1 hr after the excitotoxic insult. Pretreatment with alpha-lipoic acid, a thiol delivery agent that protects motor neurons from L-BOAA-mediated toxicity prevented the upregulation of thioltransferase and AP1 activation, presumably by maintaining thiol homeostasis. Downregulation of thioltransferase using antisense oligonucleotides prevented the recovery of complex I in motor cortex and exacerbated the mitochondrial dysfunction in lumbosacral cord, providing support for the critical role for thioltransferase in maintenance of mitochondrial function in the CNS.

Solution nuclear magnetic resonance structure of a protein disulfide oxidoreductase from Methanococcus jannaschii.

The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance. The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins. Some aspects of Mj0307's unique behavior can be explained by comparing structure-based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins. It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins. Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.

8] Protein disulfide oxidoreductase from Pyrococcus furiosus: Structural properties

The determination of the first three-dimensional structure of a protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus ( Pf PDO) has provided the first view of active site disulfides in an archaeal protein. It reveals a unique structural form compared to those of protein disulfide oxidoreductases in bacteria and eukarya. Protein disulfide oxidoreductases have been well studied in bacteria and eukarya. They are ubiquitous redox enzymes consisting of the families of thioredoxin, glutaredoxin, protein disulfide-isomerase (PDI), DsbA, and their homologs. These enzymes catalyze the formation, breakage, and rearrangement of protein disulfide bonds. These well-characterized protein disulfide oxidoreductases share a similarity in their three-dimensional structures: all of them use a common structural motif as the scaffold. The three-dimensional structure of intact PDI is not yet determined, but its amino acid sequence and tertiary domain structures have revealed it to be a multidomain protein.

7] Protein Disulfide oxidoreductase from Pyrococcus furiosus: Biochemical properties

Protein disulfide oxidoreductases (PDI) are redox enzymes that catalyze dithiol-disulfide exchange reactions in eukaryotic and bacterial cells. Several proteins belonging to this superfamily have been identified and characterized; thioredoxin, glutaredoxin, protein disulfideisomerase (PDI), disulfide bond forming (DsbA), and their homologs in prokaryotes are the most extensively studied members of this group. A highly thermostable protein disulfide oxidoreductase was first isolated from Sulfolobus solfataricus. From its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol (DTT), the protein was considered a thioredoxin. The protein showed an unusually high molecular mass of 25 kDa and from amino acid composition analysis contained four cysteine residues. A homologous protein was subsequently purified from Pyrococcusfuriosus. More recently, the protein was crystallized, and its three-dimensional (3-D) structure at high resolution revealed structural details suggesting it may be related to the multidomain eukaryotic PDI. For this reason, the protein was more correctly named “protein disulfide oxidoreductase” from P furiosus ( Pf PDO). This chapter describes the purification of the Pf PDO, the cloning, and its gene expression in E. coli .

Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells

Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase with antioxidant, cytokine, and chemotactic properties. Previously, we showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4, tumor necrosis factor alpha (TNF-alpha), and CD40-ligation induced S-phase entry and mitosis in normal B cells and B-type chronic lymphocytic leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by these protocols is high, and it has been hypothesized that the overexpression of Bcl-2 found in B-CLL protects the cells from apoptosis in vitro and in vivo. In this study, we have analyzed the response of cells derived from 12 samples of patients with B-CLL to recombinant human Trx in spontaneous apoptosis, with special reference to the Bcl-2 expression. Long-term cultures of B-CLL clones showed significantly higher viability when supplemented with human Trx (P =.031), also exemplified with clones surviving more than 2 months. Short-term cultures of B-CLL cells exposed to 1 microg/mL of Trx for 1, 5, or 12 days maintained expression or delayed down-regulation of Bcl-2 compared with control cultures containing RPMI 1640 medium and 10% fetal calf serum only (P =.032,. 002,.026, respectively). All B-CLL cells expressed constitutive Trx at varying but low levels, in contrast to adult T-cell leukemias, which overexpress Trx, as previously reported. We found that Trx added to B-CLL cells increased in a dose-dependent fashion the release of TNF-alpha, which has been suggested to be an autocrine growth factor for these cells. In conclusion, we have found that human recombinant Trx induced TNF-alpha secretion, maintained Bcl-2, and reduced apoptosis in B-CLL cells. (Blood. 2000;95:1420-1426)

Thiol oxidase and disulfide reductase activities of wheatTriticum aestivum L. caryopsis and its technological quality

Activities of oxygen-dependent thiol: O2 oxidoreductase (EC 1.8.3.2) and glutathione-dependent thiol: protein-disulfide oxidoreductase (EC 1.8.4.2) as well as technological value of seven soft spring wheat cultivars grown on different soil under contrasting climatic conditions (Krasnodar krai and Irkutsk oblast) were studied. It was found that the ratio of these enzymatic activities correlated positively with dough physical properties and flour bread-baking quality.

Chaperone Activity of DsbC

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells.

Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.