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PubMed2006

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH.

A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative by-products of respiration and peroxisomal beta-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Deltazwf1Delta strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide. Among prominent disulfide bond-containing proteins were several with known antioxidant functions. These and several other proteins were detected as multiple electrophoretic isoforms, with some isoforms containing disulfide bonds under all conditions and other isoforms exhibiting a redox-sensitive content of disulfide bonds, i.e., in the idp2Deltazwf1Delta strain and in the hydrogen peroxide-challenged parental strain. The disulfide bond content of some isoforms of these proteins was also elevated in the parental strain grown on glucose, possibly suggesting a redirection of NADPH reducing equivalents to support rapid growth. Further examination of protein carbonylation in the idp2Deltazwf1Delta strain shifted to oleate medium also led to identification of common and unique protein targets of endogenous oxidative stress.

Acetic Acid↗
PubMed2006

Protein disulfide isomerase assisted protein folding in malaria parasites.

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.

Amino Acid Sequence↗
PubMed2006

Disulfide bridges in the mesophilic triosephosphate isomerase from Giardia lamblia are related to oligomerization and activity.

Triosephosphate isomerase from the mesophile Giardia lamblia (GlTIM) is the only known TIM with natural disulfide bridges. We previously found that oxidized and reduced thiol states of GlTIM are involved in the interconversion between native dimers and higher oligomeric species, and in the regulation of enzymatic activity. Here, we found that trophozoites and cysts have different oligomeric species of GlTIM and complexes of GlTIM with other proteins. Our data indicate that the internal milieu of G. lamblia is favorable for the formation of disulfide bonds. Enzyme mutants of the three most solvent exposed Cys of GlTIM (C202A, C222A, and C228A) were prepared to ascertain their contribution to oligomerization and activity. The data show that the establishment of a disulfide bridge between two C202 of two dimeric GlTIMs accounts for multimerization. In addition, we found that the establishment of an intramonomeric disulfide bond between C222 and C228 abolishes catalysis. Multimerization and inactivation are both reversed by reducing conditions. The 3D structure of the C202A GlTIM was solved at 2.1 A resolution, showing that the environment of the C202 is prone to hydrophobic interactions. Molecular dynamics of an in silico model of GlTIM when the intramonomeric disulfide bond is formed, showed that S216 is displaced 4.6 A from its original position, causing loss of hydrogen bonds with residues of the active-site loop. This suggests that this change perturb the conformational state that aligns the catalytic center with the substrate, inducing enzyme inactivation.

Animals↗
PubMed2004

Coupling of the heme and an internal disulfide bond in human neuroglobin.

Neuroglobin displays a hexacoordination His-Fe-His in the absence of external ligands such as oxygen. The observed oxygen affinity therefore depends on the binding rates of both oxygen and the competing distal histidine. Furthermore, the binding properties depend on the presence of an internal disulfide bond. In the case of human neuroglobin, cysteines at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. For cytoglobin, the cysteine residues at positions A7 and GH4 may also form a disulfide bond. Mass spectrometry, ligand binding, and thiol accessibility studies were used to study the role influence of these disulfide bonds. Mutation of specific cysteines, or reduction to break the S-S bond, led to a large decrease in the observed oxygen affinity of human neuroglobin, mainly due to a decrease in the histidine dissociation rate. This suggests a novel mechanism for the oxygen binding; reduction of the disulfide bond would provoke the release of oxygen.

Cysteine↗
PubMed2006

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase.

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.

Amino Acid Sequence↗
PubMed2006

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides.

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.

Amino Acid Sequence↗
PubMed2005

Interdisciplinary neurotoxicity inhalation studies: carbon disulfide and carbonyl sulfide research in F344 rats.

Inhalation studies were conducted on the hazardous air pollutants, carbon disulfide, which targets the central nervous system (spinal cord) and peripheral nervous system (distal portions of long myelinated axons), and carbonyl sulfide, which targets the central nervous system (brain). The objectives were to investigate the neurotoxicity of these compounds by a comprehensive evaluation of function, structure, and mechanisms of disease. Through interdisciplinary research, the major finding in the carbon disulfide inhalation studies was that carbon disulfide produced intra- and intermolecular protein cross-linking in vivo. The observation of dose-dependent covalent cross-linking in neurofilament proteins prior to the onset of lesions is consistent with this process contributing to the development of the neurofilamentous axonal swellings characteristic of carbon disulfide neurotoxicity. Of significance is that valine-lysine thiourea cross-linking on rat globin and lysine-lysine thiourea cross-linking on erythrocyte spectrin reflect cross-linking events occurring within the axon and could potentially serve as biomarkers of carbon disulfide exposure and effect. In the carbonyl sulfide studies, using magnetic resonance microscopy (MRM), we determined that carbonyl sulfide targets the auditory pathway in the brain. MRM allowed the examination of 200 brain slices and made it possible to identify the most vulnerable sites of neurotoxicity, which would have been missed in our traditional neuropathology evaluations. Electrophysiological studies were focused on the auditory system and demonstrated decreases in auditory brain stem evoked responses. Similarly, mechanistic studies focused on evaluating cytochrome oxidase activity in the posterior colliculus and parietal cortex. A decrease in cytochrome oxidase activity was considered to be a contributing factor to the pathogenesis of carbonyl sulfide neurotoxicity.

Administration, Inhalation↗
PubMed2006

Protein disulfide isomerase: the structure of oxidative folding.

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

Animals↗
PubMed2002

Structural and functional significance of disulfide bonds in saxatilin, a 7.7 kDa disintegrin.

Saxatilin is a 7.7 kDa disintegrin that belongs to a family of homologous protein found in several snake venoms. Six disulfide bond locations of the disintegrin were determined by enzymatic cleavage and matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Functional implications of the disulfide bonds related to the biological activity of saxatilin were investigated with recombinant protein species produced by site-directed mutagenesis of saxatilin. Several lines of experimental evidence indicated that three disulfide bonds, Cys21-Cys35, Cys29-Cys59, and Cys47-Cys67, of the disintegrin are closely associated with its biological function such as its ability to block the binding of integrin GPIIb-IIIa and alpha(v)beta(3) with fibrinogen and extracellular matrix. Those disulfide linkages were also revealed to be important for maintaining the functional structure of the protein molecule. On the other hand, the disulfide bridges of Cys6-Cys15 and Cys8-Cys16 do not appear to be critical for the molecular structure and function of saxatilin.

Amino Acid Sequence↗
PubMed1990

Four disulfide bonds' allocation of Na+, K(+)-ATPase inhibitor (SPAI).

We have recently reported the primary structures of the three unique peptide inhibitors (SPAI-1, -2, and -3) against Na+, K(+)-ATPase which contained four disulfide bridges in common (Biochem. Biophys. Res. Commun. 164, 496 (1989)). The disulfide connectivities of SPAI were determined by the combination of amino acid analyses with the direct application to a gas-phase sequencer of its proteolytic fragments. The disulfide bond was identified by detection of phenylthiohydantoin derivatives of cystine and its decomposed product dehydroalanine. The four cysteine pairs were disclosed to be Cys20 to Cys49, Cys27 to Cys53, Cys36 to Cys48, and Cys42 to Cys57, all linked by disulfide bridge formation. The allocation pattern of these disulfide bonds was the same as that recently reported for human mucous proteinase inhibitor (EMBO J. 7, 345 (1988], though SPAI showed no proteinase inhibitory activity at all.

Amino Acid Sequence↗
PubMed2000

Status of glutathione and other thiols and disulfides in human plasma.

While plasma thiols, including homocysteine (HCys), glutathione (GSH), and cysteine (Cys), are being investigated as potential indicators of disease risk and health status, low levels, poor stability, and the lack of comprehensive methodologies have hampered their accurate assessment. Using our previously described HPLC with electrochemical detection method, our goal was to assess levels, stability, and distribution of biologically relevant thiols and disulfides in human plasma. In fresh plasma, processed immediately after collection, low levels of Cys, cystine, Cys-Gly, and the mixed disulfide Cys-GSH (CSSG) were consistently observed, whereas the levels of GSH and Cys-Gly disulfide were often below the limits of detection. These profiles were a consequence of poor thiol stability, as thiol standards added to human plasma were lost rapidly due to autoxidation or formation of mixed disulfides. A 75% loss of added GSH observed after 30 min was accounted for completely by the formation of GSH disulfide (24%) and CSSG (74%). Similar changes were found with other thiols when added to plasma. Thiols lost to oxidation were recovered quantitatively by reducing samples with potassium borohydride (KBH(4)) prior to analysis. In a study of 106 healthy adults, mean total thiol levels in plasma were: Cys (201 microM) > Cys-Gly (101 microM) > HCys (7 microM) > gamma-Glu-Cys (5 microM) > GSH (4 microM). All together, these results account for the poor stability of thiols in plasma and provide a method for their comprehensive and accurate determination.

Adult↗
PubMed1987

Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione.

Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.

Animals↗
PubMed1998

Maurotoxin, a four disulfide bridges scorpion toxin acting on K+ channels.

Maurotoxin, a toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus, has been purified to homogeneity by gel filtration/reversed-phase HPLC, and characterized. It is a basic and C-terminal amidated 34-residue polypeptide cross-linked by four disulfide bridges. From Edman sequencing results, only six different pairings between the first six half-cystines were retained whereas a disulfide bridge was predicted between the two half-cystines in positions 31 and 34. Modelling based on the structure of charybdotoxin favored two different pairings, one of which possessed two disulfides in common with the general motif of scorpion toxins. The solid-phase technique was used to obtain synthetic maurotoxin, sMTX. The half-cystine pairings of sMTX were determined by enzymatic cleavage and were found to be Cys3 Cys24, Cys9-Cys29, Cys13-Cys19, and Cys31-34, in agreement with experimental data obtained with natural maurotoxin. Both natural and synthetic maurotoxins were lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). They blocked the Kv1.1, Kv1.2, and Kv1.3 channels expressed in Xenopus oocytes with almost identical half-effects (IC50) in the range of 40, 0.8 and 150 nM, respectively. They also competed with 125I-apamin (SKca channel blocker) and 125I-kaliotoxin (Kv channel blocker) for binding to rat brain synaptosomes with IC50 of about 5 and 0.03 nM. As the natural and synthetic maurotoxins exhibit indistinguishable physicochemical and pharmacological properties, they are likely to adopt the same half-cystine pairing pattern which is unique among known scorpion toxins. However, this disulfide organization is different from those reported for Pandinus imperator and Heterometrus spinnifer toxins 1 (Pi1 and HsTx1), two novel four-disulfide bridged K+ channel-acting scorpion toxin sharing about 50-70% sequence identity with maurotoxin.

Amino Acid Sequence↗
PubMed2005

Gamma-glutamyltranspeptidase: disulfide bridges, propeptide cleavage, and activation in the endoplasmic reticulum.

gamma-Glutamyltranspeptidase (gammaGT) is found primarily on the apical surface of epithelial and endothelial cells, where it degrades reduced and oxidized glutathione (gamma-GluCysGly) by hydrolysis of the unique gamma-glutamyl bond. Glutathione plays a key role in disulfide rearrangement in the endoplasmic reticulum (ER) and acts as a redox buffer. Previous work has shown that overexpression of gammaGT or an inactive splice variant gammaGTDelta7 mediates a redox stress response in the endoplasmic reticulum (ER) characterized by increased levels of BiP and induction of CHOP-10. To determine whether a CX(3)C motif might be the common feature of gammaGT and gammaGTDelta7 that mediates this response, we characterized disulfide bridges in gammaGT that might form between the six highly conserved Cys residues. Using site-directed mutagenesis of gammaGT, expression in Chinese Hamster Ovary (CHO) cells, metabolic labeling, and immunoblotting, our data predict disulfide formation between Cys49 and Cys73 and between Cys191 and Cys195 (the CX(3)C motif). Potential functions for this CX(3)C motif are discussed. In the course of defining the disulfides, we also noted that propeptide cleavage correlated with enzymatic activity. Because recent reports indicate that the homologous Escherichia coli gammaGT is a member of the N-terminal nucleophile (Ntn) hydrolase family, where the amino acid at the new N-terminus functions as the nucleophile for both autocatalytic cleavage and enzymatic activity, the rat gammaGT was similarly characterized. As predicted, mutations at the propeptide cleavage site coincidentally inhibit both heterodimer formation and gammaGT enzymatic activity. Analysis of early cleavage events using cell extraction into SDS indicates that propeptide cleavage occurs while gammaGT is still within the ER. Because activation and cleavage are coincident events, this raises the new question of whether an active glutathionase is present within the ER and what role gammaGT plays in modulating ER glutathione levels that are so critical for proper redox balance and disulfide formation in this compartment.

Animals↗
PubMed2004

Flavoprotein disulfide reductases: advances in chemistry and function.

The flavoprotein disulfide reductases represent a family of enzymes that show high sequence and structural homology. They catalyze the pyridine-nucleotide-dependent reduction of a variety of substrates, including disulfide-bonded substrates (lipoamide dehydrogenase, glutathione reductase and functional homologues, thioredoxin reductase, and alkylhydroperoxide reductase), mercuric ion (mercuric ion reductase), hydrogen peroxide (NADH peroxidase), molecular oxygen (NADH oxidase), and the reductive cleavage of a carbonyl-activated carbon-sulfur bond followed by carboxylation (2-ketopropyl-coenzyme-M carboxylase?oxidoreductase). They use at least one nonflavin redox center to transfer electrons from reduced pyridine nucleotide to their substrate through flavin adenine dinucleotide. The nature of the nonflavin redox center located adjacent to the flavin varies and three types have been identified: an enzymic disulfide (most commonly), an enzymic cysteine sulfenic acid (NADH peroxidase and NADH oxidase), and a mixed Cys-S-S-CoA disulfide (coenzyme A disulfide reductase). Selection of the particular nonflavin redox center and utilization of a second, or even a third, nonflavin redox center in some cases presumably represents the most efficient strategy for reduction of the individual substrate.

Disulfides↗
PubMed1998

An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state.

Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.

Amino Acid Sequence↗
PubMed2000

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond.

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Animals↗
PubMed2003

Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate.

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.

Amino Acid Sequence↗