Formation and transfer of disulphide bonds in living cells

Protein disulphide bonds are formed in the endoplasmic reticulum of eukaryotic cells and the periplasmic space of prokaryotic cells. The main pathways that catalyse the formation of protein disulphide bonds in prokaryotes and eukaryotes are remarkably similar, and they share several mechanistic features. The recent identification of new redox-active proteins in humans and yeast that mechanistically parallel the more established redox-active enzymes indicates that there might be further uncharacterized redox pathways throughout the cell.     

Formation, isomerisation and reduction of disulphide bonds during protein quality control in the endoplasmic reticulum

Efficient protein folding and quality control in the endoplasmic reticulum (ER) require that disulphide bonds are formed in nascent proteins, isomerised during assisted folding and reduced in terminally misfolded molecules. Recent findings in yeast and mammalian cells indicate that specific protein-protein interactions underlie redox control in the ER, allowing these competing reactions to occur simultaneously during protein quality control.     

Assignment of the disulphide bonds in the sweet-tasting protein thaumatin I

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9-204, 56-66, 71-77, 121-193, 126-177, 134-149, 145-158 and 159-164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158.     

Time-dependent polymerization of beta-lactoglobulin through disulphide bonds at the oil-water interface in emulsions

Time-dependent intermolecular sulphydryl-disulphide interchange involving beta-lactoglobulin adsorbed at the oil-water interface in n-tetradecane-in-water emulsions (10 wt% oil, 0.5 wt% protein, pH 7.0) has been investigated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). While only monomers are detected in the adsorbed protein immediately after emulsion formation with pure beta-lactoglobulin, on storing the emulsion the amount of polymerized beta-lactoglobulin and the sizes of the oligomers are found to increase with time. There is no polymerization of adsorbed protein in emulsions made with pure alpha-lactalbumin after 72 h, or in emulsions made with beta-lactoglobulin in the presence of a reagent (N-ethylmaleimide) for modifying sulphydryl groups. Analysis by two-dimensional SDS-PAGE of adsorbed protein from aged emulsions made with a mixture of alpha-lactalbumin + beta-lactoglobulin shows some linking by disulphide bonds between alpha-lactalbumin and beta-lactoglobulin at the interface. Taken together with earlier time-dependent surface viscosity measurements, the results indicate the important role of free sulphydryl groups in the development of the high surface viscoelasticity of adsorbed globular proteins at the oil-water interface.     

The reactivity of the disulphide bonds of purified proteins in relationship to primary structure

1. With the aid of a coupled system involving glutathione reductase, the reaction of glutathione with the disulphide bonds of purified proteins has been studied. 2. Bovine serum albumin, conalbumin, lysozyme, trypsin inhibitors from egg white, lima bean and soya bean either did not react with glutathione or reacted only slightly. With these proteins reactivity was markedly increased by limited proteolysis. 3. Bovine and human gamma-globulins, fibrinogen and beta-lactoglobulin exhibited some reactivity (less than 15%) with glutathione and again this was increased by limited proteolysis. Pepsin, trypsin and chymotrypsin exhibited greater reactivity than the proteins previously mentioned. Di-isopropylphosphoryl-chymotrypsin exhibited less reactivity than chymotrypsin, suggesting that autolysis under the experimental conditions used contributed towards the reactivity of this protein. Proteolysis also increased the reactivity of these proteins. The three disulphide bonds of insulin were reduced by glutathione. 4. Above 35 degrees the disulphide bonds of serum albumin show a progressive increase in reactivity and at 55 degrees half of the bonds become accessible to glutathione. 5. From the results obtained with the proteins investigated, the conclusion reached is that the disulphide bonds of native proteins are structurally protected and do not react with glutathione under physiological conditions.     

On the disulphide bonds of rhodopsins.

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).     

The reactivity of the disulphide bonds of wool

1. Fully reduced and S-carboxymethylated wool samples were prepared in which either the readily reducible cystine bonds or those that could only be reduced with difficulty were specifically labelled with iodo[2-(14)C]acetate; these two cystine fractions correspond to the (A+B) and (C+D) cystine fractions respectively, of Middlebrook & Phillips (1942). 2. Radioactively labelled peptides were isolated from partial acid hydrolysates of these wool samples. 3. It appears that the (A+B) cystine residues probably owe their increased reactivity to being in a more polar environment. 4. The implication of these results for the problem of characterizing the disulphide bonds of wool is briefly discussed.     

Formation of disulfide bonds in proteins and peptides

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are crosslinked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding--a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein. This review describes basic and practical aspects of oxidative folding that should be considered while designing and optimizing manufacturing of proteins using chemical synthesis, semi-synthesis and a recombinant expression.     

Identification of the disulphide bonds in human platelet glycocalicin

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.     

Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability

It has been recently concluded that the hydrophobic effect, hitherto regarded as a major driving force in the folding of proteins, destabilizes the folded state relative to the unfolded state. We summarize the properties of the hydrophobic effect obtained from solvent transfer experiments and show that the recent conclusion is an artifact of crosslinking in the unfolded state, caused by disulphide bonds, metals or cofactors. We show that, for the proteins in the data set, crosslinks surprisingly destabilize folded structures entropically, but stabilize them enthalpically to a greater extent. We also calculate non-polar surface areas of these unfolded proteins. These surface areas are decreased by crosslinks. The unfolded state of proteins lacking constraints, such as myoglobin, is well approximated by a mixture of residues containing alpha-helical and beta-sheet dihedral angles. Surface areas of unfolded proteins cannot be obtained by summing the surface areas of individual residues, since this ignores any unavoidable side-chain-side-chain interactions.     

Adenovirus virus-associated RNAs induce type I interferon expression through a RIG-I-mediated pathway

The current study demonstrates that adenovirus virus-associated RNA (VA) is recognized by retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor, and activates RIG-I downstream signaling, leading to the induction of type I interferons (IFNs), similarly to Epstein-Barr virus-encoded small RNA. Further analysis revealed that adenovirus infection leads to biphasic type I IFN induction at 12 to 24 h and 48 to 60 h postinfection. The later induction coincided with VA expression and was reduced by virus UV inactivation or RIG-I silencing. These results suggest that VA-mediated RIG-I activation is involved in activating innate immune responses during adenovirus infection.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.     

Aspergillus nidulans hypA regulates morphogenesis through the secretion pathway

Aspergillus nidulans hypA encodes a predicted 1474 amino acid, 161.9 kDa cytoplasmic peptide. Strains with hypA1 and hypA6 alleles are wild type at 28 degrees C but have wide, slow-growing hyphae and thick walls at 42 degrees C. hypA1 and hypA6 have identical genetic lesions. hypA1 and hypA6 restrictive phenotypes have statistically similar morphometry, and strains with either allele can conidiate at 42 degrees C. hypA deletion strains require osmotic support and have aberrant morphology, but produce viable spores at 28 degrees C. hypA has full-length orthologs in filamentous fungi and yeasts and a 200 amino acid region with similarity to sequences in plants and animals. The Saccharomyces cerevisiae hypA ortholog is TRS120, a regulatory subunit in the TRAPP II complex that mediates traffic through the Golgi equivalent. Enzyme secretion is reduced in hypA1 cells at 42 degrees C. Endomembranes and cytoplasmic actin arrays in hypA1 have weak polarity at 42 degrees C and cytoplasmic microtubules have reduced number and normal distribution.