Intramolecular Disulphide Bonds Research Articles

Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core β-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the β-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

Expression and purification of human epidermal growth factor (hEGF) fused with GB1

ABSTRACTFusion expression is a promising strategy for the production of bioactive peptides in Escherichia coli. In this study, we constructed a new recombinant expression plasmid containing the coding sequence of 56-residue B1 domain of streptococcal protein G (GB1). For easy purification and cleavage of the recombinant proteins, except GB1, an engineered hexahistidine and tobacco etch virus (TEV) protease recognition sites were included in the fusion sequence. Next, we cloned the coding sequence of human epidermal growth factor (hEGF) into this new plasmid and produced the recombinant hEGF in E. coli. The bioactive hEGF is a 53-amino acid peptide and is stabilized by three intramolecular disulphide bonds. Compared with glutathione S-transferase, thioredoxin and small ubiquitin-related modifier, GB1 greatly improved the expression and solubility of hEGF. Moreover, the recombinant hEGF bound to the nickel nitrilotriacetic acid resin column, was easily cleaved by TEV protease and the free hEGF was released. The results showed that this new plasmid was appropriate for recombinant production of small bioactive peptides, such as hEGF, which contains a high proportion of hydrophobic residues and intramolecular disulphide linkages.

Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants.

Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments.

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub-cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter- and intra-molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle-specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.

Oxidation of translation factor EF-G transiently retards the translational elongation cycle in Escherichia coli.

In Escherichia coli, elongation factor G (EF-G), a key protein in translational elongation, is particularly susceptible to oxidation. We demonstrated previously that EF-G is inactivated upon formation of an intramolecular disulphide bond. However, the details of the mechanism by which the oxidation of EF-G inhibits the function of EF-G on the ribosome remain to be elucidated. When we oxidized EF-G with hydrogen peroxide, neither the insertion of EF-G into the ribosome nor single-cycle translocation activity in vitro was affected. However, the GTPase activity and the dissociation of EF-G from the ribosome were suppressed when EF-G was oxidized. The synthesis of longer peptides was suppressed to a greater extent than that of a shorter peptide when EF-G was oxidized. Thus, the formation of the disulphide bond in EF-G might interfere with the hydrolysis of GTP that is coupled with dissociation of EF-G from the ribosome and might thereby retard the turnover of EF-G within the translational machinery. When we added thioredoxin to the suppressed translation system that included oxidized EF-G, translational activity was almost immediately restored. We propose that oxidation of EF-G might provide a regulatory mechanism for transient and reversible suppression of translation in E. coli under oxidative stress.

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21-35%) but considerable motif conservation within the putative Ca(2+)-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.

C-H ... interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of β-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.

Expression of hypoallergenic Der f 2 derivatives with altered intramolecular disulphide bonds induces the formation of novel ER-derived protein bodies in transgenic rice seeds

House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy.

Effect of adding low levels of β-mercaptoethanol on the disulphide bonds of κ-casein and β-lactoglobulin solutions

Low levels of β-mercaptoethanol (up to 0.5%, v/v) were added to κ-casein and β-lactoglobulin solutions (5mgmL−1), mixtures thereof, and milk. The reduction of disulphide bonds was monitored over time for up to 24h, using micro-fluidic electrophoresis, at temperatures ranging from 20 to 50°C. Lower concentrations of β-mercaptoethanol and a shorter reaction time were required to reduce κ-casein to the same extent as β-lactoglobulin. This suggests that the intermolecular disulphide bonds of κ-casein were more accessible than the intra-molecular disulphide bonds of β-lactoglobulin. The reduction of β-lactoglobulin at any β-mercaptoethanol concentration and reaction time was enhanced as the temperature increased up to 50°C. The level of reduced κ-casein increased with increasing temperature during the first 2h; however, as the reaction time was prolonged, the level of monomeric κ-casein decreased. This suggests that monomeric κ-casein can re-form disulphide bonds via either re-oxidation or thiol-disulphide exchange reactions.

Conformational changes to deamidated wheat gliadins and β-casein upon adsorption to oil–water emulsion interfaces

The conformation of deamidated gliadins and β-casein in solution and adsorbed at the interface of oil-in-water emulsions was studied using synchrotron radiation circular dichroism (SRCD) and front-face-fluorescence spectroscopy. Deamidation led to partial unfolding of gliadins in solution. The α-helix content of the protein decreased from 35% (in the native form) to 16.3% while the percentage of β-sheet and unordered structure increased upon deamidation. The secondary structure of deamidated gliadins was largely unchanged upon adsorption to both tricaprin/water and hexadecane/water interfaces. In contrast, β-casein adopted a more ordered structure upon adsorption to these two oil/water interfaces, the α-helix content increased from 5.5% (in solution) to 20% and 22.5% respectively after adsorption to tricaprin/water and hexadecane/water interfaces. Both deamidated gliadins and β-casein have distinctive N-terminal hydrophilic and C-terminal hydrophobic domains. Unlike β-casein which contains no cysteine residue, gliadins have a large number of intramolecular disulphide bonds located in the C-terminal hydrophobic domain which constrains the conformational freedom of this protein upon adsorption to oil/water interfaces. The hydrophobicity of the oil phase also has an impact on the conformation of each protein upon adsorption to the oil/water interfaces – systematic trends were observed between oil phase polarity from: i) tryptophan fluorescence emission maxima, and ii) the α-helix content in the adsorbed state. Our results demonstrate that conformational re-arrangement of proteins upon adsorption to emulsion interfaces is dependent not only on the hydrophobicity of the oil phase, but more importantly on the conformational flexibility of the protein.

The redox state of human serum albumin in eye diseases with and without complications

To investigate the redox state of human serum albumin concerning cysteine-34 as a possible systemic redox marker in patients with different eye diseases with and without complications and with consideration of possible effects of age. Cataract (CAT), glaucoma, age-related macular degeneration (AMD), diabetes mellitus (DM), diabetic retinopathy and hypertension were the pathologies investigated. Albumin redox state concerning cysteine-34 was measured by high-performance liquid chromatography with fluorescence detection. The separation gives three fractions: the fully reduced form containing a thiol group, the disulphide form and a higher oxidized form. Statistical analysis was done by Student's t-test, analysis of variance and stepwise regression analysis. Albumin as a systemic marker for oxidative stress was shifted to a more oxidized state by DM. An even stronger shift to the oxidized form was observed in patients with proliferative diabetic retinopathy. Notably, these effects were independent from age. In contrast, CAT and AMD had no influence on serum albumin redox state. Serum albumin is not shifted to more oxidized forms by localized oxidative stress, but it is in systemic diseases like DM.

Expression and polymorphism of defensins in farm animals.

Due to their activity against bacteria, viruses, and fungi, antimicrobial peptides are important factors in the innate resistance system of humans and animals. They are called "new generation antibiotics" for their potential use in preventive and therapeutic medicine. The most numerous group of antimicrobial peptides is a family of cationic peptides which include defensins and cathelicidins. Among them the most common are peptides with a beta-sheet structure containing three intra-molecular disulphide bonds, called defensins, comprising three classes: alpha, beta, and theta. The class of beta-defensins is the largest one. Their transcripts have been found in many tissues of humans and animals. The aim of this paper is to present the current knowledge about antimicrobial peptides from the defensin family in farm animals, their expression, polymorphism, as well as the potential of their use as genetic markers of health and production traits.

Structure–function relationship in an archaebacterial methionine sulphoxide reductase B

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

Biological activity of heterologous murine interleukin-10 and preliminary studies on the use of a dextrin nanogel as a delivery system

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer with two intramolecular disulphide bonds essential for its biological activity. A mutated form of murine IL-10 was successfully expressed in E. coli, recovered and purified from inclusion bodies. Its ability to reduce tumor necrosis factor α synthesis and down-regulate class II major histocompatibility complex molecules expression on endotoxin-stimulated bone marrow-derived macrophages was confirmed, and shown to be similar to that of a commercially available IL-10. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems that can effectively deliver the protein. In this work it is shown that a dextrin nanogel effectively incorporate IL-10, stabilize, and enable the slow release of biologically active IL-10 over time. Altogether, these results demonstrate the suitability of dextrin nanogel to be used as a system for the controlled release of IL-10.

A Cys/Ser mutation of NDPK-A stabilizes its oligomerization state and enhances its activity

Nucleoside diphosphate phosphate transferase A (NDPK-A) has been shown to play critical roles in the regulation of proliferation, differentiation, growth and apoptosis of cells. Our previous study suggested that the disulphide cross-linkage between cysteine 4 (C4) and cysteine 145 (C145) of NDPK-A might be a possible regulator of its activity. To confirm this hypothesis, the C145 residue of NDPK-A was mutated to serine, and the isomerization and biological activities of the mutant were investigated and compared with those of its wild-type counterpart. It was found the C145S mutation eliminated the intramolecular disulphide bond (DB) and prevented the formation of intermolecular DB, which was known to dissociate the hexameric NDPK-A into dimeric one. We also demonstrated that the C145S mutation didn't affect the autologous hexamerization of this protein, and the mutant had increased bioactivities including phosphate transferase and DNase. These findings support the hypothesis that the formation of DBs in NDPK-A is involved in the regulation of the oligomerization and bioactivity of this multiple function protein, and that C145 is a key residue in the regulation of NDPK-A. In addition, the C145S mutant that we have constructed might be an attractive candidate for use in applications that require NDPK-A.

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

DsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PilQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.

Identification of intra‐ and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.

Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Defensins: endogenous antibiotic peptides from human leukocytes.

A variety of endogenous antimicrobial peptides equip mammals, amphibians, insects and plants to defend themselves against microbial pathogens. Defensins are small peptides of mammalian cells that contain 29-35 amino acid residues, including six invariant cysteines that form three intramolecular disulphide bonds. They are produced by the sequential proteolysis of precursors that contain approximately 95 amino acids and are synthesized by several types of cells, especially the bone marrow precursors of blood neutrophils. Defensins constitute 5-7% of the total protein in human neutrophils and are present in high concentration in the azurophil granules and phagocytic vacuoles. The production of defensins by human neutrophils amounts to approximately 10 mg/kg body weight per day. In certain mammalian species lung macrophages and specialized epithelial (Paneth) cells in the small intestine also produce defensins. Defensins are complexly folded, amphipathic, rich in antiparallel beta-sheet but devoid of alpha-helical domains. Their unusually broad antimicrobial spectrum encompasses Gram-positive and Gram-negative bacteria, many fungi, mycobacteria, spirochetes and several enveloped viruses. The antimicrobial properties of defensins result from their insertion into target cell membranes and the formation of voltage-sensitive channels. Given their abundance and broad spectrum of antimicrobial activity, defensins may contribute substantially to innate resistance to infection.

Biochemical characterization of recombinant chicken riboflavin carrier protein

Chicken Riboflavin Carrier Protein (cRCP) transports riboflavin from the maternal circulation to the egg yolk for fetal development. The cRCP is a globular protein and structurally very stable due to the presence of nine intra-molecular disulphide bonds. The cRCP comprises of two domains; the larger riboflavin binding, and the smaller, oocyte receptor binding domain. With the objective to study domain folding in cRCP, these two domains of the corresponding gene were amplified, cloned, and expressed in a eukaryotic expression system to obtain soluble product. Our studies on the biochemical characterization of the recombinant proteins indicated that though the ligand binding domain assumed near-native conformation, as determined by immunological methods, it did not bind riboflavin, suggesting the interdependence of the two domains for proper organization of the riboflavin binding pocket.