Disulphide Bond Formation Research Articles

Mechanisms and therapeutic potential of disulphidptosis in cancer.

SLC7A11 plays a pivotal role in tumour development by facilitating cystine import to enhance glutathione synthesis and counteract oxidative stress. Disulphidptosis, an emerging form of cell death observed in cells with high expression of SLC7A11 under glucose deprivation, is regulated through reduction-oxidation reactions and disulphide bond formation. This process leads to contraction and collapse of the F-actin cytoskeleton from the plasma membrane, ultimately resulting in cellular demise. Compared to other forms of cell death, disulphidptosis exhibits distinctive characteristics and regulatory mechanisms. This mechanism provides novel insights and innovative strategies for cancer treatment while also inspiring potential therapeutic approaches for other diseases. Our review focuses on elucidating the molecular mechanism underlying disulphidptosis and its connection with the actin cytoskeleton, identifying alternative metabolic forms of cell death, as well as offering insights into disulphidptosis-based cancer therapy. A comprehensive understanding of disulphidptosis will contribute to our knowledge about fundamental cellular homeostasis and facilitate the development of groundbreaking therapies for disease treatment.

Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

An insight into the pharmacology of cysteine/methionine containing peptide drugs

Since last century, peptides have emerged as potential drugs with >90 FDA approvals for various targets with several in the pipeline. Sulphur, in peptides is present either as thiol (-SH) from Cys or thioether from Met. In this review, all the peptides approved by FDA since 2000 containing sulphur have been included. Among them ∼50 % contains disulphide bridges. This clearly demonstrates the significance of disulphide bonds in peptide drugs. This can be achieved synthetically by using orthogonal protecting groups (PGs) for –SH. These PGs are compatible with Solid Phase Peptide Synthesis (SPPS), which is still the method of choice for peptide synthesis. The orthogonal PGs used for Cys thiol side chain protecting for disulphide bond formation have been included which are currently in use both by academia and industry from small scale to large scale synthesis. In addition, the details of the FDA approved drugs containing Cys and Met (or both) have also been discussed.

PDIA6 and Maspin in Prostate Cancer.

PDIA6 is a disulphide isomerase of the PDI family, known to mediate disulphide bond formation in the endoplasmic reticulum. However, PDI-related proteins also function in other parts of the cell and PDIA6 has been shown to be involved in many types of cancers. We previously identified PDIA6 as a putative Maspin interactor. Maspin has itself been implicated in prostate cancer progression. Our aim was to further explore the roles of Maspin in prostate cancer and establish whether PDIA6 is also involved in prostate cancer. RNA levels of PDIA6 and Maspin in prostate cell lines were measured using RT-PCR. Bioinformatics analysis of the TCGA database was used to find RNA levels of PDIA6 and Maspin in prostate cancer. siRNAs were used to knock-down PDIA6, and proliferation and migration assays were conducted on those cells. PDIA6 and Maspin RNA were shown to be expressed at varying levels in prostate cell lines. RNAseq data showed that PDIA6 expression was significantly increased in prostate adenocarcinoma samples, while Maspin RNA expression was decreased. When PDIA6 expression was knocked-down using siRNA in prostate cell lines, proliferation was decreased substantially in the two prostate cancer cell lines (DU145 and PC3) and also decreased in the normal prostate cell line (PNT1a), though less strongly. PDIA6 expression is higher in prostate cancer cells compared to normal prostate cells. Decreasing PDIA6 expression decreases proliferation. Thus, PDIA6 is a promising target for prostate cancer therapeutics.

Approaches for high-throughput quantification of periplasmic recombinant proteins

The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development.

An Investigation into the Mechanism of Alkaline Extraction-Isoelectric Point Precipitation (AE-IEP) of High-Thiol Plant Proteins

Hempseed protein isolate (HPI) has drawn significant attention as a promising source of plant-based protein due to its high nutritional value. The poor functionality (e.g., solubility and emulsifying properties) of HPI has impeded its food application for years. This study provides important new information on hempseed protein extraction, which may provide further insights into the extraction of other high-thiol-based plant proteins to make valuable plant-based products with improved functional properties. In this study, HPI was produced from hempseed meals using the AE-IEP method. The underlying mechanisms and extraction kinetics were investigated under different experimental conditions (pH 9.0–12.0, temperature 24–70 °C, and time 0–120 min). The results suggested that disulphide bond formation is an inevitable side reaction during hempseed protein extraction and that the protein yield and the free -SH content can be influenced by different extraction conditions. A high solution pH and temperature, and long extraction time result in increased protein yield but incur the formation of more intermolecular disulphide bonds, which might be the reason for the poor functionality of the HPI. For instance, it was particularly observable that the protein solubility of HPI products reduced when the extraction pH was increased. The emulsifying properties and surface tension data demonstrated that the functionality of the extracted hempseed protein was significantly reduced at longer extraction times. A response surface methodology (RSM) optimization model was used to determine the conditions that could maximise HPI functionality. However, a three-fold reduction in protein yield must be sacrificed to obtain the protein with this high functionality.

Thermodynamic and Experimental Substantiation of the Possibility of Formation and Extraction of Organometallic Compounds as Indicators of Deep Naphthogenesis

In this article, typomorphic associations of microelements in carbonaceous caustobioliths and oils are defined as indicators of naphthogenesis based on the analytical survey of the literature and our own research. Various approaches to the studying of crude oil genesis from the position of abiotic and complex approaches (polygenesis) are considered. Such approaches are relevant to the prospect and development of offshore oil deposits, localized at ultra-deep levels. For deep deposits, the most prominent hypothesis of oil origin is the abiogenious theory. In the foundation of that approach lays the assumption that hydrocarbons are formed mainly due to reactions of the formed mantle gases and the host rock. Key factors for these reactions to proceed are specific conditions concerning temperature, pressure, and specific catalysts. The article presents the results of thermodynamic and quantum-chemical modeling of the possibility of the organic and organometallic compound formation using the software package HSC Chemistry 6.0. Pointed out the possibility of a low molecular weight hydrocarbon formation due to the contact of ascending flows of mantle gases with cooling natural barriers. The primary synthesis proceeds with the interaction of fluid forms of H2, CO2, and H2S. The estimation of the bonding energy depending on the structure of organometallic compounds was performed using the Avogadro software package. The program used the method of bond potential energy minimization to find the most stable structure of molecules. The metals nickel and vanadium, as the main companion metals of oil, are of the most interest from the position of analysis of their form of existence in the possible formation of hydrocarbons. Vanadium’s and nickel’s accumulation in asphaltene fractions of oils, probably, is caused by complex compounds of metalloporphyrin’s formation. In addition, the high sulfur content is probably associated with polymerization of organic molecules due to the formation of di-sulphide bonds. The method of cavitation extraction of organometallic compounds from oil and complex mineral–oil raw materials has been developed for experimental confirmation of microelements the extraction capability from oil raw materials.

Enzymatic cross-linking of pea and whey proteins to enhance emulsifying and encapsulation properties

Improving the functionality of commercial plant proteins is essential for food application. Here an alternative strategy is provided to modify the structure of pea protein by microbial transglutaminase-induced cross-linking with whey protein for enhancing emulsifying and encapsulation properties. The protein structures and the physicochemical stability of β-carotene-loaded emulsions were investigated subject to protein ratio and cross-linking. Formation of disulphide and isopeptide bonds between heteroproteins according to cross-linking changed protein structures by exposing the hydrophobic sites of proteins with stiffer network. The cross-linked pea/whey protein complexes at protein ratio of 2:1 formed a stable emulsion with a droplet size of 0.10 µm, showing no phase separation for 30 days of storage and high encapsulation efficiency of 92%. These findings provided a novel strategy to design plant/dairy protein networks to protect lipophilic bioactive compounds by replacing half or more dairy protein with plant protein, leading to gradual change towards plant-based diets.

Granular Disulfide-Crosslinked Hyaluronic Hydrogels: A Systematic Study of Reaction Conditions on Thiol Substitution and Injectability Parameters

Granular polymer hydrogels based on dynamic covalent bonds are attracting a great deal of interest for the design of injectable biomaterials. Such materials generally exhibit shear-thinning behavior and properties of self-healing/recovery after the extrusion that can be modulated through the interactions between gel microparticles. Herein, bulk macro-hydrogels based on thiolated-hyaluronic acid were produced by disulphide bond formation using oxygen as oxidant at physiological conditions and gelation kinetics were monitored. Three different thiol substitution degrees (SD%: 65%, 30% and 10%) were selected for hydrogel formation and fully characterized as to their stability in physiological medium and morphology. Then, extrusion fragmentation technique was applied to obtain hyaluronic acid microgels with dynamic disulphide bonds that were subsequently sterilized by autoclaving. The resulting granular hyaluronic hydrogels were able to form stable filaments when extruded through a syringe. Rheological characterization and cytotoxicity tests allowed to assess the potential of these materials as injectable biomaterials. The application of extrusion fragmentation for the formation of granular hyaluronic hydrogels and the understanding of the relation between the autoclaving processes and the resulting particle size and rheological properties should expand the development of injectable materials for biomedical applications.

A Buried Water Network Modulates the Activity of the Escherichia coli Disulphide Catalyst DsbA

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA-substrate thiol-disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

NDP52 acts as a redox sensor in PINK1/Parkin‐mediated mitophagy

Mitophagy, the elimination of mitochondria via the autophagy‐lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin‐dependent mitophagy. We identified redox‐sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.

Self‐healing natural rubber based on dynamic disulphide exchange towards fatigue lifetime extension

AbstractDynamic disulphide exchange mechanism was introduced to promote self‐healing characteristics to natural rubber compound and the corresponding healing mechanism and mechanical properties are presented. The formation of reversible dynamic disulphide bonds within rubber molecular chains was evidenced via FTIR and the total crosslink density of dynamic disulphide crosslinks was quantitatively measured by equilibrium swelling test. Scanning Electron Microscopy (SEM) results revealed that the healed area of the fractured surface adhered well with minor scar on the contact surface, suggesting that intermolecular diffusion occur at the fracture surface. It was also found that the tensile strength and tear strength of the broken samples able to recover 91% and 103%, respectively, under thermal healing at 150°C for 10 min. The fatigue lifespan of the materials increased 154.9% compared with the conventional rubber. The successful fabrication of self‐healing natural rubber through dynamically reversible disulphide exchange mechanism would be expected to open up new opportunities for development of sustainable rubber products.

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Thiolated 2-Methyl-β-Cyclodextrin as a Mucoadhesive Excipient for Poorly Soluble Drugs: Synthesis and Characterization.

Thiolated cyclodextrins are structurally simple mucoadhesive macromolecules, which are able to host drugs and increase their apparent water solubility, as well as interact with the mucus layer prolonging drug residence time on the site of absorption. The aim of this study was to synthesize through green microwave-assisted process a freely soluble thiolated 2-methyl-β-cyclodextrin (MβCD-SH). Its inclusion complex properties with dexamethasone (Dex), a poor water soluble drug, and mucoadhesive characteristics were also determined. The product was deeply characterized through NMR spectroscopy (2D COSY, 2D HSQC, 1D/2D TOCSY, and 1D ROESY), showing a thiolation degree of 67%, a selective thiolation on the C6 residues and a monomeric structure. The association constant of MβCD and MβCD-SH with Dex resulted in 2514.3 ± 32.3 M−1 and 2147.0 ± 69.3 M−1, respectively, indicating that both CDs were able to host the drug. Microrheological analysis of mucin in the presence of MBCD-SH showed an increase of complex viscosity, G′ and G″, due to disulphide bond formation. The cytotoxicity screening on fibroblast BALB/3T3 clone A31 cells indicated an IC50 of 27.7 mg/mL and 30.0 mg/mL, for MβCD and MβCD-SH, respectively. Finally, MβCD-SH was able to self-assemble in water into nanometric structures, both in the presence and absence of the complexed drug.

In silico analyses of leptin and leptin receptor of spotted snakehead Channa punctata

The present study, in addition to molecular characterization of leptin (lepa) and its receptor (lepr) of spotted snakehead Channa punctata, is focussed on physicochemical, structural, evolutionary and selection pressure analyses which are poorly elucidated in teleosts in spite of that existence of these genes is well reported in several fish species. The putative full-length Lep and Lepr of C. punctata showed conserved structural and functional domains, especially the residues responsible for structural integrity and signal transduction. Conversely, residues predicted essential for Lep-Lepr interaction displayed divergence between teleosts and tetrapods. Impact of substitutions/deletions predicted using protein variation effect analyser tool highlighted species specificity in ligand-receptor interaction. Physicochemical properties of ligand and receptor predicted for the first time in vertebrates revealed high aliphatic and instability indices for both Lepa and Lepr, indicating thermostability of proteins but their instability under ex vivo conditions. Positive grand average of hydropathy score of Lepa suggests its hydrophobic nature conjecturing existence of leptin binding proteins in C. punctata. In addition to disulphide bonding, a novel posttranslational modification (S-126 phosphorylation) was predicted in Lepa of C. punctata. In Lepr, disulphide bond formation and N-linked glycosylation near WSXWS motif in ECD, and phosphorylation at tyrosine residues in ICD were predicted. Leptin and its receptor sequence of C. punctata cladded with its homolog from C. striata and C. argus of order Anabantiformes. Leptin system of Anabantiformes was phylogenetically closer to that of Pleuronectiformes, Scombriformes and Perciformes. Selection pressure analysis showed higher incidence of negative selection in teleostean leptin genes indicating limited adaptation in their structure and function. However, evidence of pervasive and episodic diversifying selection laid a foundation of co-evolution of Lepa and Lepr in teleosts.

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2.

The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.

Effect of cavitation jet on the structural, emulsifying properties and rheological properties of soybean protein‐oxidised aggregates

SummaryThe objective of this study was to investigate the effects of cavitation jets on the structural, emulsifying and rheological properties of soybean protein oxidation aggregates. The results showed that oxidation might induce the formation of larger particle sizes and molecular weight protein aggregates and the decrease of emulsifying properties. The cavitation jet at a short treatment time (<6 min) broke down the disulphide bonds and protein skeleton structures, which reduced the aggregate sizes and molecular weights and increased the emulsion activities, emulsion stabilities, apparent viscosity and elastic modulus. The cavitation jet at a long treatment time (>6 min) supported disulphide bond formation among molecules by intermolecular interactions to form protein aggregates. In addition, the skeleton structure showed cross‐linking aggregation. This increased the particle sizes and molecular weights and reduced the emulsion properties, consistency index K and elastic modulus. The findings showed that a cavitation jet at 6 min on oxidised aggregates of soybean protein might enhance the structural, emulsifying and rheological characteristics for the industry.

Silver Complexes as Anticancer Agents: A Perspective Review

Metal complexes gained more attention in the medical field, particularly cancer treatment, due to their enhanced potential and redox ability. These complexes exhibited more significant antiproliferation potential by triggering the generation of reactive oxygen species (ROS), altering cell membrane, changing cell redox potential and by DNA replication inhibition. Platinum-based metal complex Cisplatin was prepared. It was widely used in treating lung, breast, ovarian and testicular cancers; however, these complexes showed many adverse effects such as renal insufficiency and electrolyte abnormalities, neurotoxicity, ototoxicity, nausea, vomiting and drug resistance. To overcome these side effects and drug resistance mechanisms, silver metal is highly employed as a cytotoxic agent by DNA binding through the formation of disulphide bonds. Moreover, silver complexes exhibited more significant cytotoxicity with the lowest toxicity. This review highlighted silver complexes' anticancer and cytotoxic potential with various bioactive ligands.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro &in vivo bioactivity

Human epidermal growth factor (hEGF) is a mitogenic protein widely used in pharmaceutical and cosmetic industries, thus recombinant DNA technology has been applied to meet the high demand for hEGF. The overexpression of recombinant protein in E. coli often leads to the formation of inclusion bodies (IBs). Mild solubilisation preserves the native secondary protein structure in IBs, thereby the high recovery of active protein from IBs. The redox system also plays a pivotal role in the formation of disulphide bonds during refolding of disulphide bond-containing protein. This study aimed to recover hEGF from bacterial IBs through freeze-thawing solubilisation and glutathione-based oxidative refolding. CBD-Ssp DnaB-hEGF fusion protein was expressed as IBs in E. coli, washed with Triton X-100 and urea to remove most protein contaminants, then the solubilised fusion protein was obtained by freeze-thawing with the addition of 2 M urea. The solubilised protein was subsequently refolded by intein cleavage via a glutathione-based redox system. The refolded hEGF demonstrated heat-resistant properties, interacted with specific antibodies on ELISA, stimulated keratinocyte proliferation and possessed significant in vivo wound healing properties on the 8th day, confirming that hEGF was correctly folded. In summary, the protocol described is suitable for the recovery of refolded hEGF from bacterial IBs by mild solubilisation and oxidative refolding.

Role of Disulphide Bonds in Membrane Partitioning of a Viral Peptide.

The importance of disulphide bond in mediating viral peptide entry into host cells is well known. In the present work, we elucidate the role of disulphide (SS) bond in partitioning mechanism of membrane-active Hepatitis A Virus-2B (HAV-2B) peptide, which harbours three cysteine residues promoting formation of multiple SS-bonded states. The inclusion of SS-bond not only results in a compact conformation but also induces distorted α-helical hairpin geometry in comparison to SS-free state. Owing to these, the hydrophobic residues get buried, restricting the insertion of SS-bonded HAV-2B peptide into lipid packing defects and thus the partitioning of the peptide is completely or partly abolished. In this way, the disulphide bond can potentially regulate the partitioning of HAV-2B peptide such that the membrane remodelling effects of this viral peptide are significantly reduced. The current findings may have potential implications in drug designing, targeting the HAV-2B protein by promoting disulphide bond formation within its membrane-active region.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s00232-022-00218-0