Disulphide Bonds Research Articles

Rheological properties and in vitro digestibility of chicken breast myofibrillar protein containing various psyllium (Plantago ovata) husk concentrations

SummaryMost dietary fibres improved rheological properties but negatively impacted digestibility. However, the effect of psyllium husk powder (PHP) on digestibility has not been yet reported. Thus, this study aimed to evaluate the rheological properties and digestibility of chicken myofibrillar protein (MP) containing different concentrations of PHP (0%, 0.5%, 1.0%, 1.5%) at 0.45 m NaCl. To assess the rheological properties of MP, viscosity, hydrophobicity, disulphide bond, SDS–PAGE, cooking yield and gel strength were evaluated. The addition of PHP enhanced water retention capacity and protein–protein interactions in the MP system. To evaluate the effect of PHP on digestibility, protein digestibility, SDS–PAGE and microstructure observations were performed. It was confirmed that the addition of PHP improved the protein digestibility of MP by measuring the differences in protein content (%). Therefore, the addition of PHP into MP gel improved not only the rheological properties but also the protein digestibility during in vitro digestion.

7375 Severe Hypoglycemia In Graves’ Hyperthyroidism: A Rare Consequence Of Methimazole Induced Insulin Autoimmune Syndrome

Abstract Disclosure: J. Ratnasingam: None. N. Ismail: None. Y. Ng: None. T. Sarvanandan: None. N. Hee: None. Q. Lim: None. L. Lim: None. S. Paramasivam: None. P. Sthaneshwar: None. S. Vethakkan: None. Background: Islet autoimmune syndrome (IAS) is a rare disease characterized by elevated insulin autoantibodies (IAA), triggered by genetic, environmental or drug (sulfhydryl containing) factors. We describe a case of Graves’ hyperthyroidism who developed IAS with methimazole. Clinical Case: A 28-year-old Malaysian-Chinese woman presented with tremors and palpitations. She weighed 56 kg, had no palpable goiter or thyroid eye signs. The diagnosis of Graves’ disease was confirmed with ft4 66.5 (11.5-22.7)pmol/L and TSH <0.01 (0.55-4.78)mIU/L with elevated thyroid stimulating immunoglobulin of 5.4 (< 0.55)IU/L. She was treated with methimazole 20mg BID and Propranolol 20mg TID. After 5 weeks of methimazole, she experienced recurrent episodes of sweating and presented with a generalized tonic-clonic seizure with capillary blood glucose of 27mg/dL. Seizures aborted after resuscitation with dextrose. There was no history of fever, exposure to insulin or other drugs, weight gain, family history of diabetes or alcohol use. CT brain and other parameters were normal and she had no other apparent cause of seizures. During the hypoglycemic episode; with the plasma glucose of 32 mg/dL, she had disproportionately high insulin: 9309 (3-25)mIU/L relative to C-peptide: 18.6 (0.9-7.1)ng/mL, with appropriate cortisol levels of 958 nmol/L. Sulphonylurea screen and pancreatic imaging was negative. Low insulin recovery was documented following PEG precipitation suggesting the presence of anti-insulin antibody. Elevated serum IAA levels at >20 (<2.4)U/mL with positive HLA-DRB1*04:06, confirmed the diagnosis of IAS. Multiple small, low carbohydrate and frequent meals were prescribed to prevent hypoglycemia and methimazole was discontinued as she had mild hyperthyroidism at this point (ft4 20.6 pmol/L, TSH <0.01mIU/L). Three days after discontinuation of methimazole, hypoglycemia completely resolved without need for dextrose infusion or corticosteroids. She received immediate 131-iodine therapy and developed hypothyroidism within two months, requiring levothyroxine replacement. She had not reported any hypoglycemia since and repeated IAA titre significantly diminished to 3.2 U/mL two months after discontinuation of methimazole. Conclusion: Methimazole-induced IAS is a rare cause of insulin mediated hypoglycemia, caused by cleaving of the disulphide bonds of insulin by the drug, leading to a cascade of autoimmune response in susceptible individuals. This is the first reported Malaysian case of methimazole- induced IAS. To date, this condition have almost always been reported in East- Asians in whom the DRB1*0406 genotype is relatively common. Unprovoked hypoglycaemia in a patient on anti-thyroid medications should prompt clinicians to consider methimazole-induced IAS. Presentation: 6/1/2024

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.

Proteomic analysis revealing mechanisms of κ-carrageenan modulating the gelling properties of heat-induced microbial transglutaminase cross-linked myofibrillar protein gels

The effects of κ-carrageenan (KC, 0.2 %, w/w) on the gelling properties and microstructures of heat-treated myofibrillar protein (MP) gels mediated by various levels (0, 0.1, 0.3, and 0.5 %, w/w) of microbial transglutaminase (TG) were investigated. The results revealed that regardless of TG level, the existence of KC significantly promoted the forming of disulphide bonds but limited the generation of large-sized protein polymers in MP gel treated with TG alone, as well as subsequently enhanced the water retention ability and gel strength of mixed MP gel, especially in the sample adding 0.3% TG and 0.2% KC, which was clearly verified by the forming of more denser and continuous gel network. Moreover, TG-induced changes in protein structure led to the alteration in the protein profiles of different MP gels, and an obvious down-regulation trend of myosin (Q9TV63, Q9TV62, P79293, and Q9TV61) was identified in the TG-0.3 group. Furthermore, liquid chromatography–tandem mass spectrometry analysis demonstrated that TG mainly induces protein crosslinking in the light meromyosin domain of the myosin rod. Meanwhile, in the presence of KC, the crosslinking-site percentage of the TG-0.3 group decreased from 37.7% to 36.2%, indicating that KC potentially reduces TG-induced crosslinking in MP gels. Thus, our findings illustrate the critical mechanisms underlying the modulation of KC on the gelling properties of TG-mediated MP gels at the molecular level, which providing potential guideline for the combined applications of TG and KC in the meat industry.

Outer membrane protein assembly mediated by BAM-SurA complexes

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Secondary structure propensities of the Ebola delta peptide E40 in solution and model membrane environments

The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na+ ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles.

Proximity-driven site-specific cyclization of phage-displayed peptides

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

Biochemical and Structural Characterization of a Novel Psychrophilic Laccase (Multicopper Oxidase) Discovered from Oenococcus oeni 229 (ENOLAB 4002).

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

Modulation of pea protein isolate hydrogels by adding kappa‐carrageenan: gelling properties and formation mechanism

SummaryOur study focuses on the gelling properties of pea protein (PP), which are comparatively weaker than those of other plant proteins, thereby limiting its application. However, we found that the addition of κ‐carrageenan (κ‐CAR) significantly (P < 0.05) increased PP gels' hardness and water‐holding capacity irrespective of gel formation temperature (80 or 95 °C). Rheological and microstructural analysis confirmed a synergistic effect between PP and κ‐CAR, with higher synergy values observed in hydrogels formulated at 95 °C. Molecular force studies revealed that non‐covalent interactions were dominant, with disulphide bonds contributing only to the structure of gels formed at 95 °C. These promising findings not only enhance our understanding of the PP and κ‐CAR gel formation mechanism but also hold practical implications, potentially leading to improved gelling properties of PP and expanding its applications in the food industry.

Rational design of disulfide bonds to increase thermostability of Rhodococcus opacus catechol 1,2 dioxygenase.

Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T50, a 725% increase in half-life, a 5.5°C increase in Tm, and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase.

A dual-functional α-diketone-based disulfide monomer for visible LED photopolymerization: Mitigating volumetric shrinkage and triggering polymerization

A α-diketone-based disulfide monomer (OHS), which serves the function of both volumetric shrinkage decreases and photopolymerization initiation under the irradiation of 405 light-emitting diode (LED) was designed and synthesized. The photochemical and photoinitiating performances were study by ultraviolet–visible spectrophotometer, electron spin resonance spin trapping (ESR-ST) experiments and real-time infrared spectrometer (FTIR). Despite OHS exhibits weak absorption at approximately 405 nm, OHS can effectively produce aromatic sulfur radicals to induce photopolymerization of itself and the bisphenol A glycerolate dimethacrylate (Bis-GMA)/triethylene glycol dimethacrylate (TEGDMA) photopolymerizable system under 405 nm LED exposure. With increasing OHS addition, the final double bond conversion and maximum polymerization rate of the Bis-GMA/TEGDMA system improved, reaching up to 86.0% and 2.33% s-1, respectively. Importantly, OHS significantly reduces the volumetric shrinkage ratio of the Bis-GMA/TEGDMA photopolymerizable system, with the volumetric shrinkage ratio (5.01%) of the system containing 5% OHS declining by almost 50% compared to that (8.51%) of the control system without OHS. This reduction is attributed to reversible characteristic of the disulphide bonds and intermolecular hydrogen bondings. Furthermore, OHS has little influence on the gelation ratio, thermostability, and frictional resistance of the cured film.

Effect of curdlan on the gel properties and interactions of whey protein isolate gels

This study investigated the influence of curdlan on the gel properties of whey protein isolate (WPI). Results demonstrated that curdlan significantly improved the water-holding capacity, gel strength and rheological properties of the WPI gels. Moreover, it promoted the unfolding of the molecular structures of WPI, which was manifested by the transition from α-helix to β-sheet, an increase in free sulfhydryl content and a decrease in surface hydrophobicity. Furthermore, 4 % curdlan promoted the formation of WPI with uniform and compact elastic gel network structures, primarily attributed to disulphide bonds, hydrogen bonds and hydrophobic interactions. However, when the addition of curdlan exceeds 4 %, excessive entanglement of curdlan chains and steric hindrance effects hinder the unfolding and folding of protein structures, weaken their interaction, result in a loose network structure and affect the gel properties. In conclusion, this study demonstrates that curdlan can effectively improve the gelling properties of WPI, suggesting its potential application in low-calorie gel-based dairy products.

Incorporation of cross-linked/acetylated tapioca starches on the gelling properties, rheological behaviour, and microstructure of low-salt myofibrillar protein gels: Perspective on phase transition.

This study investigated the gelling properties, rheological behaviour, and microstructure of heat-induced, low-salt myofibrillar protein (MP) gels containing different levels (2%, 4%, 6%, and 8%, w/w) of cross-linked (CTS) or acetylated (ATS) tapioca starch. The results indicated that either CTS or ATS significantly enhanced the gel strength and water-holding capacity of low-salt MP gels (P < 0.05), an outcome verified by the rheological behaviour test results under different modes. Furthermore, iodine-staining images indicated that the MP-dominated continuous phase gradually transited to a starch-dominated phase with increasing CTS or ATS levels, and 4% was the critical point for this phase transition. In addition, hydrophobic interactions and disulphide bonds constituted the major intermolecular forces of low-salt MP gels, effectively promoting phase transition. In brief, modified tapioca starches possess considerable potential application value in low-salt meat products.

Structure–activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus

BackgroundThe liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host’s immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure–activity relationships of its intramolecular disulphide bonds.ResultsThe predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa.ConclusionsOur study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

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.

Non-esterified fatty acid palmitate facilitates oxidative endoplasmic reticulum stress and apoptosis of β-cells by upregulating ERO-1α expression

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to β-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1β, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic β-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent β-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated β-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic β-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and β-cell failure.

Vitamin K Epoxide Reductase Complex-Protein Disulphide Isomerase Assemblies in the Thiol-Disulphide Exchange Reactions: Portrayal of Precursor-to-Successor Complexes.

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme that converts vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents supplied by its redox partner through thiol-disulphide exchange reactions. The functionally related molecular complexes assembled during this process have never been described, except for a proposed de novo model of a 'precursor' complex of hVKORC1 associated with protein disulphide isomerase (PDI). Using numerical approaches (in silico modelling and molecular dynamics simulation), we generated alternative 3D models for each molecular complex bonded either covalently or non-covalently. These models differ in the orientation of the PDI relative to hVKORC1 and in the cysteine residue involved in forming protein-protein disulphide bonds. Based on a comparative analysis of these models' shape, folding, and conformational dynamics, the most probable putative complexes, mimicking the 'precursor', 'intermediate', and 'successor' states, were suggested. In addition, we propose using these complexes to develop the 'allo-network drugs' necessary for treating blood diseases.