Novel Small Molecule Inhibitors That Prevent the Neuroparalysis of Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is a protein exotoxin produced by Clostridium tetani that causes the deadly spastic neuroparalysis of tetanus. It consists of a metalloprotease light chain and of a heavy chain linked via a disulphide bond. TeNT binds to the neuromuscular junction (NMJ) and it is retro-axonally transported into vesicular compartments to the spinal cord, where it is released and taken up by inhibitory interneuron. Therein, the catalytic subunit is translocated into the cytoplasm where it cleaves its target protein VAMP-1/2 with consequent blockage of the release of inhibitory neurotransmitters. Vaccination with formaldehyde inactivated TeNT prevents the disease, but tetanus is still present in countries where vaccination coverage is partial. Here, we show that small molecule inhibitors interfering with TeNT trafficking or with the reduction of the interchain disulphide bond block the activity of the toxin in neuronal cultures and attenuate tetanus symptoms in vivo. These findings are relevant for the development of therapeutics against tetanus based on the inhibition of toxin molecules that are being retro-transported to or are already within the spinal cord and are, thus, not accessible to anti-TeNT immunoglobulins.

Poultry feather disulphide bond breakdown to enable bio-based polymer production

With oil supplies, needed for plastic production, decreasing dramatically, there is a clear driver for alterative polymers from sustainable resources. Poultry feathers, containing ∼90% keratin, are one source of natural polymer with huge potential for biopolymer production. However, the presence of crosslinks, known as disulphide bonds, hinders processability. This paper reviews techniques to enable breakage of disulphide bonds through use of reduction agents (sodium sulphite and sodium sulphate) and hydrolysis. Samples were analysed using FTIR and DSC to quantify achievable bond breakage, effect on thermal properties and changes in protein concentration. A review on the effect of particle size on disulphide bond breakage was also conducted, along with quantifying the reformation of bonds post-processing. Finally, a bicinchoninic acid (BCA) protein assay was used to quantify changes to soluble protein content, key to predicting if biopolymer formation can occur. The results showed a final disulphide bond breakage of between 48% and 67% was achievable using these techniques. It was also shown that disulphide bond content exhibited up to 60% bond reformation post treatment. These reductions in disulphide bonds increased the thermoplastic nature and apparent protein content. Despite achieving the highest bond breakage percentage, hydrolysis caused degradation of useful proteins, rendering the material unsuitable for biopolymer production. Results suggested that treatment with sodium sulphite (4.3% wt. of feathers) and use of a small particle size (0–100 µm), sufficiently altered the properties of raw feathers to enable feather biopolymer production.

A proteolytic nanobiocatalyst with built-in disulphide reducing properties

Proteolytic nanobiocatalysts are equipped with intrinsic disulphide bond reducing properties.

Analysis of disulphide bond linkage between CoA and protein cysteine thiols during sporulation and in spores of Bacillus species

ABSTRACT Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.

Squeezing out the Catalysts: The Disulphide Bond Exchange in Aryl Disulphides at High Hydrostatic Pressure

Exchange of the disulphide bond is a prominent example of equilibrium reactions. Therefore, the wide library of disulphide exchange reactions requires the application of catalysts, such as reducing agents, strong bases, ultraviolet light, or ultrasounds to stimulate higher conversion yields. We employed the pressure between 100 and 400 MPa, for promoting the exchange reactions between various homodimeric aryl disulphides and for optimized conditions obtained 100 % yields and pure single-crystal form of the heterodimer. The reactions were performed in a diamond-anvil cell, as well as in a hydraulic piston-and-cylinder press, and the products were characterized by X-ray diffraction, mass and NMR spectroscopy. <br>

Squeezing out the Catalysts: The Disulphide Bond Exchange in Aryl Disulphides at High Hydrostatic Pressure

Exchange of the disulphide bond is a prominent example of equilibrium reactions. Therefore, the wide library of disulphide exchange reactions requires the application of catalysts, such as reducing agents, strong bases, ultraviolet light, or ultrasounds to stimulate higher conversion yields. We employed the pressure between 100 and 400 MPa, for promoting the exchange reactions between various homodimeric aryl disulphides and for optimized conditions obtained 100 % yields and pure single-crystal form of the heterodimer. The reactions were performed in a diamond-anvil cell, as well as in a hydraulic piston-and-cylinder press, and the products were characterized by X-ray diffraction, mass and NMR spectroscopy. <br>

Squeezing out the Catalysts: The Disulphide Bond Exchange in Aryl Disulphides at High Hydrostatic Pressure

Exchange of the disulphide bond is a prominent example of equilibrium reactions. Therefore, the wide library of disulphide exchange reactions requires the application of catalysts, such as reducing agents, strong bases, ultraviolet light, or ultrasounds to stimulate higher conversion yields. We employed the pressure between 100 and 400 MPa, for promoting the exchange reactions between various homodimeric aryl disulphides and for optimized conditions obtained 100 % yields and pure single-crystal form of the heterodimer. The reactions were performed in a diamond-anvil cell, as well as in a hydraulic piston-and-cylinder press, and the products were characterized by X-ray diffraction, mass and NMR spectroscopy. <br>

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function

Variably reduced expression of the basement membrane component laminin-332 (α3aβ3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a β3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

The Impact of Acupuncture Treatment on dynamic thiol–disulphide homeostasis and ischemia-modified albumin levels to assess the oxidative stress in Migraine Patients

The aim of this study was to investigate the effect of acupuncture on dynamic thiol–disulphide homeostasis and ischemia-modified albumin (IMA) levels as a novel oxidative stress parameter in migraine patients. Acupuncture treatment was applied to the 22 volunteer migraine patients. The acupuncture treatment consists of 5 sessions with 2 sessions per week. Blood samples have been collected before performing acupuncture, after the 1st session and after the 5th session of the acupuncture. And for the control group blood samples were collected only once. A total of 44 participants were included in the study, and 24 of whom were diagnosed with migraine. The total thiol, native thiol and ischemia-modified albumin (IMA) levels in the serum were measured in all patients and healthy individuals. The dynamic disulphide bond and ischemia-modified albumin (IMA) levels were calculated from these values. The total and native thiol levels of migraine patients participating in the study were found to be significantly higher than the total and native thiol levels of healthy individuals. The total thiol, native thiol, and dynamic disulphide bond levels, and ischemia-modified albumin (IMA) were correlated with attack frequency, pain intensity, or migraine type. Thiol-disülfide homeostasis can play roles in the etiology and severity of Migraine.

Cysteine immobilisation on the polyethylene terephthalate surfaces and its effect on the haemocompatibility

Nitric oxide (NO) is an important signalling molecule involved in haemostasis. NO, present as endogenous S-nitrosothiols, is released by cysteine through a transnitrosation reaction. To exploit this mechanism, cysteine was immobilised onto the different carboxylated polyethylene terephthalate (PET) surfaces using 1-step EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) crosslinking mechanism. Immobilised cysteine concentration and NO release were dependent on the surface carboxyl density. Stability studies showed that the immobilised cysteine concentration and NO release reduced within 6 h. Immobilisation of cysteine derivatives eliminated the possibility of formation of polycysteine and its electrostatic interaction with the carboxylated PET. The immobilised cysteine concentration did not recover after DTT treatment, eliminating the possibility of disulphide bond formation. Further, cysteine was immobilised using a 2-step EDC crosslinking mechanism. Although the cysteine concentration reduced during stability studies, it recovered upon DTT treatment, indicating that cysteine forms amide bonds with the carboxylated PET and the observed decrease in cysteine concentration is probably due to the formation of disulphide bonds. The haemocompatibility of the cysteine immobilised PET surfaces showed similar results compared to the carboxylated PET. The loss of thiol groups due to the disulphide bond restricts the transnitrosation reaction. Hence, these materials can be used primarily in short-term applications.