The biotechnological applications of transglutaminases

Transglutaminases catalyse a diverse range of reactions leading to the modification of proteins and peptides such that their physical, chemical and biological properties become changed. They are found in many different living organisms and as a consequence display subtle differences in their biochemical and physical properties. it is therefore not surprising that this group of enzymes have been exploited as applied biocatalysts in a wide range of commercial sectors varying from the textile industry to the highly lucrative cosmetic industry. in addition the pathophysiological importance of this group of enzymes has increased significantly over the last decade with their involvement noted in a number of human diseases. As a consequence their identification as therapeutic targets or as monitoring aids for a range of different diseases has caused significant interest from the diagnostics and pharmaceutical industries. This review describes some of the current applications of transglutaminases; together with their potential strategic importance and future uses.

Recent developments in particulate-based vaccines

Vaccines remain a key tool in the defence against major diseases. However, in the development of vaccines a trade off between safety and efficacy is required with newer vaccines, based on sub-unit proteins and peptides, displaying improved safety profiles yet suffering from low efficacy. Adjuvants can be employed to improve their potency, but currently there are only a limited number of adjuvant systems licensed for clinical use. Of the new adjuvants being investigated, particulate systems offer several advantages including: passive targeting to the antigen-presenting cells within the immune system, protection against adjuvant degradation, and ability for sustained antigen release. There has been a range of particulate vaccine delivery systems outlined in recent patents including polymer-based microspheres (which are generally more focused on the use of synthetic polymers, in particular the polyesters) and surfactant-based vesicles. Within these formulations, several patented systems are exploiting the use of cationic lipids which, despite their limitations in gene therapy, clearly offer strong potential as adjuvants. Within this review, the current range of particulate system technologies being investigated as potential adjuvants are discussed with regard to both their respective advantages and the potential hurdles which must be overcome for such systems to be converted into successful pharmaceutical products.

Contact lens interactions with the tear film

Biochemical changes brought about by the influence of the contact lens on the tear film are conveniently split into two categories. Firstly, the lens can remove or reduce the levels of specific components in the tear film, and secondly, the lens can augment the tear film, by stimulating the influx of new components or increasing the level of existing components. The most obvious tear film components for study in this context are lipids, proteins, mucins and electrolytes. The interactions are affected by the properties of the lens, the characteristics of the individual wearer and the wear schedule. An additional complicating factor is the fact that the lens is many times thicker than the tear film and any immobilised tear components will be more extensively exposed to oxygen and UV radiation than is the case in the absence of a lens. It is arguably the lipoidal components that are most markedly affected by lens wear, since their immobilisation on the lens surface markedly increases their susceptibility to autoxidative degradation. The limited information that is available highlights the importance of subject specificity and suggests that lipid oxidation phenomena are potentially important in contributing to the 'end of day' discomfort of symptomatic contact lens patients. It is clear that tear lipids, although regarded as relatively inert for many years, are now seen as a reactive and potentially important family of compounds in the search for understanding of contact lens-induced discomfort. The influence of the lens on tear proteins shows the greatest range of complexity. Deposition and denaturation can stimulate immune response, lower molecular weight proteins can be extensively absorbed into the lens matrix and the lens can stimulate cascade or upregulation processes leading either to the generation of additional proteins and peptides or an increase in concentration of existing components. Added to this is the stimulating influence of the lens on vascular leakage leading to the influx of plasma proteins such as albumin. The evidence from studies of mucin expression in tears is not consistent and conclusive. This is in part because sample sources, lens materials and methods of analysis vary considerably, and in some cases the study population numbers are low. Expression levels show mucin and material specificity but clear patterns of behaviour are elusive. The electrolyte composition of tears is significantly different from that of other body fluids. Sodium and potassium dominate but potassium ion concentrations in tears are much higher than in serum levels. Calcium and magnesium concentrations in tears are lower than in serum but closer to interstitial fluids. The contact lens provides the potential for increased osmolarity through enhanced evaporation and differential electrolyte concentrations between the anterior and posterior tear films. Since the changes in ocular biochemistry consequent upon contact lens wear are known to be subject-dependent - as indeed is wearer response to the lens - pre-characterisation of individual participant tear chemistry in clinical studies would enhance understanding of these complex effects. © 2013 Elsevier Ltd.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

Healthy ageing and depletion of intracellular glutathione influences T cell membrane thioredoxin-1 levels and cytokine secretion

Background: During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Intracellular thiols are maintained in their reduced state by a network of redox regulating peptides, proteins and enzymes such as glutathione, thioredoxins and thioredoxin reductase. Here we have investigated whether any relationship exists between age and secreted or cell surface thioredoxin-1, intracellular glutathione concentration and T cell surface thioredoxin 1 (Trx-1) and how this is related to interleukin (IL)-2 production.Results: Healthy older adults have reduced lymphocyte surface expression and lower circulating plasma Trx-1 concentrations. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show that cell surface Trx-1 is lowered, secretion of Trx-1 is decreased and the response to the lectin phytohaemagglutinin measured as IL-2 production is also affected. These effects are recapitulated by another glutathione depleting agent, diethylmaleate.Conclusion: Together these data suggest that a relationship exists between the intracellular redox compartment and Trx-1 proteins. Loss of lymphocyte surface Trx-1 may be a useful biomarker of healthy ageing. © 2013 Carilho Torrao et al.; licensee Chemistry Central Ltd.

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Histidine hydrogen bonding in MHC at pH 5 and pH 7 modeled by molecular docking and molecular dynamics simulations

Hydrogen bonds play important roles in maintaining the structure of proteins and in the formation of most biomolecular protein-ligand complexes. All amino acids can act as hydrogen bond donors and acceptors. Among amino acids, Histidine is unique, as it can exist in neutral or positively charged forms within the physiological pH range of 5.0 to 7.0. Histidine can thus interact with other aromatic residues as well as forming hydrogen bonds with polar and charged residues. The ability of His to exchange a proton lies at the heart of many important functional biomolecular interactions, including immunological ones. By using molecular docking and molecular dynamics simulation, we examine the influence of His protonation/deprotonation on peptide binding affinity to MHC class II proteins from locus HLA-DP. Peptide-MHC interaction underlies the adaptive cellular immune response, upon which the next generation of commercially-important vaccines will depend. Consistent with experiment, we find that peptides containing protonated His residues bind better to HLA-DP proteins than those with unprotonated His. Enhanced binding at pH 5.0 is due, in part, to additional hydrogen bonds formed between peptide His+ and DP proteins. In acidic endosomes, protein His79β is predominantly protonated. As a result, the peptide binding cleft narrows in the vicinity of His79β, which stabilizes the peptide - HLA-DP protein complex. © 2014 Bentham Science Publishers.