Identification of the Primary Factors Determining the Specificity of Human VKORC1 Recognition by Thioredoxin-Fold Proteins

Redox (reduction–oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol–disulphide exchange reactions between PDI and hVKORC1.     

Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase

The observables associated with protein intrinsic fluorescence – spectra, time decays, anisotropies – offer opportunities to monitor in real time and non-invasively a protein‘s functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.     

Cancer Cell Response to Anthracyclines Effects: Mysteries of the Hidden Proteins Associated with These Drugs

A comprehensive proteome map of T-lymphoblastic leukemia cells and its alterations after daunorubicin, doxorubicin and mitoxantrone treatments was monitored and evaluated either by paired comparison with relevant untreated control and using multivariate classification of treated and untreated samples. With the main focus on early time intervals when the influence of apoptosis is minimized, we found significantly different levels of proteins, which corresponded to 1%–2% of the total amount of protein spots detected. According to Gene Ontology classification of biological processes, the highest representation of identified proteins for all three drugs belong to metabolic processes of proteins and nucleic acids and cellular processes, mainly cytoskeleton organisation and ubiquitin-proteasome pathway. Importantly, we observed significant proportion of changes in proteins involved in the generation of precursor metabolites and energy typical for daunorubicin, transport proteins participating in response to doxorubicin and a group of proteins of immune system characterising response to mitoxantrone. Both a paired comparison and the multivariate evaluation of quantitative data revealed daunorubicin as a distinct member of the group of anthracycline/anthracenedione drugs. A combination of identified drug specific protein changes, which may help to explain anti-cancer activity, together with the benefit of blocking activation of adaptive cancer pathways, presents important approaches to improving treatment outcomes in cancer.     

Antimicrobial effect of an Undaria pinnatifida composite film containing vanillin against Escherichia coli and its application in the packaging of smoked chicken breast

To develop a value‐added product, we used the under‐utilised seaweed Undaria pinnatifida as a source material for the fabrication of a biodegradable film by extracting U. pinnatifida protein (UPP). UPP/gelatine composite films with different constituent ratios were prepared. In addition, the UPP/gelatine composite films containing vanillin (0.05%, 0.1% and 0.5%) were prepared as antimicrobial packaging material. To evaluate the utility of the UPP composite film containing 0.5% vanillin as food packaging material, smoked chicken breast samples inoculated with Escherichia coli were packed with the film and stored at 4 °C for 10 days. It was observed that packaging of smoked chicken breast with the UPP composite film containing vanillin decreased the population of the inoculated E. coli by 1.12 log CFU g^?1 compared with that in the control sample. Thus, the UPP/gelatine composite film with added vanillin can be utilised as a packaging material for smoked chicken breast.     

Spermatozoa Transcriptional Response and Alterations in PL Proteins Properties after Exposure of Mytilus galloprovincialis to Mercury

Mercury (Hg) is an environmental pollutant that impacts human and ecosystem health. In our previous works, we reported alterations in the properties of Mytilus galloprovincialis protamine-like (PL) proteins after 24 h of exposure to subtoxic doses of toxic metals such as copper and cadmium. The present work aims to assess the effects of 24 h of exposure to 1, 10, and 100 pM HgCl₂ on spermatozoa and PL proteins of Mytilus galloprovincialis. Inductively coupled plasma–mass spectrometry indicated accumulation of this metal in the gonads of exposed mussels. Further, RT-qPCR analyses showed altered expression levels of spermatozoa mt10 and hsp70 genes. In Mytilus galloprovincialis, PL proteins represent the major basic component of sperm chromatin. These proteins, following exposure of mussels to HgCl₂, appeared, by SDS-PAGE, partly as aggregates and showed a decreased DNA-binding capacity that rendered them unable to prevent DNA damage, in the presence of CuCl₂ and H₂O₂. These results demonstrate that even these doses of HgCl₂ exposure could affect the properties of PL proteins and result in adverse effects on the reproductive system of this organism. These analyses could be useful in developing rapid and efficient chromatin-based genotoxicity assays for pollution biomonitoring programs.     

Improved microstructure and properties of the gelatin film produced through prior cross‐linking induced by horseradish peroxidase,glucose oxidase and glucose

The effects of prior enzymatic cross‐linking of bovine gelatin via horseradish peroxidase, glucose oxidase and glucose on microstructure and properties of the target film (cross‐linked gelatin film) were assessed. The cross‐linked gelatin film exhibited similar film thickness and moisture content, lower water solubility and higher opacity than the gelatin film directly prepared with bovine gelatin. The cross‐linked gelatin film also demonstrated improved thermal stability and mechanical properties, characterised by higher melting point and glass transition temperature, enhanced tensile strength and elongation at break and greater storage modulus. Prior gelatin cross‐linking resulted in 30.2% and 68.6% reductions in water vapour and CO₂ permeability of the cross‐linked gelatin film, respectively, but did not affect oil permeability. Furthermore, the cross‐linked gelatin film possessed smaller cross‐sectional voids (diameter 100?360 nm vs. 200?595 nm) than the bovine gelatin film. This study shows that cross‐linking can efficiently improve film microstructure and properties of the gelatin‐like products.     

Evolution of oxidative and structural characteristics of proteins,especially lipid transfer protein 1 (LTP1) in beer during forced-ageing

The evolution of oxidative and structural characteristics of proteins, especially lipid transfer protein 1 (LTP1), in beer during forced-ageing was examined. The oxidative characteristics of beer and proteins were evaluated by DPPH radical scavenging activity and ABTS radical cation scavenging activity. Results showed that the levels of proteins, thiols, LTP1 and antioxidant activity decreased gradually. This was accompanied by the degradation of macromolecular proteins in beer during forced-ageing. Results from circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), surface hydrophobicity (S₀) and ζ-potential further indicated that the secondary and tertiary structure of LTP1 changed drastically during forced-ageing, with the reduction of the S₀, α-helix and β-sheet contents and the increase in negative ζ-potential and random coil. Thus, the proteins, especially LTP1, might play important roles in maintaining oxidative stability of beer.     

Cell-envelope proteinases from lactic acid bacteria: Biochemical features and biotechnological applications

Proteins displayed on the cell surface of lactic acid bacteria (LAB) perform diverse and important biochemical roles. Among these, the cell-envelope proteinases (CEPs) are one of the most widely studied and most exploited for biotechnological applications. CEPs are important players in the proteolytic system of LAB, because they are required by LAB to degrade proteins in the growth media into peptides and/or amino acids required for the nitrogen nutrition of LAB. The most important area of application of CEPs is therefore in protein hydrolysis, especially in dairy products. Also, the physical location of CEPs (i.e., being cell-envelope anchored) allows for relatively easy downstream processing (e.g., extraction) of CEPs. This review describes the biochemical features and organization of CEPs and how this fits them for the purpose of protein hydrolysis. It begins with a focus on the genetic organization and expression of CEPs. The catalytic behavior and cleavage specificities of CEPs from various LAB are also discussed. Following this, the extraction and purification of most CEPs reported to date is described. The industrial applications of CEPs in food technology, health promotion, as well as in the growing area of water purification are discussed. Techniques for improving the production and catalytic efficiency of CEPs are also given an important place in this review.     

Mechanisms of molecular and structural interactions between lentil and quinoa proteins in aqueous solutions induced by pH recycling

There has been a growing interest in plant proteins due to their beneficial health effects, low cost and variety of applications in food industries. The low solubility of lentil proteins (LPs) is one of the significant factors that limit their use in food applications. Quinoa proteins (QPs), which have high water solubility, were combined with LPs at pH 12 to generate LP-QP complexes to generate pH-based soluble protein compounds. The LP-QP complexes demonstrated a large surface charge with an increase solubilisation of the protein complexes by more than 85%, together with resistance to protein aggregation. The combination of LPs to QPs led to a significant increase (P < 0.05) in unique tertiary and secondary protein structures as determined by the protein–protein interaction (PPI) technique involving pH recycling. Interactions between LPs and QPs affected the surface morphology of the protein complexes formed. Electrostatic interactions, hydrophobic forces and hydrogen bonding were indicated to play key roles in the PPIs. The capacity of pH cycling to illustrate the above protein interactions shows that this is a robust approach for assessing the emulsion and foaming properties of food proteins.