Protein and Peptide Letters - Volume 29, Issue 8, 2022

In recent years, the antibiotic resistance of pathogenic bacteria has become an increasing public health threat. Finding alternatives to antibiotics may be an effective solution to the problem of drug resistance. Antimicrobial peptides are small peptides produced by various organisms that are considered to be effective antibiotic substitutes because of their strong, broad-spectrum antibacterial activity, stability, and diversity, and because target strains do not easily develop resistance. Recent research on antimicrobial peptides has shown they have broad potential for applications in medicine, agriculture, food, and animal feed. The source, classification, acquisition methods, and mechanisms of action of antimicrobial peptides are very complex. This review presents an overview of research on the sources, isolation, expression and purification, and classification of antimicrobial peptides; the relationship between their structural conformation and function; their mechanisms of action; and application prospects.

Background: To recognize the action of pharmacologically approved anticancer drugs in biological systems, information regarding its pharmacokinetics, such as its transport within the plasma and delivery to its target site, is essential. In this study, we have tried to collect and present complete information about how these drugs bind to human serum albumin (HSA) protein. HSA functions as the main transport protein for an enormous variety of ligands in circulation and plays a vital role in the efficacy, metabolism, distribution, and elimination of these agents. Methods: Therefore, this study includes information about the quenching constant, the binding constant obtained from Stern-Volmer and Hill equations, and molecular docking. Results: Molecular docking was carried out to detect the binding models of HSA–anticancer drugs and the binding site of the drugs in HSA, which further revealed the contribution of amino acid residues of HSA in the drug complex binding. Conclusion: This review study showed that site I of the protein located in domain II can be considered the most critical binding site for anticancer drugs.

Nuclear factor erythroid-2-related factor 2 (Nrf2), an inducible transcription factor in phase II metabolic reactions, as well as xenobiotic response pathway, is referred to as ‘master regulator’ in anti-oxidant, anti-inflammatory, and xenobiotic detoxification processes. The activity of Nrf2 is tightly regulated by KEAP1, which promotes ubiquitination, followed by degradation under homeostatic conditions and also allows Nrf2 to escape ubiquitination, accumulate within the cell, and translocate in the nucleus upon exposure to the stresses. The Nrf2 pathway has shown an intrinsic mechanism of defense against oxidative stress (OS). It emerged as a promising therapeutic target as both inducers and as there is an increasing number of evidence for the protective role of the Nrf2-ARE pathway towards exacerbations of ROS generation as well as OS, mitochondrial dysfunction as well as prolonged neuroinflammation is a prevalent pathophysiological process rooted in brain-related disorders. Elevated concentrations of ROS generation and OS have been linked to the pathophysiology of a diverse array of brain related disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Friedrich’s ataxia, multiple sclerosis, and epilepsy. Further, it not only modulates the articulation of anti-oxidant genes but has often been associated with implicating anti-inflammatory consequences as well as regulating mitochondrial functionalities and biogenesis. Therefore, Nrf2 can be considered a potential therapeutic target for the regimen of various brain-related disorders.

Background: Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. Objective: In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. Methods: Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. Results: Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. Conclusion: ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Introduction: Streptococcus pneumoniae is a Gram-positive diplococci bacteria that causes infectious diseases such as otitis, meningitis, and pneumonia. Streptococcus pneumoniae has various virulence factors, one of which is pilus. In addition to being immunogenic, pilus S. pneumoniae also plays a role in bacterial adhesion to host cells and biofilm formation. The S. pneumoniae pilus found in this study consisted of several proteins with various molecular weights, one of which was a 67 kDa protein. Objective: This study aimed to determine the characteristics of the 67 kDa pilus protein, including its capacity as hemagglutinin and adhesin and its amino acid sequence (AA). Methods: The LCMS/MS method is used to determine the AA sequence of the 67 kDa pilus protein. The AA structure was analyzed through BLASTP by matching it with the sequence of the protein data bank of S. pneumoniae (taxid: 1313). The ProtParam tool from ExPASY was used to calculate various physical and chemical parameters of the protein, while for evaluating its immunogenicity, the VaxiJen V2.0 online server was used. Results: The results of this study indicate that the 67 kD a pilus protein, is an anti-hemagglutinin protein and has a role as an adhesin protein. Adhesion tests show the action between protein concentration and the number of bacteria attached to enterocyte cells. LCMS/MS test results obtained by BLASTP showed that the 67 kDa pilus protein had three AA sequences (ITYMSPDFAAPTLAGLDDATK, AEFVEVTK, and LVVSTQTALA), which had similarities with the A backbone chain of S. pneumoniae pilus. The physicochemical test showed that the protein is hydrophilic and nonpolar, while the antigenicity test showed that the protein is antigenic. Conclusion: Based on these characteristics, it can be concluded that the 67 kDa S. pneumoniae pilus protein can be used as a vaccine candidate for pneumococcus.

Introduction: A comprehensive study of enzymes of the antioxidant system (AOS) and phenolic metabolism is an actual subject of biochemical research; changes in the activity of these enzymes can be used as a diagnostic sign. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions. The article presents the chemical kinetics study of reactions catalyzed by superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues (Pinus sylvestris L.). The dependence of the enzyme reaction rate on the enzyme concentration and the substrate concentration is presented, and the pH-optimum for each reaction is established. Background: Determination of AOS enzyme activity and PAL activity in woody plants has many difficulties. The chemical composition of pine trunk tissues affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics. Objective: This study aimed at determining the AOS enzyme activity in 40-year-old Scots pine (Pinus sylvestris L.) plants growing in the Karelian (Russia) forest seed plantation. Methods: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4 °C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 15-min of extraction, the homogenate was centrifuged at 12000 g for 10 min (MPW-351R centrifuge, Poland). The supernatant was purified on 20 cm3 columns with Sephadex G-250. Aliquots with the highest protein amount were collected. In tissues, the protein concentration was 10-50 μg/ml. Proteins in the extracts were quantified by a Bradford assay. The enzyme activity was determined spectrophotometrically on a SpectroStar Nano plate spectrophotometer (BMG Labtech, Germany). Results: Our study made it possible to modify the methods for determining the activity of superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues. The enzymatic reaction rate dependence on the enzyme concentration and the substrate concentration was determined, and pH-optimum was also noted. This methodological article also provides formulas for calculating the activities of the enzymes. Conclusion: We found that determining AOS enzyme activity and PAL activity in woody plants is challenging. The chemical composition of the xylem and phloem of pine affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics.

Background: The fight against cancer has started since its discovery and has not subsided to nowadays. Currently, the hybrid molecules have become a promising alternative to the standard chemotherapeutics for the treatment of multi-causal diseases, including cancers. Objective: Herein, we report the synthesis, biological evaluation, mathematical docking calculations and hydrolytic stability of the new bioconjugates of monofluorinated analogues of BIM-23052, containing second pharmacophore naphthalimide, caffeic acid or the tripeptide Arg-Gly-Asp. Methods: All new molecules are obtained using standard peptide synthesis on solid support. Anticancer potential is studied against a panel of tumor cell lines included human mammary carcinoma cell lines MCF-7 (ER+, PR+ and Her-2-); MDA-MB-231 (ER-, PR- and Her-2-), as well as cell lines BALB 3T3 (mouse embryonic fibroblasts) and MCF-10A (human breast epithelial cell line). Results: The IC50 values found in the MCF-10A cell line assay were used to calculate the selective index (SI). The highest SI relative to MCF-7, with a value of 2.62 is shown by the compound Npht- Gly-D-Phe-Phe(4-F)-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2. In MCF-10 cells, the weakest antiproliferative effect was caused by the same compound (IC50 = 622.9 ± 23.91 μM), which makes this analogue a good candidate for the new anticancer medical drug. Unfortunately, the hydrolytic stability studies reveal that this bioconjugate is the most unstable of hydrolysis under physiological conditions in the body. Conclusion: Even with lower anticancer activity and selectivity in comparison with Npht-Gly-DPhe- Phe(4-F)-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2, the compound Arg-Gly-Asp-D-Phe-Phe(4-F)-Phe- D-Trp-Lys-Thr-Phe-Thr-NH2 is the best candidate between three investigated bioconjugates for practical application due to combination of activity and stability profiles. Mathematical docking calculation also reveals that synthesized bioconjugates show selectivity according to different somatostatin receptors on the surface of different cell lines.