Protein and Peptide Letters最新文献

The bacterial cell wall is composed of a wide variety of intricate proteins in addition to lipids, glycolipids, and polymers. Given the diversity of cell wall proteins among bacterial species, they are a feasible target for biomarker identification and characterization in clinical research and diagnosis of the disease. The slow growth rate of Mycobacterium leprae poses a major hurdle in the accurate diagnosis of leprosy before the onset of peripheral neuropathy. The use of biomarker- based diagnostic methods can help in preventing the spread and manifestation of leprosy. Despite many advances in research methods and techniques, there remains a knowledge gap regarding the cell wall proteomes of M. leprae that can be used as biomarkers. The cell wall and secretory proteins of M. leprae are the major focus of this review article. This article enfolds the characteristics and functions of M. leprae cell wall proteins and gives an insight into those cell wall proteins that are yet to be established as biomarkers. Tools and techniques used in cell wall extraction and biomarker identification can also be explored in this article.

Background: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue.

Objectives: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system.

Results: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system.

Conclusion: Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Introduction: Bacterial biofilm is known as the main cause of periodontal disease. Generally, the anaerobic Gram-negative, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are considered the most identified bacteria.

Objective: This study aimed to investigate the antimicrobial effect and cytotoxicity of two experimental composites containing chitosan-silver oxide (CH-Ag₂O) particles.

Materials and methods: Four experimental groups, including Ag2O and CH, along with two composites of CH-Ag₂O 20 and CH-Ag₂O 60 mg, were prepared. Antimicrobial activity was performed against Porphyromonas gingivalis (ATCC#33277) and Fusobacterium nucleatum (ATCC#25586) using the agar dilution method. Moreover, the cytotoxicity assay was performed on human gingival fibroblasts (HGF) by the use of the MTT method. The obtained data were analyzed with descriptive methods, one-way ANOVA, and Tukey's LSD tests.

Results: The antibacterial activity of both composites was higher than both CH and Ag₂O, and the greatest antibacterial properties were presented in CH-Ag₂O 60. In all three measurements (24, 48, and 72 h), the greatest cytotoxicity was seen in Ag₂O, followed by CH, CH-Ag₂O 20, and CHAg₂O 60 in descending order, respectively. The cytotoxicity of these components was related to the concentration and not to the time of exposure. The results showed that Ag₂O in 3.7 and 7.5 μg/ml concentrations and CH-containing groups in 250 and 500 μg/ml were toxic to the cultured HGF.

Conclusion: The experimental composite containing CH-Ag₂O 60 showed the greatest antibacterial properties against two periodontal pathogens evaluated. In order to clarify the clinical significance of composite cytotoxicity, further clinical studies are necessary.

Background: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH).

Methods: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2.

Results: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (k_cat/K_m) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K⁺, Na⁺, Mg²⁺ and Cu²⁺-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg²⁺ was observed to enhance the enzyme's activity by 27% at a concentration of 300 mM. Neither K⁺ nor Na+ had a significant influence on activity. Only Cu²⁺ was found to reduce enzyme activity.

Conclusion: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

Background: We studied UPBEAT1 (UPB1) which regulated superoxide radical / hydrogen peroxide ratio together with peroxidase (POD) activity and PAL genes expression under different ways of apical meristem development during the xylem structural elements' formation in unique woody plants B. pendula var. pendula with straight-grained wood and B. pendula var. carelica with figured wood. The differentiation process predominanced in straight-grained wood (B. pendula var. pendula) or proliferation - in the figured wood. The investigation was conducted in the radial row (cambial zone - differentiating xylem - mature xylem) during the active cambial growth period.

Objective: The study aimed to study the xylogenesis processes occurring in the 16-year-old straight-grained silver birch (Betula pendula Roth) and Karelian birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti) with figured wood.

Methods: Hydrogen peroxide and superoxide radical contents and peroxidase activity were determined spectrophotometrically. Gene expression for PAL family genes and the UPBEAT1 gene was assessed using qRT-PCR.

Results: Principal component analysis has confirmed trees with straight-grained and figured wood to be different according to UPBEAT1-ROS-POD-PAL system functioning.

Conclusion: The higher superoxide radical/hydrogen peroxide ratio in figured Karelian birch, along with UPBEAT1 transcription factor and PAL genes upregulation, distinguished it from straight-grained silver birch. This metabolic picture confirmed the shift of Karelian birch xylogenesis towards proliferation processes, accompanied by ROS and phenolic compounds' flow and POD activity.

Background: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (C^I-C^IV, C^II-C^V, C^III-C^VI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks.

Aim: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments.

Objective: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 μM) and non-potent (MIC > 20 μM) cyclotides for antifungal activity.

Methodology: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico.

Results: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability.

Conclusion: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.

Background: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies).

Objectives: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed.

Methods: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy.

Results: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions.

Conclusion: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

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Necrotrophic phytopathogens pose a serious challenge to the productivity of several crops causing seedling damage, pre- and post-emergence damping-off and root rot thus reducing plant growth and yield. They are known to gain nutrition by secreting a diverse array of hydrolytic enzymes and thereby causing extensive host plant tissue maceration. Amongst the diverse hydrolases, proteases play a pivotal role in the necrotrophic mode of nutrition and thereby in determining pathogenic virulence. Host plants often counteract the necrotrophic proteolysis events by proteins (peptides), particularly through protease inhibitors (PIs). PIs play an important role in host innate immunity function by functioning as anti-metabolic proteins inhibiting the activity of phytopathogenic secretory proteases. Their abundance in plant storage organs explains their anti-nutritional interaction which stalls pathogenic invasion. PIs, therefore, constitute potential candidates that can be deployed as effective antimicrobials in agriculture, particularly against necrotrophic soil-borne pathogens. The present review traces the progress made in the identification of PIs from plants, and their inhibitory potential against necrotrophic phytopathogens and explores prospects of utilizing these molecules as effective anti-necrotrophic formulations for disease management.