Enzymatic browning is a biochemical process catalyzed by polyphenol oxidase (PPO), employing endogenous phenolic compounds and molecular oxygen as substrates.This intriguing reaction leads to the formation of brown or black pigments, commonly referred to as melanins, specifically occurring on the surfaces of certain fruits and vegetables.This research focuses on investigating the kinetic parameters of PPO, also known as tyrosinase, extracted from Agaricus bisporus (Paris mushroom) and Terfezia leonis (Desert truffle).The study explores the effects of pH, temperature, and substrate concentration (L-tyrosine) on PPO activity from both sources.The results demonstrate that the activity of PPO from Agaricus bisporus reaches its maximum at pH 5 and a temperature of 45 °C, with inhibition observed in the presence of excess substrate.The kinetic parameters, Vmax and Km, for Agaricus bisporus PPO were determined to be 0.07587 ΔAbs.Min^(-1) and 0.1386 mmol.l^(-1),respectively.On the other hand, the activity of PPO from Terfezia leonis reaches its peak at pH 5 and a temperature of 40 °C, and the corresponding kinetic parameters are Vmax = 66.35 μM/min and Km = 0.17 mM.Enzymatic browning has been a subject of interest for numerous scientists who have explored various techniques to inhibit or eliminate the compounds responsible for the reaction, such as oxygen, copper, substrate, or the enzyme itself.Overall, this study provides valuable insights into the kinetic behavior of PPO from Agaricus bisporus and Terfezia leonis, shedding light on potential strategies for inhibiting enzymatic browning to preserve the visual appeal and quality of food.
Terrestrial D-amino acids have been found as either free amino acids or the components of peptides and proteins.The proteins originally comprising L-amino acids sometimes change into the D-amino acid-comprising form following natural epimerization.The resulting peptides and proteins have different physical, chemical, and/or biological properties from the original forms.Looking at the oligopeptide region of proteins, the hydrophobicity of oligopeptides has been calculated using semiempirical methods.The oligopeptides partly comprising D-amino acids mostly have more hydrophobic regions than the oligopeptides comprising only L-amino acids.This research focuses on the epimerization and isomerization of aspartic acid residues of the segments in the wild type and Italian mutant of Amyloid β42 to estimate the relationship between the epimerization and the hydrophobicity change.Higher hydrophobicity is known to facilitate the aggregation and toxicity of Amyloid β42 and its mutants.This research used a linear relationship between experimental log P and calculated log P (Clog P) in which log P is the logarithm of the partition coefficient P in 1-octanol to water.The calculation using semiempirical methods (PM5) validated the relationship between the structure change and the hydrophobicity during the epimerization of the oligopeptides.The epimerization at the two common aspartic residues, D 1 and D 7 for the wild type and the Italian mutant, gave smaller Clog P values than non-epimerized ones.The epimerization at another common residue D 23 showed larger Clog P for the hexapeptide segments.Clog P values of the epimerized hexapeptide segments of the Italian mutant were larger than those of the wild type.The results suggest that the original and D-epimer at D 23 of the Italian mutant must be more hydrophobic than those of the wild type.The estimation agrees with the experimental results of aggregation.
The most common antimicrobial polypeptides in breast milk are lactoferrin (LF), lactalbumin (LA), lysozyme (LC), and lactoperoxidase (LP).The aim of the present study was to evaluate the combined effect of these polypeptides on the cells of Candida albicans, Staphylococcus aureus, and Escherichia coli in vitro by spectrophotometric method.The antimicrobial effect of LP on the microbial cells was tested without connection with the lactoperoxidase system.It was shown that LA alone did not demonstrate any antimicrobial activity.However, the LF, LP, and LC in the concentration range from 2.5 mg/ml to 20 mg/ml exhibited a direct microbicidal effect in a significantly dose-dependent manner.The combined effect of the preparations was studied at a concentration of 5 mg/ml.The total antimicrobial activity of the combination of LF and LC on S. aureus and E. coli was significantly lower than the sum of the activities of individual preparations, i.e., there was an antagonistic effect, whereas there was a slight synergy towards C. albicans.For the LF and LP pairs, the experimental and calculated values of the total activity turned out to be almost the same against both types of bacteria, whereas in the case of yeast, there was a significant antagonistic effect.For the pair LP and LC in both species of bacteria, there was an antagonistic effect, and in relation to C. albicans, these results were practically the same.Thus, the combined actions of all the three antimicrobial peptides have shown a prominent antagonistic effect.
Most peptides found in nature possess homochirality, that is, they contain almost exclusively L-amino acids.Nonetheless, D-amino acids have been discovered in peptides from various organisms.This study investigated the modes of posttranslational D-amino acid incorporation in the ribosomal peptides, dermorphin and contryphan-R, through molecular modeling and molecular docking simulation techniques.The structures and interactions of the propeptides with appropriate isomerizing enzymes (racemases) were explored.Depending on the position and interaction with the bound prodermorphin, the dimeric alanine racemase adopted either a closed or an open conformation.On the other hand, regardless of the position of the procontryphan-R, a tryptophantargeting racemase with broad substrate specificity assumed only the closed conformation.Analysis of the total energies from the different interaction types involved in the enzyme-substrate models revealed that the total energy present in the dimeric configuration of the racemase was reduced in the presence of the propeptide.A complex combination of these interactions occurred at specific racemase conformations, which suggest possible energy tradeoffs associated with the association of the racemase and the propeptide.These conformational changes may represent different mechanisms through which different racemases may act on specific substrates.Analysis of the structural bases for the D-amino acid incorporation provides insights into the rare occurrence of these non-proteinogenic amino acids in peptides.The post-translational L-to Damino acid isomerization process may be further explored for the functional benefits that it offers, including its application for the development of novel peptide-based therapies.
Proteomics is an advanced approach that aims to characterize a large number of proteins (proteome) in a single assay, or single experiment.As opposed to single protein analysis using antibodybased techniques, such as western blotting, enzyme-linked immunosorbent assay (ELISA) and fluorescent microscopy, proteomics offers a valuable opportunity to study proteins "in bulk" in order to draw a clearer picture of the molecular biology of healthy and/or diseased cells.This review discusses the fundamentals of proteomics with respect to proteome complexity, reducing sample complexity by fractionation and separation methods, and protein identification based on peptide-mass and/or amino acid sequence.This review also presents a number of common proteomics workflows, the combination of some of which is likely to reveal larger proteome coverage.Both proteomics and transcriptomics enable researchers to comprehensively study the dynamic biological activities of living cells.However, each one of them has its limitations and advantages; therefore, combining the two approaches has the potential to better elucidate cellular biological processes.
It is known that human biological fluids contain antimicrobial protective factors that include antimicrobial peptides (AMP). Concentrations of AMP vary in different biofluids and tissues. The aim of the present study is to estimate the total antimicrobial activity of different human biofluids in comparison with the activity of their AMP fractions as markers of local immunity. The antimicrobial activities of serum (SE), vaginal fluid (VF), saliva (SA), water-soluble part of skin secretion (SS) and urine (UR) of 6 healthy women, 22-25 years old, were measured by the spectrophotometric method based on the ability of the above-mentioned biofluids to disrupt the cytoplasmic membrane of Candida albicans [Patent RF No 2602298]. The results were evaluated as the percent of dye penetrated into dead cells of treated yeast cell suspensions compared to the control ones. The medians of total and AMP activities are SE – 86.6% and 33.7%; VF – 24.8% and 21.9%; SA – 18.3% and 10.9%; SS – 12.8% and 2.8%; UR – 10.0% and 0.0%, respectively. The highest level of immune defense occurs in SA whereas the lowest in UR. The antimicrobial activity of AMP in VF is maximal compared to the other biofluids. Levels of total and AMP activities of SA, SS and UR have high positive correlation between each other (Pirson’s correlation coefficients r ≥ 0,700), while none of them correlated with the activities of VF (r ≤ 0,300). Using the most appropriate method of antimicrobial activity estimation the differences between biofluids of healthy human tissues of different localization are demonstrated for the first time.
Activity of antimicrobial peptide fractions of human serum and saliva against clinically important yeasts
Cardiovascular disease, specifically atherosclerosis, is exacerbated by hypercholesterolemia. Current therapies that target lipid lowering, however, are not effective in all patients. Apolipoprotein E (apoE) plays an important role in mediating the clearance of plasma cholesterol and also exerts numerous cytoprotective responses. Our laboratory has synthesized novel therapeutics that mimic the ability of apoE to decrease plasma cholesterol. The apoE mimetic peptide AEM-2 is a dual domain peptide composed of an amphipathic helical region that binds phospholipids and a positively charged region that mediates the hepatic clearance of lipoproteins. Administration of AEM-2 to apoE null mice reduced plasma cholesterol concentration by 80% one hour post-administration. Since apoE is also known to exert anti-inflammatory effects that are independent of its ability to lower cholesterol, we tested effects of AEM-2 on lipopolysaccharide-induced responses in human THP-1 macrophages. Pre-treatment of THP-1 cells with AEM-2 significantly reduced the LPS-induced secretion of IL-6 and TNFα. Since LPS administration is associated with an increase in mitochondrial injury, we monitored effects of AEM-2 on mitochondrial function. AEM-2 significantly reduced mitochondrial superoxide formation, prevented the LPS-induced decrease in mitochondrial membrane potential and attenuated the release of cytochrome c. AEM-2 also inhibited the activities of initiator caspases 8 and 9 and effector caspase 3. The attenuation of apoptosis in AEM-2 treated cells was associated with an increase in cellular autophagy. These data suggest that AEM-2 attenuates cellular injury in LPS-treated THP-1 macrophages and facilitates the removal of cellular debris and damaged organelles via induction of autophagy.
Dipeptidyl peptidases III (DPPIII) are zinc- dependent peptidases and constitute the M49 family of metalloproteases. These enzymes are presumed to play a general role in peptide catabolism in the cell cytoplasm and may also have more specific roles in mammalian physiology. Here, we describe the recombinant production and characterization of DPPIII from the ringless honey mushroom Armillariella tabescens (ArtaDPPIII). The purified protein possessed all characteristic features expected of an enzyme of the M49 family, such as cleavage of dipeptidyl-2-naphthylamides and inhibition by the peptide tynorphin. Previous reports that the enzyme is capable of oxidizing aflatoxin B1 and thus acts as a detoxifying enzyme could not be reproduced. In accordance with its enzymatic properties, the amino acid sequence of ArtaDPPIII exhibits all characteristics established for this protein family and lacks any features that could rationalize additional enzymatic activities, such as the putative aflatoxin oxidase activity. Thus, in conclusion, ArtaDPPIII is a canonical member of the M49 family of metalloproteases and earlier claims that the enzyme carries novel and very unusual oxidative activities are not supported by our study.
The mating pheromone a-factor is a lipidated dodecapeptide found in the budding yeast S. cerevisiae. The biosynthesis of this peptide encompasses the same three-step processing pathway (farnesylation of C-terminal cysteine, C-terminal proteolysis and C-terminal methyl esterification) as Ras proteins, mutated forms of which have been found in ~30% of human cancers. For the mating of two haploid yeast cells into a diploid cell to happen, the a-factor pheromone travels to the cell surface of the opposite mating cell, where it binds and activates a G-protein coupled receptor. This lipidated-peptide/protein interaction has caught the attention of researchers studying protein prenylation, and studies have shown that this peptide can be used as a model system to understand the role of prenyl groups in protein-protein interactions. Here, we review the different methods used for the synthesis of a-factor and a-factor analogs containing C-terminal cysteine esters and the assays developed for detecting pheromone bioactivity and quantitation of pheromone efficiency. Also, we review crucial peptide modifications that have been used to investigate relationships between the structure and activity of this lipopeptide with its receptor Ste3p. Finally, we aim to discuss recent and future applications of a-factor as a chemical biology tool to study protein prenylation. These include the use of photo crosslinking reactions to map peptide/receptor interactions, the addition of fluorophore tags to visualize the peptide binding, and the use of bio-orthogonal reactions for protein labeling and protein purification.
Migration of vascular smooth muscle cells is a key element in remodeling during pulmonary arterial hypertension (PAH). We are observing key alterations in the migratory characteristics of human pulmonary artery smooth muscle cells (HPASMC) isolated from transplanted lungs of subjects with PAH. Using wound migration and barrier removal assays, we demonstrate that the PAH cells migrate under quiescent growth conditions and in the absence of pro-migratory factors such as platelet derived growth factor (PDGF). Under the same conditions, in the absence of PDGF, non-PAH HPASMC show negligible migration. The dysregulated migration initiates, in part, through phosphorylation events signaled through the unstimulated PDGF receptor via focal adhesion kinase (FAK) whose total basal expression and phosphorylation at tyrosine 391 is markedly increased in the PAH cells and is inhibited by a motif mimicking cell-permeable peptide (MMCPP) targeting the Tyr751 region of the PDGF receptor and by imatinib. However, exposure of the PAH cells to PDGF further promotes migration. Inhibition of p21 activated kinases (PAK), LIM kinases (LIMK), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) reduces both the dysregulated and the PDGF-stimulated migration. Immunofluorescence microscopy confirms these observations showing activated JNK and p38 MAPK at the edge of the wound but not in the rest of the culture in the PAH cells. The upstream inhibitors FAK (PF-573228) and imatinib block this activation of JNK and p38 at the edge of the site of injury and correspondingly inhibit migration. MMCPP which inhibit the activation of downstream effectors of migration, cofilin and caldesmon, also limit the dysregulated migration. These results highlight key pathways which point to potential targets for future therapies of pulmonary hypertension with MMCPP.
The fibronectin matrix provides mechanical and biochemical information to regulate homeostatic and pathological processes within tissues. Fibronectin consists of independently-folded modules termed Types I, II and III. In response to cellular contractile force, Type III domains unfold to initiate a series of homophilic binding events which result in the assembly of a complex network of intertwining fibrils. The unfolding of Type III modules provides elasticity to the assembled fibronectin matrix allowing it to function as a dynamic scaffold which provides binding sites for cellular receptors, growth factors and other matrix molecules. Access to bioactive sites within the fibronectin matrix is under complex regulation and controlled through a combination of mechanical and proteolytic activity. Mechanical unfolding of Type III modules and limited proteolysis can alter the topographical display of bioactive sites within the fibronectin fibrils by exposing previously cryptic sites and disrupting functional sites. In this review we will discuss cryptic activity found within the first Type III module of fibronectin and its impact on tissue angiogenesis and inflammation.
Peptide libraries are useful tools to investigate the relationship between structure and function of proteins. The creation of peptide libraries with free C-termini presents unique synthetic challenges. In this review, methods for creating peptide libraries using either solid-phase peptide synthesis or phage display are described. Methods for screening such libraries and their application in studying several important biological problems are also reported.
Human immunodeficiency virus type 1 (HIV-1) infection remains to be one of the major global health problems. It is thus necessary to identify novel therapeutic molecules to combat HIV-1. Natural antimicrobial peptides (AMPs) have been recognized as promising templates for developing topical microbicides. This review systematically discusses over 80 anti-HIV peptides annotated in the antimicrobial peptide database (http://aps.unmc.edu/AP). Such peptides have been discovered from bacteria, plants, and animals. Examples include gramicidin and bacteriocins from bacteria, cyclotides from plants, melittins and cecropins from insects, piscidins from fish, ascaphins, caerins, dermaseptins, esculentins, and maximins from amphibians, and cathelicidins and defensins from vertebrates. These peptides appear to work by different mechanisms and could block viral entry in multiple ways. As additional advantages, such anti-HIV peptides may possess other desired features such as antibacterial, antiparasital, spermicidal, and anticancer activity. With continued optimization of peptide stability, production, formulation and delivery methods, it is anticipated that some of these compounds may eventually become new anti-HIV drugs.
