Search Results (65)

Sheep milk is a rich source of bioactive compounds with significant potential in functional foods and biomedical applications. It contains high levels of proteins, peptides, and fatty acids with numerous health-promoting properties for the human body. Key components such as lactoferrin, proline, orotic acid, and conjugated linoleic acid (CLA) support the prevention and treatment of chronic diseases such as diabetes, cardiovascular disease, obesity, cancer, and neurodegenerative disorders. Bioactive peptides from sheep milk regulate blood glucose levels by inhibiting enzymes such as dipeptidyl peptidase-IV (DPP-IV) and α-glucosidase, while conjugated linoleic acid improves lipid metabolism and reduces inflammation. The high-quality proteins in sheep milk are essential for tissue regeneration and maintaining muscle mass, which is particularly beneficial for the elderly and infants who are allergic to cow milk. Recently, there has been an increasing interest in hydrogel dressings enriched with bioactive substances from sheep milk, which support wound healing by supporting collagen synthesis, reducing inflammation, and having antimicrobial properties. Such hydrogels are particularly promising for the treatment of chronic wounds, burns, and diabetic ulcers, making them a valuable tool in regenerative medicine. The aim of this manuscript is to review the current reports on bioactive components of sheep milk and their potential for biomedical applications. Full article

Background: The well-studied 18-residue-long proline-rich antimicrobial designer peptide Api137 utilizes at least two lethal intracellular mechanisms that target the bacterial 70S ribosome. First, Api137 stalls the ribosome by binding to the peptidyl-transferase center, trapping the release factor, and inhibiting protein expression. Second, Api137 disrupts the assembly of the large 50S subunit of the ribosome, resulting in partially assembled pre-50S dead-end particles that are unable to form the functional 70S ribosome. Methods: All six proline residues in Api137 were substituted with 4S- and 4R-fluoro-l-proline (Fpr), which promote the cis- and trans-conformer ratio of the preceding Xaa-Pro-bond, respectively. The effect on the antibacterial activity was studied using Escherichia coli. The underlying mechanisms were investigated by studying 70S ribosome binding, inhibition of in vitro translation, and ribosome profile analysis. Results: Interestingly, the analogs were equipotent to Api137, except for the 4S-Fpr11 and 4S-Fpr16 analogs, which were four times more or less active, respectively. The most active 4S-Fpr11 analog competed the least with Api137 for its ribosome binding site, suggesting a shifted binding site. Both Fpr14 and the 4S-Fpr16 analogs disturbed 50S subunit assembly less than Api137 or not at all. The strongest effect was observed with the 4R-Fpr16 analog resulting in the lowest 70S ribosome content and the highest pre-50S particle content. This peptide also showed the strongest competition with Api137 for its binding site. However, its antibacterial activity was similar to that of Api137, possibly due to its slower cellular uptake. Conclusions: Api137 inhibits protein translation and disrupts 50S assembly, which can be adjusted by substituting specific proline residues with fluoroproline. 4R-Fpr16 potently inhibits ribosome assembly and offers a novel, unexploited clinical mechanism for future antibiotic development. Full article

Dipeptidyl peptidase-4 (DPP-4) inhibitors play a critical role in the management of type 2 diabetes; however, some synthetic drugs may cause adverse effects. Natural peptides derived from rice offer a promising alternative due to their favorable biocompatibility and development potential. In this study, an AI-assisted virtual screening pipeline integrating machine learning, molecular docking, and molecular dynamics (MD) simulations was established to identify and evaluate rice-derived DPP-4 inhibitory peptides. A random forest classification model achieved 85.37% accuracy in predicting inhibitory activity. Peptides generated by simulated enzymatic hydrolysis were screened based on machine learning and docking scores, and four proline-rich peptides (PPPPPPPPA, PPPSPPPV, PPPPPY, and CPPPPAAY) were selected for MD analysis. The simulation results showed that PPPSPPPV formed a stable complex with the DPP-4 catalytic triad (Ser592–Asp670–His702) through electrostatic and hydrophobic interactions, with low structural fluctuation (RMSF < 1.75 Å). In vitro assays revealed that PPPPPY exhibited the strongest DPP-4 inhibitory activity (IC₅₀ = 153.2 ± 5.7 μM), followed by PPPPPPPPA (177.0 ± 6.0 μM) and PPPSPPPV (216.3 ± 4.5 μM). This study presents an efficient approach combining virtual screening and experimental validation, offering a structural and mechanistic foundation for the development of natural DPP-4 inhibitory peptides as candidates for functional foods or adjunct diabetes therapies. Full article

Proline-rich antimicrobial peptides (PrAMPs) primarily exert their antimicrobial effects intracellularly, inhibiting protein synthesis. B7-005, a synthetic 16-amino acid PrAMP, has a broader antimicrobial spectrum compared to native counterparts, despite shorter PrAMPs typically exhibiting reduced activity. This study aimed to enhance B7-005’s potency by extending it with 6 or 11 amino acids derived from the C-terminal sequences of cetacean Tur1A and Lip1 PrAMPs, as well as bovine Bac7(1-35). Six chimeric derivatives were evaluated for antimicrobial and bactericidal potency, cytotoxicity, bacterial membrane permeabilization, and in vitro inhibition of protein synthesis. Extending B7-005 with sequences from other PrAMPs increased its activity against most ESKAPE+E pathogens, reducing minimum inhibitory concentration (MIC) values by 2- to 8-fold, with notable differences among bacterial species, without increasing cytotoxicity toward the A549 cell line. All chimeras retained the ability to inhibit protein synthesis in Escherichia coli and to modestly perturb the E. coli membranes like B7-005. These novel chimeric PrAMPs, particularly the 22-mer derivatives, hold promise for developing new antimicrobial agents. The study also highlights variability in bacterial responses to PrAMPs and underscores how minor sequence differences can significantly impact efficacy against specific microorganisms. PrAMPs thus represent a valuable scaffold to rationally design derivatives targeting high-priority pathogens. Full article

Antioxidants play an important role in maintaining health and enhancing food stability by neutralizing free radicals. This study aimed to extract antioxidant peptides from white-feathered chicken bones through enzymatic hydrolysis, optimize the enzymatic hydrolysis conditions, and further investigate the relevance between the amino acid composition, molecular weight, and antioxidant activity of the resulting chicken bone hydrolysate. Alcalase was the most effective enzyme for hydrolyzing cooked chicken bones compared with papain, pepsin, and trypsin, yielding hydrolysates with the highest DH and ABTS radical scavenging activity. The enzymatic conditions were optimized using single-factor experiments and response surface methodology (RSM). The optimal conditions were a substrate concentration of 10%, an enzyme-substrate ratio of 502.75 U/g, a hydrolysis temperature of 48.48 °C, and a hydrolysis time of 1.13 h. Under these conditions, the ABTS radical scavenging activity reached 83.43%. Amino acid composition analysis revealed that peptides from chicken bones were rich in glycine, glutamic acid, alanine, proline, and aspartic acid, which were associated with antioxidant functions. Among these peptides, those with a molecular weight below 3 kDa exhibited the highest antioxidant effects through membrane filtration. In summary, chicken bone hydrolysate exhibits potent antioxidant activity, nominating them for potential application as natural antioxidants investible in novel functional foods and pharmaceuticals. Full article

Antimicrobial peptides (AMPs) constitute a large and diverse group of molecules with antibacterial, antifungal, antiviral, antiprotozoan, and anticancer activity. In animals, they are key components of innate immunity involved in fighting against various pathogens. Proline-rich (Pr) AMPs are characterized by a high content of proline (and arginine) residues that can be organized into Pro-Arg-Pro motifs. Such peptides have been described in many invertebrates (annelids, crustaceans, insects, mollusks) and some vertebrates (mammals). The main objective of this review is to present the diversity of invertebrate PrAMPs, which are associated with the presence of cysteine-rich domains or whey acidic protein domains in the molecular structure, in addition to the presence of characteristic proline-rich regions. Moreover, PrAMPs can target intracellular structures in bacteria, e.g., 70S ribosomes and/or heat shock protein DnaK, leading to the inhibition of protein synthesis and accumulation of misfolded polypeptides in the cell. This unique mechanism of action makes it difficult for pathogens to acquire resistance to this type of molecule. Invertebrate PrAMPs have become the basis for the development of new synthetic analogues effective in combating pathogens. Due to their great diversity, new highly active molecules are still being searched for among PrAMPs from invertebrates. Full article

Fish by-products can be converted into high-value-added products like fish protein hydrolysates (FPHs), which have high nutritional value and are rich in bioactive peptides with health benefits. This study aims to characterise FPHs derived from salmon heads (HPSs) and Cape hake trimmings (HPHs) using Alcalase for enzymatic hydrolysis and Subcritical Water Hydrolysis (SWH) as an alternative method. All hydrolysates demonstrated high protein content (70.4–88.7%), with the degree of hydrolysis (DH) ranging from 10.7 to 36.4%. The peptide profile of FPHs indicated the breakdown of proteins into small peptides. HPSs showed higher levels of glycine and proline, while HPHs had higher concentrations of glutamic acid, leucine, threonine, and phenylalanine. Similar elemental profiles were observed in both HPHs and HPSs, and the levels of Cd, Pb, and Hg were well below the legislated limits. Hydrolysates do not have a negative effect on cell metabolism and contribute to cell growth. HPSs and HPHs exhibited high 2,2′–azino-bis(3 ethylbenzthiazoline-6)-sulfonic acid (ABTS) radical scavenging activity, Cu²⁺ and Fe²⁺ chelating activities, and angiotensin-converting enzyme (ACE) inhibitory activity, with HPHs generally displaying higher activities. The α-amylase inhibition of both FPHs was relatively low. These results indicate that HPHs are a promising natural source of nutritional compounds and bioactive peptides, making them potential candidates for use as an ingredient in new food products or nutraceuticals. SWH at 250 °C is a viable alternative to enzymatic methods for producing FPHs from salmon heads with high antioxidant and chelating properties. Full article

Introduction. The proline-rich decapeptide 10c (Bj-PRO-10c; ENWPHPQIPP) from the Bothrops jararaca snake modulates argininosuccinate synthetase (AsS) activity to stimulate L-arginine metabolite production and neuroprotection in the SH-SY5Y cell line. The relationships between structure, interactions with AsS, and neuroprotection are little known. We evaluated the neuroprotective effects of Bj-PRO-10c and three other PROs (Bn-PRO-10a, <ENWPRPKIPP; Bn-PRO-10a-MK, <ENWPRPKIPPMK; and, Bn-PRO-10c, <ENWPRPKVPP) identified from Bitis nasicornis snake venom, with a high degree of similarity to Bj-PRO-10c, on oxidative stress-induced toxicity in neuronal PC12 cells and L-arginine metabolite generation via AsS activity regulation. Methods. Cell integrity, metabolic activity, reactive oxygen species (ROS) production, and arginase activity were examined after 4 h of PRO pre-treatment and 20 h of H₂O₂-induced damage. Results. Only Bn-PRO-10a-MK and Bn-PRO-10c restored cell integrity and arginase function under oxidative stress settings, but they did not reduce ROS or cell metabolism. The MK dipeptide in Bn-PRO-10a-MK and valine (V8) in Bn-PRO-10c are important to these effects when compared to Bn-PRO-10a. Bj-PRO-10c is not neuroprotective in PC12 cells, perhaps because of their limited NMDA-type glutamate receptor activity. The PROs interaction analysis on AsS activation can be rated as follows: Bj-PRO-10c > Bn-PRO-10c > Bn-PRO-10a-MK > Bn-PRO-10a. The structure of PROs and their correlations with enzyme activity revealed that histidine (H5) and glutamine (Q7) in Bj-PRO-10c potentiated their affinity for AsS. Conclusions. Our investigation provides the first insights into the structure and molecular interactions of PROs with AsS, which could possibly further their neuropharmacological applications. Full article

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases. Full article

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a–Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals. Full article

Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112—Bac(1–10, R/Y)—and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1–10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1–10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1–10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test. Full article

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein–protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain–PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain–PRM interactions and be utilized as a general approach for other domain–peptide interactions. Full article

Impaired wound healing is a complication of diabetes, which constitutes a serious problem in clinical practice. Currently, there is a high demand on the market for local treatment options for difficult-to-heal wounds caused by diabetes. The development of dressings that accelerate wound healing has recently been the subject of much research. Sheep and camel milk is gaining importance due to the content of many bioactive substances with health-promoting effects, such as insulin, LF, proline, or CLA. Sheep and camel milk proteins are a promising source of insulin, antidiabetic, and antihypertensive peptides. Numerous studies show that local administration of insulin has a significant impact on the healing of diabetic wounds. Sheep and camel milk, due to the highest LF content among ruminants, reduces autoimmune inflammatory processes and protects against bacterial and viral infections in the wound environment. Sheep’s milk has the highest content of proline and CLA, and their addition to a hydrogel dressing can help in the development of an effective dressing material. The production of hydrogel dressings containing sheep and camel milk, which are naturally rich in the bioactive substances presented in this review, may be a promising step in the market of specialized dressings for difficult-to-heal diabetic wounds. Full article

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins’ gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5′-3′ untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes. Full article

The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species. Full article