Proline-rich Peptides Research Articles

Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin.

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonasentomophila

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the 103 but not 105 dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction.

Immunomodulatory and neurotropic activities of synthetic peptides in a model of brain injury in rats

Treatment of consequences of traumatic brain injury (TBI) remains one of the current problems of medicine. To increase the effectiveness of treatment of post-traumatic complications, various drugs are recommended, including the peptide with neuromodulatory activity Semax.The present study aims to determine the presence of neuro- and immunoprotective properties of the synthetic peptide PR5, composed of fragments of proline-rich antimicrobial peptides.The work was performed on male Wistar rats weighing 300-350 g. The “falling weight” model of mechanical brain injury was used, which mainly causes diffuse brain damage. The synthesized peptide PR5, composed of fragments of known proline-rich peptides of animal neutrophils, and the peptide preparation Semax in the form of a 1% aqueous solution were used. The drugs were administered intranasally 1 hour after TBI, then twice a day for 4 days at a dose of 100 mg/kg body weight. Control animals received physiological saline in the same regimen as the peptide preparations.TBI led to a significant decrease in body weight, but in rats receiving the peptide preparation Semax, the decrease in body weight was significantly less than in control animals, and the PR5 preparation completely prevented the decrease in body weight after TBI. After TBI, the proliferative activity of lymphocytes was suppressed and the cytotoxicity of NK cells decreased. In animals treated with peptide preparations, there was no significant suppression of NK cell cytotoxicity, and the proliferative activity of lymphocytes was restored to the level of control animals by day 14 after TBI. Both peptide preparations used contributed to higher locomotor activity, and in animals treated with the PR5 peptide, this type of activity reached the parameters of control animals. The reduction in freezing duration in groups treated with peptide preparations indicates the presence of a sedative effect.The peptide preparation PR5 was active in this series of experiments, showing immunotropic and neuroprotective activity comparable to the Semax preparation. Further studies aimed at confirming the identified types of activity of the peptide preparation PR5 may justify its prospects for clinical use as a new nootropic agent.

Enhanced Molecular Networking Shows Microbacterium sp. V1 as a Factory of Antioxidant Proline-Rich Peptides

Two linear proline-rich peptides (1-2), bearing an N-terminal pyroglutamate, were isolated from the marine bacterium Microbacterium sp. V1, associated with the marine sponge Petrosia ficiformis, collected in the volcanic CO2 vents in Ischia Island (South Italy). Peptide production was triggered at low temperature following the one strain many compounds (OSMAC) method. Both peptides were detected together with other peptides (3-8) via an integrated, untargeted MS/MS-based molecular networking and cheminformatic approach. The planar structure of the peptides was determined by extensive 1D and 2D NMR and HR-MS analysis, and the stereochemistry of the aminoacyl residues was inferred by Marfey's analysis. Peptides 1-8 are likely to arise from Microbacterium V1 tailor-made proteolysis of tryptone. Peptides 1 and 2 were shown to display antioxidant properties in the ferric-reducing antioxidant power (FRAP) assay.

Antibiotic synergist OM19r reverses aminoglycoside resistance in multidrug-resistant Escherichia coli.

The continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action. Utilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin. A proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2. Our study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

Genomic Insights into Bacterial Resistance to Proline-Rich Antimicrobial Peptide Bac7

Proline-rich antimicrobial peptides (PrAMPs) having a potent antimicrobial activity and a modest toxicity toward mammalian cells attract much attention as new templates for the development of antibiotic drugs. However, a comprehensive understanding of mechanisms of bacterial resistance development to PrAMPs is necessary before their clinical application. In this study, development of the resistance to the proline-rich bovine cathelicidin Bac71-22 derivative was characterized in the multidrug-resistant Escherichia coli clinical isolate causing the urinary tract infection. Three Bac71-22-resistant strains with ≥16-fold increase in minimal inhibitory concentrations (MICs) were selected by serially passaging after four-week experimental evolution. It was shown that in salt-containing medium, the resistance was mediated by inactivation of the SbmA transporter. The absence of salt in the selection media affected both dynamics and main molecular targets under selective pressure: a point mutation leading to the amino acid substitution N159H in the WaaP kinase responsible for heptose I phosphorylation in the LPS structure was also found. This mutation led to a phenotype with a decreased susceptibility to both the Bac71-22 and polymyxin B. Screening of antimicrobial activities with the use of a wide panel of known AMPs, including the human cathelicidin LL-37 and conventional antibiotics, against selected strains indicated no significant cross-resistance effects.

Structural basis for translation inhibition by the glycosylated drosocin peptide

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated drosocin on the ribosome at 2.0–2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, our study reveals novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

Inhibition of translation termination by the antimicrobial peptide Drosocin.

The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.

Computational design of cyclic peptides to inhibit protein-peptide interactions

Many protein-protein interactions result from the binding of one folded protein with one short peptide segment, such as complexes formed by SH3 or PDZ domains. These transient protein-peptide interactions are notably involved in cellular signaling pathways and generally have low affinities, which opens the possibility to design competitive inhibitors of these complexes. We present and assess here our computational approach, called Des3PI, to design de novo cyclic peptides with potential high affinity for protein surfaces involved in interactions with peptide segments. The results were not conclusive for two receptors, the αVβ3 integrin and the CXCR4 chemokine receptor, but were promising in the case of SH3 and PDZ domains: For the former, Des3PI was able to find at least one cyclic sequence with six hotspots that binds a SH3 domain with a better theoretical affinity to the known proline-rich RLP2 peptide. For the latter, Des3PI could identify at least four cyclic sequences with four or five hotspots that have lower binding free energies computed by the MM-PBSA method than the reference peptide GKAP.

Novel Arginine- and Proline-Rich Candidacidal Peptides Obtained through a Bioinformatic Approach

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated.

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 μg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 μg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

Structural and Functional Characteristics of the Proline-Rich Antimicrobial Peptide Minibactenecin from Leukocytes of Domestic Goat Capra hircus.

We performed structural and functional studies of minibactenecin mini-ChBac7.5Nα, a natural proline-rich cathelicidin from domestic goat Capra hircus. To identify the key residues important for the biological action of the peptide, a panel of its alanine-substituted analogues was produced. The development of E. coli resistance to the natural minibactenecin, as well as to its analogues carrying substitutions for hydrophobic amino acids in the C-terminal residues was studied. The data obtained indicate the possibility of rapid development of the resistance to this class of peptides. The main factors in the formation of the antibiotic resistance are various mutations leading to inactivation of the SbmA transporter.

Directed chemical dimerisation enhances the antibacterial activity of the antimicrobial peptide MSI-78(4–20)

Antimicrobial resistance (AMR) is on the rise, leading to 700 000 deaths worldwide in 2020. Antimicrobial peptides (AMPs) are antibiotic agents that are active against multi-drug resistant pathogens and also have a reduced risk of AMR development. Previous studies have shown that dimerisation of the proline-rich antibacterial peptide (PrAMP) Chex1–Arg20 can enhance its antimicrobial activity while also reducing its toxicity. To determine if dimerisation via a simple disulfide bond can similarly improve other classes of AMPs, the α-helical cationic peptide MSI-78(4–20) was used as a model. The monomer alone, an S-carboxamidomethyl-capped N-terminal Cys–MSI-78(4–20) analogue and the disulfide-linked dimer were successfully synthesised and their antimicrobial activity and toxicity were determined. It was shown that dimerisation enhanced antimicrobial activity against the Gram-positive opportunistic pathogen Staphylococcus aureus ATCC 29213, the Gram-negative bacteria Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 47615. The peptides showed no significant haemolytic activity with red blood cells and only induced 50% lactate dehydrogenase (LDH) release in mammalian cells at the highest tested concentration, 15 µM. The MSI-78(4–20) dimer was less cytotoxic than the monomer and S-alkyl monomer. Together, the data support the strategy of AMP chemically directed dimerisation as a means of producing potentially more therapeutically useful antimicrobial agents.

Bactericidal Properties of Proline-Rich Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF).

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1-13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1-13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1-13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1-13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195–0.39 μM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

Evaluation of Therapeutic Efficiency of Stylicin against Vibrio parahaemolyticus Infection in Shrimp Penaeus vannamei through Comparative Proteomic Approach.

The shrimp immune system defends and protects against infection by its naturally expressing antimicrobial peptides. Stylicin is a proline-rich anionic antimicrobial peptide (AMP) that exhibits potent antimicrobial activity. In this study, stylicin gene was isolated from Penaeus vannamei, cloned into vector pET-28a ( +), and overexpressed in Escherichia coli SHuffle T7 cells. The protein was purified and tested for its antibiofilm activity against shrimp pathogen Vibrio parahaemolyticus. It was resulted that the recombinant stylicin significantly reduced the biofilm formation of V. parahaemolyticus at a minimum inhibitory concentration (MIC) of 200µg. Cell aggregation was observed by using scanning electron microscopy and confocal laser scanning microscopy, and it was resulted that stylicin administration significantly affects the cell structure and biofilm density of V. parahaemolyticus. In addition, real-time PCR confirmed the downregulation (p < 0.05) of genes responsible for growth and colonization. The efficacy of stylicin was tested by injecting it into shrimp challenged with V. parahaemolyticus and 7days after infection, stylicin-treated animals recovered and survived better in both treatments (T2-100µg stylicin, - 68.8%; T1-50µg stylicin, 60%) than in control (7%) (p < 0.01). Comparative proteomic and mass spectrometry analysis of shrimp hemolymph resulted that the expressed proteins were involved in cell cycle, signal transduction, immune pathways, and stress-related proteins representing infection and recovery, and were significantly different in the stylicin-treated groups. The result of this study suggests that the stylicin can naturally boost immunity and can be used as a choice for treating V. parahaemolyticus infections in shrimp.

Identifying ligand-binding specificity of the oligopeptide receptor OppA from Bifidobacterium longum KACC91563 by structure-based molecular modeling

As a ATP-binding cassette (ABC) transporter the OppA receptor plays key roles in protecting the host organism and transporting nutrients across the intestine by the oligopeptide transporter from symbiotic bacteria and directs maturation of the host immune system. Among lactic acid bacteria, Bifidobacterium longum KACC91563, isolated from fecal samples of healthy Korean neonates, has the capability to alleviate food allergy effects. Operating as a peptide importer, the extracellular OppA receptor from gram-positive B. longum KACC91563 translocates nutrients, specifically peptides, from the outside environment of the intestinal tract to the inside of symbiotic cells. In the present study we attempt to explicate the relationship between the substrate’s specificity from the OppA importer and the probiotic effects of B. longum KACC91563 in the host intestine. It was first identified in this study the specialized structure–function relationship from the OppA importer of B. longum KACC91563 with its structural and functional determinants. This could provide insights into substrate specificity of unique immunological properties and a key switch for the substrate’s metabolism to reprogramming immune responses in the host intestine by structure-based molecular modeling. The probiotic effects of oligopeptide substrate (such as a proline-rich peptide containing at least one branched residue of leucine, isoleucine, and valine) and its metabolism for the OppA from B. longum KACC91563 are attributed to enhancement of the epithelial barrier by several different strain specific pathways to prevent the strong adhesion of pathogens.

Computer-aided design of proline-rich antimicrobial peptides based on the chemophysical properties of a peptide isolated from Olivancillaria hiatula

The chemophysical properties of a peptide isolated from Olivancillaria hiatula were combined with computational tools to design new antimicrobial peptides (AMPs). The in silico peptide design utilized arbitrary sequence shuffling, AMP sequence prediction and alignments such that putative sequences mimicked those of proline-rich AMPs (PrAMPs) and were potentially active against bacteria. Molecular modelling and docking experiments were used to monitor peptide binding to some intracellular targets like bacteria ribosome, DnaK and LasR. Peptide candidates were tested in vitro for antibacterial and antivirulence activities. Chemophysical studies of peptide extract suggested hydrophobic, acidic and proline-rich peptide properties. The amino acid signature of the extract matched that of AMPs that inhibit intracellular targets. Two of the designed PrAMP peptides (OhPrP-3 and OhPrP-5) had high affinity for the ribosome and DnaK. OhPrP-1, 2 and 4 also had favorable interactions with the biomolecular targets investigated. Peptides had bactericidal activity at the minimum inhibitory concentration against Pseudomonas aeruginosa. The designed peptides docked strongly to LasR suggesting possible interference with quorum sensing, and this was corroborated by in vitro data where sub-inhibitory doses of all peptides reduced pyocyanin and pyoverdine expression. The designed peptides can be further studied for the development of new anti-infective agents.

Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.