Molecular Mass Peptides Research Articles

Effects of applied current modes on the migration selectivity of peptides from a porcine cruor hydrolysate during electrodialysis with ultrafiltration membrane

Pepsic hydrolysis of porcine hemoglobin produces various antimicrobial peptides, however, within such complex hydrolysates, certain peptide bioactivities remain unexpressed. Electrodialysis with ultrafiltration membrane (EDUF) addresses this by separating peptides from hydrolysates based on peptide charge and molecular mass, resulting in fractions with increased bioactivity. This study aims to assess the impacts of different current modes (continuous current (CC), pulsed electric field (PEF) and polarity reversal (PR)) on peptide migration selectivity, which has never been reported. Different combinations of pulse and pause or reversed pulse were tested: 1 s/1 s and 10 s/1 s. This investigation focused on the 31 most migrating peptides and their 6 main physicochemical characteristics. Three groups of current conditions with similar performances were observed: (1) CC, PEF 10 s/1 s and PR 10 s/1 s (Averaged final migration rate of 5.0 g/m2h and energy consumption of 48.7 Wh/g of peptide); (2) PEF 1 s/1 s (9.0 g/m2h and 61.0 Wh/g of peptide), and (3) PR 1 s/1 s (3.3 g/m2h and 95.6 Wh/g of peptide). CC, PEF, and PR 10 s/1 s facilitated the migration of mainly cationic peptides with low molecular masses and were the most selective conditions. PR 1 s/1 s allowed the migration of some anionic peptides with lower molecular masses, due to the short polarity reversal. This study demonstrated for the first time the effects of the electrical current mode and condition associated on migration selectivity of peptides during EDUF and their performances. Modifying the electrical current mode offers a new way to optimize separation selectivity and generate specific peptide populations in the recovery compartment with potentially different bioactivities.

Chemical analysis and protein enzymatic hydrolysis of poly-floral bee pollen

This study assessed chemical components and enzymatic hydrolysis of bee pollen (BP) as an approach to modify its structure and generate bioactive peptides which can be used as functional ingredients. The food grade proteases Alcalase 2.4L and Protamex were used at optimized parameters. The degree of hydrolysis (DH), protein content and molecular mass (MM) distribution of the soluble peptides in hydrolysates were determined as indicators for enzymatic hydrolysis. The highest DH was reached by Alcalase 2.4L (10.39 %), whereas Protamex showed the lowest (9.31 %). The total protein content recovered from the hydrolysates was 16.01 % w/w for Alcalase 2.4 L and 15.51 % w/w for Protamex. Electrophoretic profile for Alcalase 2.4L and Protamex showed a range of low molecular (4.5 kDa) and low – medium mass peptides (5.8-51 kDa), respectively. Protein recovery and analysis of peptides MM distribution provides data for further research, taking into account the trend of using BP peptides in food products.

De novo profiling of insect-resistant proteins of rice via nanopore peptide differentiation

Nanopore is used as a single-molecule detector for proteins, peptides and amino acids identification of biomedical importance. We, on the first try, extended its profiling application for peptidome encoded by the insect-resistance gene in rice. Taking brown planthopper (Bph) for example, we previously cloned and verified Bph32 gene, which encodes SCR (“Bph32”) protein as direct executor against this injurious insect. However, the homology protein expressed in susceptible line doesn't have this antibiosis resistance. Hence, profiling the structural modulars (peptide domains) of Bph32 proteins may provide essential basis to understand rice in response to insects. Herein, we combined approaches of bioinformatics, biochemistry and nanopore analysis to profile the rice peptides with diverse properties. Bph32 proteins were theoretically modeled into 24 functional peptide domains using Swiss-Model workspace. Next, 22 water-soluble peptides were identified by biuret-chemistry amplified nanopore current modulations. Among those, 16 ones were distinguished at one amino acid resolution via reading the current modulation spectrum, consequently providing the peptidome fingerprints. In addition, the current modulations were evidenced as quadratic function of peptide's molecular masses. These findings suggest that nanopore may work as a new generation of mass detector for more omics analysis, especially in agricultural field where demands strongly.

Characterization of a Novel Protease Inhibitor from the Edible Mushroom Agaricus bisporus.

Protease inhibitors inhibit the activity of protease enzymes; hence, they are essentially involved in the regulation of the metabolic processes involving protease enzymes and the protection of the host organism against external damage due to proteases. These inhibitors are abundantly present in all living organisms but have not been much reported in mushrooms. Mushrooms are one of the major food components of humans, with delicious taste and high nutritional value. Mushrooms also have therapeutic and economic significance. The edible mushrooms with medicinal properties are much in commercial demand. To date, the presence of protease inhibitors has not been reported much in edible mushrooms. The present study reports the characterization of a protease inhibitor isolated from the common white button mushroom Agaricus bisporus. The objective of the present study is to characterize the novel protease inhibitor from Agaricus bisporus in order to determine its nature and activity under varying environmental conditions. The protease inhibitor was characterized through SDS PAGE, gel filtration chromatography, and de novo sequencing in order to determine its molecular mass and sequence, respectively. The optimum pH, temperature, and thermal stability were studied to determine the optimum working range of the protease inhibitor. The protease inhibitory activity (%) was determined in the presence of metal ions, surfactants, oxidizing agents, and reducing agents. The kinetic parameters and the type of inhibition exhibited by the protease inhibitor were determined using casein and trypsin protease enzyme. The protease inhibitor was found to be a low molecular mass compound of 25 kDa. The de novo sequencing matched the inhibitor against a 227 amino acid containing peptide molecular mass of 24.6 kDa molecular mass. The protease inhibitory activity (%) was found highest at pH 7.0 and temperature 50 °C, and the inhibitor was stable from pH 4.0-9.0 and temperature 30-80 °C. In the presence of metal ions, the residual protease inhibitory activity (%) enhanced in the presence of Na+, Mg2+, and Fe3+. The residual activity increased in the presence of the surfactant SDS slightly in comparison to control, while it decreased in the case of Triton-X and Tween 20. The presence of oxidizing agents, hydrogen peroxide and dimethyl sulfoxide decreased the residual inhibitory activity. The protease inhibitor's activity was unaffected by the reducing agents, dithiothreitol and β-mercaptoethanol, at up to 2mM concentration, but it decreased at higher concentrations. The inhibitor exhibited uncompetitive inhibition against trypsin with an inhibitory constant of 166 nM, indicating a strong affinity towards the protease, with a half-life of 93.90 minutes at 37 °C. Protease inhibitors isolated from mushrooms are generally small in size, more stable, and tolerant towards varying external conditions. The protease inhibitor isolated from Agaricus bisporus also exhibited similar characteristics.

Preparation process of chicken small peptide by enzymatic hydrolysis

The study optimized the enzymatic hydrolysis conditions for the production of small peptides composed of 2~3 amino acids from chicken. Experiment adapted 4 factors 3 levels orthogonal design, and result analysis showed that enzymatic hydrolysis condition has an effect on total nitrogen (T-N), total amino nitrogen (A-N), degree of hydrolysis (DH), nitrogen recovery (NR), average peptide chain length (APL) and average peptides molecular mass (APM). By main effect analysis, enzymolysis time was the main influencing factor of the DH, APL and APM. Solid-liquid ratio was the main influencing factor of NR, A-N and T-N. From quadratic curve between temperature and NR, optimal temperature can be obtained. The small peptides samples were analyzed using HPLC to find out the peptide content. These results indicated that method with simple equipment, mild reaction, and easy control, this provides a further theoretical basis for the production of meat peptides.

Scale-Up and Long-Term Study of Electrodialysis with Ultrafiltration Membrane for the Separation of a Herring Milt Hydrolysate.

Electrodialysis with ultrafiltration membrane (EDUF) was selected to separate a herring milt hydrolysate (HMH) in a scale-up and long-term study for the recovery of bioactive peptides. The scale-up was performed to maximise peptide recovery by placing a total membrane area of 0.08 m2 for each anionic and cationic compartment. Twelve consecutive runs were carried out, for a total of 69 h, with minimal salt solution cleaning in between experiments. The final peptide migration rate showed that cationic peptides had a higher average migration rate (5.2 ± 0.8 g/m2·h), compared to anionic peptides (4.7 ± 1.1 g/m2·h). Migration was also selective according to peptide identifications and molecular mass distribution where only small molecular weights were found (<1000 Da) in both recovery compartments. The areal system resistance slightly decreased during each run and the averaged values were stable in between experiments since they were all found in the 95% confidence interval. In addition, total relative energy consumption was quite consistent with an average value of 39.95 ± 6.47 Wh/g all along the 12 consecutive runs. Finally, according to membrane characterization, there was no visual fouling on the different membranes present in the EDUF cell after 69 h of treatment. This may be due to the salt cleaning in between experiments which allowed removal of peptides from the membranes, thus allowing recovering initial system working parameters at the beginning of each run. The entire process was revealed to be very consistent and repeatable in terms of peptide migration, global system resistance, and energy consumption. To the best of our knowledge, this is the first time such EDUF conditions (membrane surface, duration, and minimal salt cleaning between experiments) are being tested on a complex hydrolysate.

Enzymatic Modification of Porphyra dioica-Derived Proteins to Improve their Antioxidant Potential.

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve® and Prolyve® plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components <1 kDa. Antioxidant activity was assessed using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) inhibition, and reactive oxygen species scavenging ability, i.e., oxygen radical absorbance capacity (ORAC) and hypochlorous acid (HOCl) scavenging assays. In general, enzymatic hydrolysis of P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 μmol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS·+ assays). Such hydrolysates may be of value as functional food ingredients.

SpBOK inhibits WSSV infection by regulating the apoptotic pathway in mud crab (Scylla paramamosain)

B-cell lymphoma 2 (Bcl-2) related ovarian killer (BOK) is a member of the Bcl-2 family, which has a similar function to BAX and BAK in the process of apoptosis. However, how BOK activates the intrinsic (mitochondrial) apoptotic pathway remains poorly understood in invertebrates. In this study, SpBOK identified in mud crab is an important effector responsible for the anti-WSSV (White Spot Syndrome Virus) infection by activating the apoptotic pathway. The SpBOK gene encoded a 282 amino acid peptides (molecular mass of 29 kD), which contained four distinct Bcl-2 family homology (BH) domains. SpBOK was widely expressed in all tested tissues and up-regulated after WSSV infection in vivo. The role of SpBOK on the anti-WSSV response in mud crab was investigated by using the RNAi approach in vivo. SpBOK exerted a regulatory role in changing the mitochondrial membrane potential (⊿ψm) and activating the caspase signaling and thus induced apoptosis. Moreover, the results showed that WSSV replication in mud crab could be effectively inhibited by SpBOK. Therefore, the results of this study demonstrated that SpBOK can inhibit WSSV infection by regulating the intrinsic apoptosis pathway in mud crab.

Identification and characterization of phage protein and its activity against two strains of multidrug-resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen with a capacity to develop antibiotic resistance, which underlies a larger proportion of hospital-acquired infections and higher morbidity and mortality, compared to other bacterial infections. Effective novel approaches for treatment of infections induced by this pathogen are therefore necessary. Phage therapy represents a promising alternative solution to eradicate antibiotic-resistant pathogens. Here, we investigated phage protein efficacy against multi-drug resistant (MDR) P. aeruginosa PAR21 and PAR50 strains isolated from diabetic foot ulcer patients. The results obtained using spot assay, zymography, spectrophotometry and scanning electron microscopy at low voltage (SEM-LV) indicate that the phage protein, PA-PP, exerts activity against P. aeruginosa PAR50 while having no impact on the PAR21 strain. Using LC-MS-MS/MS and comparative analysis of the peptide molecular mass with the protein sequence database, PA-PP was identified as a member of the serine protease family, a result corroborated by its ability to digest casein. We additionally showed a capacity of PA-PP to digest porin protein on the bacterial outer membrane (OM). Moreover, synergistic activity between PA-PP protein and piperacillin led to higher sensitivity of bacterial cells to this antibiotic. Our collective findings suggest that PA-PP targets porin protein on PAR50 OM, thereby increasing its sensitivity to specific antibiotics. The adverse effects observed on bacterial cells using SEM-LV suggest further roles of this protein that remain to be established.

Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells

The venoms of wasps are a complex mixture of biologically active compounds, such as low molecular mass compounds, peptides, and proteins. The aim of the study was to evaluate the action of wasp venoms, Polybia occidentalis and Polybia fastidiosa, on the DNA of human leukocytes and on the cell cycle and genetic material of the plant model Lactuca sativa L. (lettuce). The cultured leukocytes were treated with the venoms and then evaluated by the comet assay. On another assay, seeds were exposed to a venom solution; the emitted roots were collected and the occurrence of cell cycle alterations (CCAs) and DNA fragmentation were evaluated by agarose gel electrophoresis and TUNEL assay. The results demonstrated that the venom of both wasps induces several CCAs and reduces the mitotic index (MI) on treated cells. They induced damage on human leukocytes DNA. High frequencies of fragments were observed in cells exposed to P. occidentalis venom, while those exposed to P. fastidiosa showed a high frequency of non-oriented chromosome. Both venoms induced the occurrence of various condensed nuclei (CN). This alteration is an excellent cytological mark to cell death (CD). Additionally, CD was evidenced by positive signals in TUNEL assay, by DNA fragmentation in agarose gel electrophoresis with vegetal cells, and by DNA fragmentation of the human leukocytes evaluated. Furthermore, human leukocytes exposed to the venom of P. fastidiosa had high rate of damage. The data demonstrate that both vegetal and human cells are adequate to evaluate the genotoxicity induced by venoms.

Isolation of Antimicrobial Peptides from Fritillaria pallidiflora

Polypeptide fractions from seeds of Fritillaria pallidiflora Schrenk were isolated and purified. The peptide compositions and molecular masses were determined using SDS-PAAG electrophoresis and LC-MS analysis. The results showed that the most active fraction of biocidal peptides contained greater than 20 constituents with molecular masses in the range of 1050-4727.2 Da. The isolated peptide fractions exhibited antimicrobial activity against the fungus Candida albicans.

Isolation, Identification of Lactobacillus mucosae AN1 and its Antilisterial Peptide Purification and Characterization.

Lactobacillus mucosae strain AN1 isolated from sheep milk and characterized for its probiotic suitability. In vitro evaluation of critical gut endurance properties of this strain were assessed by different screening methods such as bile salt, gastric acid, lysozyme tolerance assays, hemolytic, cholesterol reduction properties, and HT-29 cell line adhesion assay. Antibacterial peptide from this strain was purified using ammonium sulphate precipitation, gel filtration chromatography and reverse-phase HPLC. The molecular mass of peptides was determined by Tricine-SDS-PAGE and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-MS). Purified peptide was named as AN1 having a molecular mass of 10.66kDa. Helical structures of peptide were determined using circular dichroism spectroscopy. Stability of peptide AN1 towards different parameters such as pH, temperature, organic solvents, proteolytic, and glycolytic enzymes was also analyzed.

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2–6 × 108M−1s−1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.

ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using Bacillus subtilis: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution.

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC50 values of hydrolysates for ACE-inhibitory and 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500-800Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500-800Da and 1200-1500Da.

Heterologous expression and purification of plantaricin NC8, a two-peptide bacteriocin against Salmonella spp. from Lactobacillus plantarum ZJ316

Bacteriocin, which is produced by lactic acid bacteria (LAB), has the potential to act as natural preservatives in the food industry. To develop strategies to overproduce such peptides, plantaricin NC8, a class IIb LAB bacteriocin that consists of two peptides, PLNC8α and PLNC8β, was successfully heterologously expressed in Escherichia coli BL21 (DE3). PLNC8α and PLNC8β peptides were expressed as His6-tag fusion proteins and were separated by Ni2+ chelating affinity chromatography. To get the PLNC8α and PLNC8β peptides without extra amino acids in the N-terminus, the fusion proteins were cleaved by enterokinase and further purified using the Ni-NTA Sefinose™ Resin Kit. The molecular masses of peptides were checked using Tricine-SDS-PAGE and MALDI-TOF-MS. The yield of purified PLNC8α was around 2–2.5 mg/L, and the yield of PLNC8β was around 1.5–2 mg/L. The antimicrobial spectrum of cleaved peptides was detected and the synergistic action of PLNC8α and PLNC8β was preliminarily confirmed. It was found that E. coli was a suitable host for heterologous expression of plantaricin NC8 with a significant yield. Importantly, the bacteriocin appeared to be very active for controlling and inhibiting the food-borne pathogenic Gram-negative bacteria Salmonella spp., and might be useful as a natural preservative candidate.

Impact of commercial precooking of common bean (Phaseolus vulgaris) on the generation of peptides, after pepsin-pancreatin hydrolysis, capable to inhibit dipeptidyl peptidase-IV.

The objective of this research was to determine the bioactive properties of the released peptides from commercially available precook common beans (Phaseolus vulgaris). Bioactive properties and peptide profiles were evaluated in protein hydrolysates of raw and commercially precooked common beans. Five varieties (Black, Pinto, Red, Navy, and Great Northern) were selected for protein extraction, protein and peptide molecular mass profiles, and peptide sequences. Potential bioactivities of hydrolysates, including antioxidant capacity and inhibition of α-amylase, α-glucosidase, dipeptidyl peptidase-IV (DPP-IV), and angiotensin converting enzyme I (ACE) were analyzed after digestion with pepsin/pancreatin. Hydrolysates from Navy beans were the most potent inhibitors of DPP-IV with no statistical differences between precooked and raw (IC50 = 0.093 and 0.095 mg protein/mL, respectively). α-Amylase inhibition was higher for raw Red, Navy and Great Northern beans (36%, 31%, 27% relative to acarbose (rel ac)/mg protein, respectively). α-Glucosidase inhibition among all bean hydrolysates did not show significant differences; however, inhibition values were above 40% rel ac/mg protein. IC50 values for ACE were not significantly different among all bean hydrolysates (range 0.20 to 0.34 mg protein/mL), except for Red bean that presented higher IC50 values. Peptide molecular mass profile ranged from 500 to 3000 Da. A total of 11 and 17 biologically active peptide sequences were identified in raw and precooked beans, respectively. Peptide sequences YAGGS and YAAGS from raw Great Northern and precooked Pinto showed similar amino acid sequences and same potential ACE inhibition activity. Processing did not affect the bioactive properties of released peptides from precooked beans. Commercially precooked beans could contribute to the intake of bioactive peptides and promote health.

AHTPDB: a comprehensive platform for analysis and presentation of antihypertensive peptides

AHTPDB (http://crdd.osdd.net/raghava/ahtpdb/) is a manually curated database of experimentally validated antihypertensive peptides. Information pertaining to peptides with antihypertensive activity was collected from research articles and from various peptide repositories. These peptides were derived from 35 major sources that include milk, egg, fish, pork, chicken, soybean, etc. In AHTPDB, most of the peptides belong to a family of angiotensin-I converting enzyme inhibiting peptides. The current release of AHTPDB contains 5978 peptide entries among which 1694 are unique peptides. Each entry provides detailed information about a peptide like sequence, inhibitory concentration (IC50), toxicity/bitterness value, source, length, molecular mass and information related to purification of peptides. In addition, the database provides structural information of these peptides that includes predicted tertiary and secondary structures. A user-friendly web interface with various tools has been developed to retrieve and analyse the data. It is anticipated that AHTPDB will be a useful and unique resource for the researchers working in the field of antihypertensive peptides.

Physicochemical Characterization of Protein Hydrolysates Produced by Autolysis of Jumbo Squid (Dosidicus gigas) Byproducts

The physicochemical characteristics of protein hydrolysates of jumbo squid (Dosidicus gigas) byproducts (JSBP) produced by endogenous proteases at two different pH values (5.0 and 7.0) were studied using destructive and nondestructive methods. Reaction mixture samples were collected at different interval times during hydrolysis to monitor changes in the level of hydrolysis (DH) using the OPA method, the protein peptide molecular masses (MM), and SDS-PAGE. The DH increased from 3.5 to 11.2 % at pH 5.0 and from 4.8 to 17.5 % at pH 7.0. Both pH treatments exhibited similar degradation patterns with progressive proteolysis and, after 120 min of hydrolysis, yielded hydrolysates that contained MM <45 kDa proteins. It was detected lower hydrophobic amino acid exposure for the protein hydrolysates prepared at pH 5.0 compared with the hydrolysates at pH 7.0. In several wavebands, higher wavenumbers were observed in the FT-IR spectra for the pH 5.0 hydrolysates. Nine different equivalent protons were observed in the NMR spectra for both hydrolysates; these protons might belong to amino acid side chains. SEM showed substantially lower particle size for the JSBPs after hydrolysis at pH 7.0. The hydrolysate zeta potentials were −29.4 mV at pH 7.0 and −10.5 mV at pH 5.0. The pH 5.0 hydrolysates exhibited lower endothermic resistance and hydrophobicity (SoANS) compared with the pH 7.0 hydrolysates. This biophysical characterization enhances the understanding of jumbo squid byproduct hydrolysate physiochemical properties, which will aid in determining the proper use of these hydrolysates.

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC50 values differed by up to ~10-fold (5.3×106−4.5×105 conidia/ml) and LT50 values by ~40% (9.8−7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.