Showing papers in "International Journal of Peptide Research and Therapeutics in 2018"

Apoptotic Effect of Melittin Purified from Iranian Honey Bee Venom on Human Cervical Cancer HeLa Cell Line.

Abstract: Melittin, an amphipathic 26-residue peptide, is the main component of honey bee venom. Studies have been demonstrated that melittin has an inhibitory effect on proliferation of cancer cells. However, the precise mechanism of action is not completely understood. In the present study we have shown that purified melittin from Iranian honey bee venom shows anti-cancer effects on human cervical cancer cell line through induction of apoptosis. The venom was collected from Iranian honey bee (Apis mellifera meda) and melittin isolated using reversed phase HPLC. Biological activity of melittin was analyzed by hemolytic test on human red blood cells. In order to investigate whether melittin inhibits proliferation of cervical cancer cells, the viability of the melittin treated HeLa cell line was measured via MTT assay. Finally, cell death analysis was performed using Propidum iodide and Annexin V-FITC dual staining. The results showed that the half hemolytic concentration (HD50) induced by mellitin was 0.5 µg/ml in free FBS solution. IC50 obtained after 12 h at 1.8 µg/ml by MTT assay. According to flow cytometric analysis, melittin induced apoptosis at concentrations more than 1 µg/ml. These results suggest that melittin induces apoptotic cell death in cervical cancerous cells as observed by flow cytometric assay. It is concluded that melittin could be regarded as a potential candidate in future studies to discovery of new anticancer agents.

Influence of Whey Protein Concentrate on the Production of Antibacterial Peptides Derived from Fermented Milk by Lactic Acid Bacteria

Abstract: In the study, growth, proteolysis and antimicrobial activity of lactic acid bacteria were evaluated in skim milk medium supplemented with different concentration of whey protein concentrate (WPC 70). Lactobacillus helveticus (V3) showed maximum pH reduction with 1% WPC. Lactobacillus rhamnosus (NS4) also produced maximum lactic acid production and viable cells counts at 1 and 1.5% WPC, respectively. However, V3 showed maximum proteolytic activity with 1.5% WPC. Streptococcus thermophilus (MD2) was found to exhibit maximum antimicrobial activity with 1.5% WPC. Peptides formed during fermentation were purified by RP-HPLC and identified using RP-LC/MS analysis. Antimicrobial peptide was identified as lactoferrin, which was found in fermented milk supplemented with 1.5% WPC by NS4.

Bioactive Peptides from Marine Ascidians and Future Drug Development–A Review

Abstract: Marine ascidians are considered as one of the richest sources of bioactive compounds. The extraction and utilization of marine peptides have been attracted much attention owing to their potential health benefits. Most of the bioactive compounds from marine ascidians are already in different phases of the clinical and preclinical pipeline. They can be used in different functional and nutraceutical values due to their antineoplastic, antihypertensive, antioxidant, and antimicrobial properties. The screening in vivo and in vitro bioassays are coupled to the purification process for the exploration of its biological interest which is of great value. The growing significance to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs and other drugs are in clinical/pre-clinical trials. The present review highlights the recent research progress in marine ascidians’ peptides and its prospects for the future pharmaceutical development.

In Vitro and In Silico Analysis of Novel ACE-Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

Abstract: In the study, two Lactobacillus cultures i.e. L. casei (NK9) and L. fermentum (LF) were studied for their proteolytic activity, di and tripeptidase activity, ACE-inhibitory activity and peptides production under optimized growth condition from fermented goat milk (Capra aegagrus hircus). NK9 and LF were found to be a strong proteolytic culture with 2.0% rate of inoculation after 48 h. LF (10 kDa retentate) produced maximum peptides among all the retentates of the fermented goat milk. Goat milk fermented with NK9 (10 kDa permeates) exhibited peptide sequence i.e. AFPEHK which had ACE inhibitory activity, matched with goat milk protein databases of AHTPDB. However, L. casei (NK9) and L. fermentum (LF) could be explored for the production of ACE inhibitory peptides from fermented goat milk.

Isolation, Purification and Characterization of Antimicrobial Peptides Produced from Saccharomyces boulardii

Abstract: Saccharomyces boulardii was used for antimicrobial peptides production. Separation process of produced antimicrobial peptides was conducted using ultrafiltration technique through dialysis membranes with porous 10 (MWCO) kDa. The inhibition activity was determined against four bacterial isolates. As a result, higher inhibition zone against Bacillus cereus were 26, 29 and 33 mm after adding 50, 75 and 100 µL of concentrated peptide, respectively. After that, peptide passed through the Sephadex G-50 column to achieve purified peptide using gel filtration. The high activity of purified peptide was confirmed based on the second peak reaching to 37 mm of bacterial inhibition zone while other peaks did not show any inhibition against tested bacteria. Some of the important characteristics of purified bioactive peptide were applied. Antimicrobial peptides stability was studied and found to be stable at pH range from 5 to 7 values studied in addition to its inhibition activity reached to 100%. Regarding thermal stability, it was observed that the peptide was fully activity at a both 60–80 °C for 30 min. Moreover, molecular weight of a peptide was identified using electrophoresis technique with SDS measured at 5792 Dalton.

Kinetics Study of Protein Hydrolysis and Inhibition of Angiotensin Converting Enzyme by Peptides Hydrolysate Extracted from Walnut

Abstract: Bioactive peptides are defined as protein-based components having nutritional value and have proved roles important for the human health. In this study inhibition of angiotensin converting enzyme (ACE) by protein-based hydrolysate extracted from walnut (Juglanse regia. L.) seeds was evaluated. The peptide fraction obtained by enzymatic hydrolysis with trypsin showed higher ACE-inhibitory and lower IC50 value (0.39 ± 0.05 mg/mL) than obtained by hydrolysis with chymotrypsin and proteinase K. The study of kinetics showed that by increasing the concentration of the trypsin hydrolysate from 0.01–0.5 mg/mL, Km increased, while Vmax decreased. Also the value of Ki was found to be 0.17 ± 0.01 mg/mL, which means that binding affinity for the substrate decreased in the presence of inhibitor. The structural studies of ACE demonstrated that, in comparison with a commercial antihypertension drug (enalapril), the trypsin hydrolysate had no effect on secondary structure and less tertiary structure changes of protein was observed.

Effects of Dietary Supplementation with Fish Scales-Derived Collagen Peptides on Skin Parameters and Condition: A Randomized, Placebo-Controlled, Double-Blind Study

Abstract: Fish scales-derived collagen peptides (CPs) are characterized by their specific amino acid composition with a high concentration of glycine, proline and hydroxyproline. These amino acids have been known to exert beneficial effects on human skin. The aim of the present study was to examine the effects of collagen peptides obtained from fish scales on changes in periorbital wrinkles, facial skin hydration, and skin elasticity in healthy women aged 30–60 years. In the present randomized, placebo-controlled, double-blind trial, 71 subjects consumed a 20 mL beverage containing 3000 mg of CPs or placebo once per day over 12 weeks. Significant decreases in periorbital wrinkles (p < 0.05) were observed in the treatment group after 12 weeks of CPs ingestion compared to the control group. This study also demonstrated a consistent trend of enhanced facial skin moisture (p < 0.001) and skin elasticity (p < 0.001) by dietary intake of CPs without any side effects or adverse events. These findings indicate that fish-derived CPs hold great promise as a natural supplement with cutaneous anti-aging properties.

Modulation of Oral Bioavailability and Metabolism for Closely Related Cyclic Hexapeptides.

Abstract: Recently, a variety of studies concerned with the permeability and oral bioavailability of cyclic peptides have been reported. In particular, strategies aiming at modifying peptides to maintain or to enhance solubility while enabling permeability constitute a significant challenge, but are of high interest to ensure a smooth drug discovery process. Current methodologies include N-methylation, matching of hydrogen bonding acceptors and donors across the macrocycle, and additional masking of polarity. In this study, we investigate further the pivotal effects of shielding on permeability and studied the metabolism of the corresponding peptides in more detail by comparing peptide concentrations in the portal versus the jugular vein in rats. Interestingly, minor changes in one particular side chain impacts both permeability and liver metabolism.

Target Specific Anticoagulant Peptides: A Review

Abstract: Anticoagulant drugs are of crucial importance for the treatment and prophylaxis of thrombotic disorders. The use of traditional anticoagulants like heparin and warfarin is majorly associated with bleeding complications. In the quest for safer anticoagulation therapy, the interest for the isolation of novel anticoagulant compounds has shifted towards natural sources. Peptides can be considered as better alternative due to their therapeutic potential in the treatment of diseases. Peptides from hematophagous (blood-feeding) and venomous organisms have been recognized as potential anticoagulant agents. Of late, peptides derived from the hydrolysis of food proteins, including edible seaweeds, milk and seed proteins, have also shown to possess promising in vitro anticoagulant activity. To overcome the problems associated with regular anticoagulants, peptides targeting vital steps in the clotting cascade have been studied. This review focuses on anticoagulant peptides with known targets, inhibiting crucial factors in the coagulation cascade such as FXa, FXIa, FXIIa and FVIIa/TF complex, as well as peptides with unknown targets.

In Silico Analyses of Staphylococcal Enterotoxin B as a DNA Vaccine for Cancer Therapy

Abstract: Immunotherapy has been suggested as a compelling alternative approach for conventional breast cancer treatment methods. Despite the paramount rolesof T cells in this approach, insufficient numbers of them in the combat against progressive tumor growth still remain to be dealt with. Super antigens are a class of antigens, capable of eliciting T cell proliferation response against desired antigens. Staphylococcal enterotoxin B (SEB) is categorized as a super antigen, its anti-tumor properties has been previously reported. However, to the best our knowledge, SEB has not been ever administered as a DNA construct. In the present study, we exploited bioinformatics tools to assess the immunoreactivity of a SEB-coding DNA construct that serves as a DNA vaccine for breast cancer therapy. Potential B and T (MHC class I and II binders) cell epitopes of the hypothetically expressed protein, along with its sub cellular localization were predicted. Moreover, probable glycosylation and phosphorylation sites within the protein sequence were determined. The gene sequence was optimized according to murine model codon bias and its mRNA stability was analyzed. Employing an integrative in silico approach, we revealed that apparently the construct could be efficiently expressed in mouse model. Moreover, the hypothetically expressed protein could act as an amenable adjuvant in cancer immunotherapy.

Purification and Characterization of Antioxidative Peptides Derived From Fermented Milk (Lassi) by Lactic Cultures

Abstract: Fermentation of milk with lactic acid bacteria is the most suitable approach to enrich the bioactive peptides in fermented milk products. So in the present study, two sets of fermented milk (lassi) were prepared. The one lassi was prepared using standard Dahi culture NCDC-167(BD4) and the other one was made with the same Dahi culture combined with Lactobacillus acidophilus NCDC-15 as an adjunct culture. The preparation steps i.e. preheat treatment and incubation period were optimized by using response surface methodology to obtain maximum antioxidant activity. Lassi prepared with adjunct culture using optimized conditions showed an antioxidant activity of 0.66 ± 0.01 µM Trolox/mg protein which was significantly higher than that control (0.22 ± 0.01 µM Trolox/mg protein). Out of 59 peptide fragments of β casein fermented by L. acidophilus and 24 peptides from control have been identified by LC–MS/MS. Most of the peptides showed the antioxidant activity. The therapeutic potential of fermented milk products could be improved by increased production of bioactive peptides through controlled fermentation using appropriate proteolytic starter strain.

Antibacterial Natural Peptide Fractions from Indian Ganoderma lucidum

Abstract: Natural peptides are emerging as a leading alternative to conventional drugs and antibiotics, owing to their remarkable potency, better stability and less toxicity. Such peptides encompass numerous healing properties such as antimicrobial, anti-inflammatory, immunomodulatory, etc. Though plant- derived peptides have been widely studied for their therapeutic benefits, however, fungal peptides are still lesser explored. Ganoderma lucidum, a highly medicinal oriental mushroom comprises a vast array of phytoconstituents, namely flavonoids, phenolics, terpenoids, polysaccharides, proteins, glycolipids, etc and hence, is being used since several decades in traditional Chinese medicine (TCM) for its various ameliorative effects e.g. anti-inflammatory, antimicrobial, anti-proliferative and antioxidant properties. This study presents the isolation and characterization of antibacterial peptide fractions from fruiting body (GLF) and mycelium (GLM) of Indian G. lucidum. Representative amide bonds were identified in the fractions using established standard techniques. Peptide mass fingerprinting and HPLC confirmed the presence of cationic and hydrophobic amino acids in the peptide fractions which are known to be major structural features of antimicrobial peptides. Secondary structure prediction showed abundance of α-helices and random coils in GLF and GLM fractions respectively. The fractions exhibited appreciable antioxidant potential. Besides, these also possessed substantial antibacterial activity against Escherichia coli and Salmonella typhi wherein it was observed that generation of reactive oxygen species and induction of intracellular protein leakage within the bacterial cells were the possible mechanisms of inhibitory action.

Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides

Abstract: Biocompatibility, low toxicity and high selectivity towards bacterial cells has been the hallmark of peptide-based antibiotics. The innate immune system has been employing such molecular systems against invading pathogens as a successful defense strategy. In this study, we attempt to develop topologically constrained antimicrobial peptides with syndiotactic stereochemical arrangement, by incorporating L and D amino acids successively in its amino acid sequence. Acetylated versions of the designed peptides were also examined for its influence on bactericidal potency, against Gram-positive and Gram-negative bacteria. Syndiotactic stereochemical arrangement of the polypeptide main chain mimics stereochemistry of Gramicidin, a naturally occurring antimicrobial peptides. Gramicidin is a class of penta-deca-peptides isolated from soil bacteria Bacillus brevis, but their utility as antibiotic was limited to topical use due to high levels of hemotoxicity. Activity profiles of the four de novo designed peptide variants show higher specificity towards Gram-positive bacteria than Gram-negative variants, matching earlier reports on the therapeutic potential of gramicidin as a broad spectrum antibiotic. Significantly, our hemolytic assay confirms very low (<1%) levels of toxicity for the designed peptides unlike gramicidin. Earlier reports confirm that incorporation of D amino acids effectively negates the possibility of proteolytic degradation, thus pointing to the potential utility of de novo designed peptides with diversified stereochemistry as a promising new approach in the generation of novel antibiotic peptides.

Recombinant Production and Antimicrobial Assessment of Beta Casein- IbAMP4 as a Novel Antimicrobial Polymeric Protein and its Synergistic Effects with Thymol

Abstract: There is a growing research interest on products with antimicrobial activity. Antimicrobial polymers are one of the most surefire procedures to combat microbes. In the present study, the ability of Βeta-casein- one of the milk major self assembly proteins with high polymeric film production capability—as a fusion partner of Ib-AMP4 antimicrobial peptide was investigated. Also, the antimicrobial activities of Βeta-casein- IbAMP4 fusion protein antimicrobial against common food pathogens were assessed. The pET21a-BCN-Ib-AMP 4 construct was transformed to Escherichia coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg mL−1 fusion protein by ultrafiltration. 5 μg mL−1 H2O2 was applied for accelerating the formation of two necessary disulfide bonds. Antimicrobial assays were performed against E. coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus and Candida albicans. Results of antimicrobial tests confirmed the efficiency of BCN-IbAMP4 against all tested microorganisms. Overall, the combination of thymol plus BCN-IbAMP4 increased their antimicrobial activities. MIC, MBC, MFC, FICI and FBCI values showed strong synergistic activity between the two examined compounds. Time kill and growth kinetic studies indicated significant reduction of cell viability during first period of exposure to BCN-IbAMP4 and thymol combination.

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge Xestospongia testudinaria , with Characterization of Stability in Human Serum

Abstract: Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC50 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC50 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2 ± 0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.

Identification of PP2A/Set Binding Sites and Design of Interacting Peptides with Potential Clinical Applications

Abstract: Protein phosphatase 2A (PP2A) is known to be a negative regulator of several survival and proliferating pathways that are frequently altered in cancer. In addition to chemical enzymatic inhibitors of the PP2A activity, the oncoprotein SET has been described as a physiological PP2A inhibitor by forming a complex with PP2A catalytic subunit (PP2Ac). Increased SET protein levels therefore directly reduce the tumor suppressor function of PP2A and promote tumor progression. We have used the PEP-Scan approach to identify the binding site between the serine/threonine phosphatase PP2A and the oncoprotein SET. For in vivo validation of the peptides, we have used chronic lymphocytic leukemia (CLL) xenograft models. In this manuscript we describe the identification of amino acid sequences involved in the complex formation, both at the PP2Ac and SET sides. The amino acid sequences of the binding sites were coupled to an optimized penetrating peptide in order to generate chimeras (Mut3DPT-PP2A and Mut3DPT-SET) able to target the PP2A/SET interaction. We demonstrate that these peptides have an in vitro apoptotic effect on breast and lung cancer cell lines, as well as an antitumoral effect on CLL and lymphoma xenograft models. The new generated chimeric peptides allow the modulation of the PP2Ac/SET interaction and might have a potential as a new therapeutic approach for cancer treatment.

Peptide Therapeutics Versus Superbugs: Highlight on Current Research and Advancements

Abstract: Antibiotics have saved several millions of lives, but its persistent use of antibiotics in the treatment of various infections, whether bacterial, fungal, viral or parasitic has lead to the development of antibiotic resistance. The rapid emergence of antibiotic resistant strains poses a serious challenge to existing antimicrobial therapies. Due to the increase in drug-resistant pathogens and failure of antibiotics the urgent need for the discovery of novel antimicrobials has been continuously emphasized in the global forum. Here we review about antimicrobial peptides (AMPs), their structural insights and recent developments. We had summarized the major classes, mechanism of action and biophysical parameters that modulate therapeutic potency of AMPs. Also, we had briefed the challenges involved in developing therapeutic peptides and the global market potential for peptide therapeutics.

The Effect of Anti-aging Peptides on Mechanical and Biological Properties of HaCaT Keratinocytes

Abstract: Atomic force microscopy (AFM) and fluorescence microscopy was applied to determine the influence of the anti-aging peptides on the morphology and the mechanical properties of keratinocytes. Immortalized human keratinocytes (HaCaT) were treated with two anti-aging bioactive peptides: Acetyl Tetrapeptide-2 and Acetyl Hexapeptide-50 (Lipotec). The AFM measurement of the keratinocyte stiffness were carried after 48 h exposure at an indentation depth of 200 nm. AFM analysis showed increase of the cell stiffness for cells treated with Acetyl Tetrapeptide-2 (P1) in concentration range. Acetyl Hexapeptide-50 (P2) at concentration of 0.05 µg/ml also increased the stiffness of HaCaT cells but at higher concentrations 0.5 and 5 µg/ml cell stiffness was lower as compared to untreated control. Fluorescence microscopy revealed remodeling of actin filaments dependent on the concentration of P2 peptide. The mechanical response of HaCaT cells treated with P2 peptide corresponds to change of transcription level of ACTN1 and SOD2 which activity was expected to be modulated by P2 treatment.

Characterization of Antimicrobial and Antioxidative Peptides Synthesized by L. rhamnosus C6 Fermentation of Milk

Abstract: Lactobacillus rhamnosus C6 was used for milk fermentation with the aim of synthesizing antimicrobial and antioxidant peptides rich preparations. The proteolysis was checked for an incubation period of 72 h to check the extent of both bioactivities in fermentate. The 36 h incubated fermentate showed higher inhibition zone diameter against E. coli ATCC 25922 as well as antioxidant activity. Ultrafiltrate was further purified by solid phase extraction and then subjected to reverse phase chromatography. Among 12 fractions collected, higher activity containing fractions were sequenced through LC–MS and characterized. Total 49 peptide sequences identified including 13 novel sequences rich in proline with helix forming ability. Higher antimicrobial activity containing fractions have potent previously reported Casicidin-17 peptides along with a series of proline rich peptides. Antioxidant rich peptides profile contains 21 peptide of smaller sequence of mainly 9–12 amino acids with lower molecular weight. This study demonstrates the capacity of L. rhamnosus C6 to release antioxidative and antimicrobial peptide by proteolysis of milk proteins through peptide profiling and characterization.

Effect of In Ovo Feeding of Folic Acid on Subsequent Growth Performance and Blood Constituents Levels in Broilers

Abstract: This study designed to determine effect of in ovo feeding of folic acid on subsequent growth performance and blood constituents levels in broilers. A total of 1000 fertile broiler eggs were divided into four groups. Control group (1) received no injection. In group 2, eggs received in ovo feeding of distiller water (40 µg). Group 3 received in ovo feeding of folic acid (40 µg). Groups 4 and 5 were similar to Group 3, except eggs injected with 80 and 120 µg of folic acid. All eggs were incubated and after hatch chickens were randomly assigned into their experimental groups. On days 1 and 42 post-hatch, chicken randomly selected and blood constituents, carcass characteristics, food intake, body weight gain and food conversion ratio were determined. According to the results, no significant difference detected on hatchability rate of the in ovo injected eggs (P > 0.05). Dose dependent increase observed in glucose and folic acid levels in chicken in ovo injected with folic acid on day 1 post hatch (P = 0.001). Blood glucose, folic acid and phosphorous levels increased (P = 0.001) while cholesterol, triglyceride, HDL and LDL, calcium and alkaline phosphatase decreased in ovo injected with folic acid on day 42 post hatch (P = 0.001). Food conversion ratio increased by in ovo injection of the folic acid (P = 0.001). These results suggest folic acid had positive effects in broiler chicken.

Citropin 1.1 Trifluoroacetate to Chloride Counter-Ion Exchange in HCl-Saturated Organic Solutions: An Alternative Approach

Abstract: In view of the increasing interest in peptides in various market sectors, a stronger emphasis on topics related to their production has been seen. Fmoc-based solid phase peptide synthesis, although being fast and efficient, provides final products with significant amounts of trifluoroacetate ions in the form of either a counter-ion or an unbound impurity. Because of the proven toxicity towards cells and peptide activity inhibition, ion exchange to more biocompatible one is purposeful. Additionally, as most of the currently used counter-ion exchange techniques are time-consuming and burdened by peptide yield reduction risk, development of a new approach is still a sensible solution. In this study, we examined the potential of peptide counter-ion exchange using non-aqueous organic solvents saturated with HCl. Counter-ion exchange of a model peptide, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), for each solvent was conducted through incubation with subsequent evaporation under reduced pressure, dissolution in water and lyophilization. Each exchange was performed four times and compared to a reference method—lyophilization of the peptide from an 0.1 M HCl solution. The results showed superior counter-ion exchange efficiency for most of the organic solutions in relation to the reference method. Moreover, HCl-saturated acetonitrile and tert-butanol provided a satisfying exchange level after just one repetition. Thus, those two organic solvents can be potentially introduced into routine peptide counter-ion exchange.

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, Antheraea assamensis

Abstract: Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.

In silico designing of peptide inhibitors against pregnane X receptor: the novel candidates to control drug metabolism

Abstract: Drug-induced pregnane X receptor (PXR) activation may be unavoidable with some treatments. Besides, drug-induced PXR activation may lead to drug interactions with a number of treatments. Human PXR antagonists represent a means to counteract such interactions. Indeed, the ideal small molecule would have good drug-like properties, but be devoid of nonspecific cell-target effects and ability to induce cytotoxicity. Researchers assess on the possibility of deriving inhibitory peptides, which are derived from interfaces of “protein–protein interactions” (PPIs) could inhibit interactions of their origin domain by mimicking its mode of binding to cognate partners. Such peptides could serve as promising leads for rational design of inhibitory drugs. In this study, In silico designing of peptide inhibitors and peptidomimetics against PPIs was done to prevent unwanted drug metabolisms caused by PXR activation. In sum, no designed peptides had considerable higher energy of interactions and more H-bonds with the receptor compared to the SRC-1 peptides. Hence, none of the designed peptides are predicted to be more active against the target receptor. However, compared to the SRC-1 peptide, a designed peptidomimetic (mimetic3) had considerable higher energy of interactions with the receptor. It formed five H-bonds with PXR that was equal to the number of H-bonds that SRC-1 formed with the receptor. Therefore, by designing such mimetics, it would be probable to have a realistic chance of achieving peptides with improved binding affinity compared to SRC-1 and consequently prevent the unwanted drug metabolisms caused by PXR activation.

Improvement of the Antioxidant Activity of Fenugreek Protein Isolates by Lactococcus lactis Fermentation

Abstract: This study investigated the antioxidant capacity of fenugreek protein isolate and its improvement by Lc. lactis fermentation through bioactive peptides production and the effect of molecular weight variation on fenugreek fractions antioxidant activity. Fenugreek protein isolate showed a significant increase of antioxidant and radical scavenging activity after 24 h of fermentation, by about 23.7, 42.9 and 40% for respectively antioxidant activity coefficient AAC, DPPH and ABTS radical scavenging activity. FI fermentation led to a hydrolysis of peptide bands with MW > 35 kDa and a generation of new bands with a MW < 25 kDa. A significant reduction in molecular-mass distribution of hydrolysates and a great increase of total free amino acids content, especially an increase on isoleucine, leucine, glutamic acid, serine, histidine, glutamine and lysine was noted. The infrared results showed that different reactions may take place after fermentation, and showed an increase of proteins, amides and aromatic compounds. However, fenugreek fraction (F2) with MW 15–50 kDa presented the highest activity instead of fraction (F1) with lower MW. Lc. lactis had the ability to degrade and convert fenugreek proteins into bioactive peptides that contribute positively in the improvement of antioxidant activity of FI and fractions. FI presents a significant antioxidant activity and thus, can be considered as a potential source of high added value natural antioxidants and may be employed as a functional food ingredient with good potential applications in food products.

Biochemical Characterization of HL-7 and HL-10 Peptides Identified from Scorpion Venom of Hemiscorpius lepturus

Abstract: In this study, the scorpion venom of H. lepturus was fractionized by reversed phase high-pressure liquid chromatography (RP-HPLC). The sequences of two peptide fractions were identified by tandem mass spectrometry and named as HL-10 (AFPYYGHHLG) and HL-7 (YLYELAR), respectively. Antioxidant activity and cellular effect of synthetic peptides on A549 cell line were investigated. Results showed that the two peptides had high activities in radical scavenging and inhibition of lipid peroxidation in a concentration-dependent manner. The HL-10 and HL-7 peptides demonstrated cytotoxicity on A549 without any hemolytic effect. By increasing of peptide concentrations induced significantly (P ≤ 0.01) activities of catalase and glutathione peroxidase. Our results showed that HL-7 peptide had higher antioxidant potency, whereas the HL-10 peptide revealed a great cytotoxicity on A549 cell line by MTT assay. Our results suggested that the peptides of H. lepturus, possessed free radicals scavenging likewise increased antioxidant enzyme activities in A549 cells.

A Peptide Based Pro-drug Disrupts Alzheimer’s Amyloid into Non-toxic Species and Reduces Aβ Induced Toxicity In Vitro

Abstract: Aggregation of Amyloid β (Aβ) in the interneuronal spaces is a major etiopathological factor for onset and progression of Alzheimer’s disease (AD) Since the mechanism of aggregation is not fully understood, control and modulation of the aggregation process is a challenging task Although, several strategies were developed for the past few decades, yet there is no proper therapeutics available Herein, we report a peptide based pro-drug, termed as a conformational Pro-Drug peptide (PDp), which disrupts existing Aβ fibrils, but does not produce toxic soluble oligomers, through a series of spontaneous chemical reactions resulting in in situ generation of β-sheet destabilizing factors Furthermore, PDp reduces Aβ mediated toxicity examined on an in vitro model consisting of the human neuroblastoma SH-SY5Y cells PDp also disrupts fibrils originated from AD affected human cerebrospinal fluid These findings will help to understand the process of amyloidogenesis better and also indicate a novel approach for therapeutically important peptide design

Computationally Design of Inhibitory Peptides Against Wnt Signaling Pathway: In Silico Insight on Complex of DKK1 and LRP6

Abstract: Wnt signaling pathway plays a major role in the regulation of cell proliferation, migration, tissue homeostasis, tumor progression and cancer. This pathway can be antagonized by different proteins such as DKK proteins, which disrupt the initiatory complex (Frizzled–LRP6 complex). Therefore, interruption of its formation could be a promising strategy for the design of Low-density lipoprotein receptor-Related Protein 6 (LRP6) inhibitors. A computational study was conducted in order to assist in the design of inhibitory peptides against LRP6 as co-receptor of frizzled. Twelve fragments as peptide derivatives of natural ligand of LRP6 receptor (DKK1) were designed using the information from the analysis of the DKK1_C/LRP6 complex, hot spot residues and the secondary structure. These fragments were based on cys2 domain of DKK1. The designed peptides were energy minimized by molecular dynamics simulations in the presence and absence of LRP6 receptor and their binding affinities were investigated via molecular docking using ClusPro, HADDOCK and PRODIGY webservers. Finally, the stability and free energy of binding in peptides were calculated by FoldX software. The results showed that four designed peptides had the highest affinity (the interaction energy: −10.2867, −10.1388, −7.94339 and −7.57536 kcal/mol) to interact with the receptor which showed the most interacting residues and the lowest free energy of binding. Also, the RMSD, RMSF and RoG of the protein–peptide complex exhibited less structural fluctuations which can be linked to the stability of peptides associated to the receptor. These peptides may be considered as candidates for inhibiting Wnt signaling pathway through LRP6 receptor.

Fish Heat Shock Cognate 70 Derived AMPs CsHSC70 A1 and CsHSC70 A2

Abstract: Heat shock cognate 70 (HSC70) is an important evolutionary conserved protein that plays a major role in maintaining the homeostasis and immunity of many organisms. In this study, a HSC70 from Channa striatus was identified from its cDNA library and characterized using bioinformatics and molecular biology tools. CsHSC70 cDNA was 1953 base pair (bp) in length along with an open reading frame which encoded a polypeptide of 650 amino acid residues. Tissue distribution results showed that CsHSC70 was considerably expressed in gill, to a lesser extent in head kidney, blood, spleen and liver and at low level in other tissues. Using C. striatus gill as cell model, effects of fungal, bacterial and poly I:C stimulant on the mRNA levels of CsHSC70 was examined. We also described the antimicrobial features of two peptides namely CsHSC70 A1and CsHSC70 A2 derived from the N-terminal of CsHSC70 protein. CsHSC70 A1 peptide (40 µg/ml) exhibited potent bactericidal activity against Micrococcus luteus cells. Flow cytometric analysis revealed that the M. luteus cells stained with propidium iodide, upon treated with CsHSC70 A1 at the concentration of 40 µM/ml showed 38% survival compared to its control (99.6%). It seems that CsHSC70 A1 peptide shows antimicrobial activity against M. luteus through membrane disruption. Additionally, scanning electron microscope (SEM) observation confirmed that CsHSC70 A1 peptide treatment completely damaged and destructed the M. luteus cells. Taken together, these findings suggest that CsHSC70 A1 peptide could be a safe and potential therapeutic molecule substitute to antibiotics in various clinical fields.

Purity Measurement of Human C-Peptide by High Performance Liquid Chromatography and Quantitative Nuclear Magnetic Resonance

Abstract: Purity determination of human C-peptide (hCP) with metrological traceability is fundamental to accurate and comparable clinical analysis of hCP. Based on high performance liquid chromatography and quantitative nuclear magnetic resonance (qNMR) with a purified hCP, a method was reported to determine the purity of an hCP sample used in an intercomparison. The composition of deuterium solvent for qNMR was optimized to improve the separation of the β-proton of an aspartic acid residue in the sequence of hCP, which were used for NMR integration. The calculated mean and uncertainty (77.86 ± 3.85%) are consistent with the results (80.18 ± 0.62%) of different methods reported by many metrological laboratories. This method extended the application of qNMR to peptides with high molecular weight of about 3020.

Catechol Oxidase and SOD Mimicking by Copper(II) Complexes of Multihistidine Peptides

Abstract: Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis–Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis–Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {Nim/2Nim,2N−} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range.