Absorption Of Peptides Research Articles

Antioxidant and angiotensin I-converting enzyme (ACE) inhibitory peptides of rainbow trout (Oncorhynchus mykiss) viscera hydrolysates subjected to simulated gastrointestinal digestion and intestinal absorption

The objective of this work was to evaluate in vitro bioaccessibility, intestinal absorption, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of peptides from rainbow trout viscera hydrolysate (H). Rainbow trout Viscera (V) was hydrolyzed by Alcalase® 2.4L and a degree of hydrolysis (DH) of 44.8 ± 2.5% was achieved. Viscera and its hydrolysate were subjected to simulated gastrointestinal digestion (SGID) and intestinal absorption across Caco-2/TC7 cell monolayers. After the hydrolysis with Alcalase® 2.4L and the SGID of V, the species between 60.6 kDa and 13.0 kDa were decreased, causing an increase in species less than 6.51 kDa. The SGID of H did not modify the oxygen radical absorbance capacity (ORAC) or ACE inhibitory values but caused a significant decrease in the hydroxyl radical antioxidant capacity (HORAC) (30.2%). It also produced an increase in ABTS radical cation (ABTS assay) scavenging activity and ferric reducing antioxidant power (FRAP) (9.46% and 20.2%, respectively). Bioactive peptides in H were stable after SGID and they were partially able to cross Caco-2/TC7 cell monolayer, which demonstrates their possible intestinal absorption and their potential to act inside the organism.

Effects of Ghrelin on intestinal cell proliferation, the expression of protein absorption and metabolism factors in juvenile grass carp (Ctenopharyngodon idella)

Ghrelin can stimulate pituitary growth hormone (GH) release, and has a key role in the regulation of food intake and adiposity in vertebrates. At present, no relevant studies have reported the effects of Ghrelin on intestinal cell proliferation, protein absorption and metabolism. In this paper, firstly we established the intestinal epithelial cell lines of juvenile grass carp (Ctenopharyngodon idella) that can be passed stably and identified by the intestinal specific expression proteins E-cadherin and ZO-1. And we used this cell line as a model to investigate the effects of Ghrelin, Ala-Gln, Ala-Gln+Ghrelin on the proliferation of juvenile grass carp intestinal cells. We found that the use of Ghrelin, Ala-Gln and Ala-Gln+Ghrelin could promote the proliferation of intestinal cells of juvenile grass carp, but Ala-Gln+Ghrelin had the strongest promoting effect. Further, we investigated the effects of Ghrelin on the expression of cdx2, PepT1 and mTOR which are related to intestinal cell proliferation, protein absorption and metabolism of grass carp in vitro and in vivo experiments. And we studied the effects of different protein sources on the expression of Ghrelin, PepT1 and mTOR in grass carp at individual level. The results suggested that Ghrelin can promote grass carp intestinal cell proliferation, and affect the expression of genes which are related to intestinal peptide absorption and metabolism in juvenile grass carp.

Studies on the oral absorption of cyclic peptides from Pseudostellaria heterophylla and interacting with membrane receptors

Pseudostellaria heterophylla (Miq.) Pax. (Taizishen, TZS) contains a variety of natural active cyclic-peptide compounds (CP). In this article, four kinds of CP monomers were isolated by HPLC and the structures were identified by mass spectrometry. The in vivo absorption of CP was detected by UPLC-MS/MS. The interaction between CP and membrane receptor was analyzed by SPR. As a result, the relative absorption rate of CP was Pesudostellarin B > Heterophyllin B > Pesudostellarin C > Pesudostellarin E. The difference in absorption rate of CP in vivo was related to its interaction with membrane receptors. The absorption mechanism of CP might be different. This is the first report that in vivo absorption study of different CP from TZS and explore its absorption mechanism, laying a theoretical foundation for the research and development of its oral drugs, and providing new ideas for the study of the absorption mechanism of CP from traditional Chinese medicine.

Preparation, Characterization and Evaluation of Guar Films Impregnated with Relaxing Peptide Loaded into Chitosan Microparticles

Background: Biological molecules such as therapeutic proteins and peptides have provided ample opportunities for further improvements in health and wellbeing. However, such biomacromolecules face stability challenges regarding premature processing and digestion, whenever any enteric delivery route is considered. Hence, oral guar films entrapping peptide-containing chitosan microparticles have been developed as a new delivery system to carry and protect therapeutic relaxing peptides. Results: The composite films were characterized regarding physicochemical properties using FTIR as well as peptide encapsulation efficiency, cytotoxicity, and peptide permeability through buccal and intestinal cell-based models. The composite films with 0.18% (w/w) of peptide demonstrated ca. 0.63% of peptide loading efficiency into the chitosan microparticles and to be safe for buccal and intestinal epithelial cells up to 24 h of contact. The permeability assays through TR146 cells to mimic buccal mucosa epithelium and Caco-2/HT29-MTX to mimic duodenum conditions showed that the incorporation of peptide chitosan microparticles into guar-gum oral films offered a faster permeability, when compared with the free peptide and peptide-chitosan-microparticles-loaded chitosan. Conclusion: This study suggests that peptide-loaded chitosan microparticles into guar-gum oral films might be a promising carrier for enhancement of the buccal and intestinal absorption of a relaxing peptide.

What are the digestion and absorption models used to reproduce gastrointestinal protein processes?: A protocol for systematic review.

Background:Animal, cell, and in vitro studies have been applied to simulate the human gastrointestinal tract (GIT) and evaluate the behavior of biomolecules. Understanding the peptides and/or proteins stability when exposed to these physiological conditions of the GIT can assist in the application of these molecules in the treatment of diseases such as obesity. This study describes a protocol of systematic reviews to analyze the methodologies that mimic the digestive and absorptive processes of peptides and/or proteins.Methods:The protocol follows the guidelines described by Preferred Reporting Items for Systematic Reviews and Meta-Analyzes Protocols (PRISMA-P). The search strategies will be applied in the electronic databases PubMed, ScienceDirect, Scopus, Web of Science, Evidence portal, Virtual Health Library, and EMBASE. The intervention group will be formed by in vivo, in cells, and in vitro (gastrointestinal simulating fluids) studies of digestion and absorption of peptides and/or proteins presenting a schedule, duration, frequency, dosages administered, concentration, and temperature, and the control group consisting in studies without peptides and/or proteins. The selection of studies, data extraction, and assessment of the risk of bias will be carried out independently by 2 reviewers. For animal studies, the risk of bias will be assessed by the instrument of the Systematic Review Center for Experimentation with Laboratory Animals (SYRCLE) and the Office of Health Assessment and Translation (OHAT) tool will be used to assess the risk of bias in cell studies.Results:This protocol contemplates the development of 2 systematic reviews and will assist the scientific community in identifying methods related to the digestive and absorptive processes of peptides and/or proteins.Conclusion:Both systematic reviews resulting from this protocol will provide subsidies for the construction of research related to the clinical application of bioactive peptides and/or proteins. In this context, they will make it possible to understand the gastrointestinal processes during administering these molecules, as the gastrointestinal environment can affect its functionality. Therefore, validating the effectiveness of these protocols is important, as it mimics in vitro biological conditions, reducing the use of animals, being consistent with the reduction, refine and replace program.

New Nanocarrier System for Liposomes Coated with Lactobacillus acidophilus S-Layer Protein to Improve Leu-Gln-Pro-Glu Absorption through the Intestinal Epithelium.

The present study describes the development of a novel liposome nanocarrier system. The liposome was coated with Lactobacillus acidophilus CICC 6074 S-layer protein (SLP) to improve the intestinal absorption of the cholesterol-lowering peptide Leu-Gln-Pro-Glu (LQPE). The SLP-coated liposomes were prepared and characterized with morphology, particle size, zeta potential, membrane stability, Fourier transform infrared spectroscopy, and dual-channel surface plasma resonance. The results showed that SLP could successfully self-assemble on liposomes. Then, LQPE liposomes and SLP-coated LQPE liposomes (SLP-L-LQPE) were prepared. SLP-L-LQPE not only showed better sustained release properties and gastrointestinal tolerance in vitro but also increased the retention time in mice intestine. Transepithelial transport experiment indicates that the transshipment of LQPE increased significantly after being embedded by liposomes and coated with SLP. The research provides a theoretical basis for the study of SLP-coated liposomes and a potential drug delivery system for improving the intestinal absorption of peptides.

A Pharmacological and Clinical Overview of Oral Semaglutide for the Treatment of Type 2 Diabetes.

Oral semaglutide (Rybelsus®) is a glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1RA) with 94% homology to human GLP-1. It is the first GLP-1RA developed for oral administration, and it comprises a co-formulation of the peptide semaglutide with the absorption enhancer sodium N-(8-[2-hydroxybenzoyl] amino) caprylate, which overcomes the challenges of peptide absorption in the acidic conditions of the stomach. Oral semaglutide is indicated for use as an add-on combination therapy (with other glucose-lowering agents, including insulin) or as a monotherapy (in patients who are intolerant to metformin) for type 2 diabetes when diet and exercise do not provide adequate glycemic control. In an extensive phase III clinical program including patients from across the disease spectrum, treatment with oral semaglutide resulted in effective glycemic control, reductions in body weight, and decreases in systolic blood pressure when used as monotherapy or in combination with other glucose-lowering therapies. Studies showed that oral semaglutide was well tolerated, with a safety profile consistent with the GLP-1RA drug class. The risk of hypoglycemia was low, and the most common adverse events were gastrointestinal, with nausea and diarrhea generally being the most frequently reported manifestations. Cardiovascular (CV) safety was shown to be noninferior to placebo and observations suggest that the CV profile of oral semaglutide is likely to be similar to that of subcutaneous semaglutide. The evolution of the GLP-1RA class to include an oral agent could facilitate the use of these agents earlier in the diabetes treatment cascade owing to wider acceptance from patients and healthcare professionals.Video abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s40265-021-01499-w.

Intracellular antioxidant activity and intestinal absorption of amaranth peptides released using simulated gastrointestinal digestion with Caco-2 TC7 cells

The capacity of amaranth peptides generated using simulated gastrointestinal digestion to exert intracellular antioxidant activity and its potential modification and/or passage through the intestinal cells were studied. The digested protein isolate (APId) and its fractions (gel filtration chromatography) were analysed. The effect of amaranth peptides pre-treatment of Caco-2 TC7 cells treated with 500 μM H2O2 was measured using different biomarkers. All samples led to a partial or complete decrease of reactive oxygen species and different behaviours regarding the enzymatic antioxidant activities (superoxide dismutase and glutathione peroxidase) and reduced glutathione levels. Fractions showed different behaviours after modification by and/or passage across Caco-2 TC7 monolayers suggesting potential activity at local intestinal and/or systemic sites. These results suggested that the APId was a source of bioactive peptides showing antioxidant activity at the intracellular level.

Properties and regulation of Gly-Sar uptake and transport in bovine intestinal epithelial cells.

Intestinal absorption of peptides is vital for the overall health and productivity of dairy cows. This study investigated the regulation, uptake and transport of dipeptides in bovine intestinal epithelial cells (BIECs). We also evaluated the effects of time, pH, concentration of the dipeptides, temperature, presence of diethylpyrocarbonate (DEPC)-an inhibitor of PepT1, and other dipeptides (Met-Met, Lys-Lys or Met-Lys), on the uptake and transport of Gly-Sar-FITC, which was a common fluorophore-labelled dipeptide. Under controlled experiments, BIECs were treated with 25μM LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) and 25μM Perifosine (a protein kinase B (AKT) inhibitor). The subsequent expression of PepT1 in the BIECs was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. It was found that the uptake and transport of Gly-Sar-FITC were significant high at 37℃ than that at 4℃. The optimal pH for transport and uptake of Gly-Sar-FITC was 6.0-6.5, whereas the two properties decreased significantly in the presence of DEPC, Met-Met, Lys-Lys and Met-Lys (p<0.05). The apical-to-basolateral transport was also found to be significantly higher than the reverse transport (p<0.05). PI3K and AKT inhibitors were found to significantly suppress the expression of PepT1, thus impairing uptake and transport of Gly-Sar-FITC. Findings of this study thus suggest that the uptake and transport of Gly-Sar-FITC in BIECs are mediated by PepT1, and the PI3K/AKT signalling pathway regulates the absorption of small peptides.

Asterias pectinifera derived collagen peptide-encapsulating elastic nanoliposomes for the cosmetic application

Asterias pectinifera, a starfish that has been known to ruin the aquaculture industry owing to its voracious appetite, has recently been identified as an eco-friendly source of non-toxic and highly water-soluble low-molecular weight collagen peptides, which promotes wound healing, bone regeneration, and skin protection. Although they have potential applications in biomedical applications, including pharmaceuticals and cosmetic products, it remains unclear how to improve the in vivo absorption of collagen peptides. Here, we present a novel method to enhance the absorption rate of collagen peptides using a lipid-based nanocarrier. We prepared an elastic nanoliposome by controlling the composition ratio of phospholipids and low-molecular weight collagen peptides. Our results indicate that low-molecular weight collagen peptides extracted from Asterias pectinifera have higher encapsulation efficiency than the collagen peptides extracted from pork and fish, which have traditionally been considered as a conventional source of collagen. Moreover, we demonstrate that the elastic nanoliposome containing the collagen peptide of Asterias pectinifera can reduce MMP-1 expression caused by ultraviolet radiation-induced photoaging. Therefore, the combination of Asterias pectinifera-derived low-molecular-weight collagen peptides and elastic nanoliposomes may be a promising formulation as an eco-friendly source of materials for anti-aging cosmetics.

Activity and bioavailability of food protein-derived angiotensin-I-converting enzyme-inhibitory peptides.

Angiotensin-I-converting enzyme (ACE) inhibitory peptides are able to inhibit the activity of ACE, which is the key enzymatic factor mediating systemic hypertension. ACE-inhibitory peptides can be obtained from edible proteins and have the function of antihypertension. The amino acid sequences and the secondary structures of ACE-inhibitory peptides determine the inhibitory activities and stability. The resistance of ACE-inhibitory peptides to digestive enzymes and peptidase affect their antihypertensive bioactivity in vivo. In this paper, the mechanism of ACE-inhibition, sources of the inhibitory peptides, structure-activity relationships, stability during digestion, absorption and transportation of ACE-inhibitory peptides, and consumption of ACE-inhibitory peptides are reviewed, which provide guidance to the development of new functional foods and production of antihypertensive nutraceuticals and pharmaceuticals.

Effect of paracellular permeation enhancers on intestinal permeability of two peptide drugs, enalaprilat and hexarelin, in rats

Transcellular permeation enhancers are known to increase the intestinal permeability of enalaprilat, a 349 Da peptide, but not hexarelin (887 Da). The primary aim of this paper was to investigate if paracellular permeability enhancers affected the intestinal permeation of the two peptides. This was investigated using the rat single-pass intestinal perfusion model with concomitant blood sampling. These luminal compositions included two paracellular permeation enhancers, chitosan (5 mg/mL) and ethylenediaminetetraacetate (EDTA, 1 and 5 mg/mL), as well as low luminal tonicity (100 mOsm) with or without lidocaine. Effects were evaluated by the change in lumen-to-blood permeability of hexarelin and enalaprilat, and the blood-to-lumen clearance of 51chromium-labeled EDTA (CLCr-EDTA), a clinical marker for mucosal barrier integrity. The two paracellular permeation enhancers increased the mucosal permeability of both peptide drugs to a similar extent. The data in this study suggests that the potential for paracellular permeability enhancers to increase intestinal absorption of hydrophilic peptides with low molecular mass is greater than for those with transcellular mechanism-of-action. Further, the mucosal blood-to-lumen flux of 51Cr-EDTA was increased by the two paracellular permeation enhancers and by luminal hypotonicity. In contrast, luminal hypotonicity did not affect the lumen-to-blood transport of enalaprilat and hexarelin. This suggests that hypotonicity affects paracellular solute transport primarily in the mucosal crypt region, as this area is protected from luminal contents by a constant water flow from the crypts.

Nutrition and Functions of Amino Acids in Aquatic Crustaceans.

Crustaceans (e.g., shrimp and crabs) are a good source of protein-rich foods for human consumption. They are the second largest aquaculture species worldwide. Understanding the digestion of dietary protein, as well as the absorption, metabolism and functions of amino acids (AAs) and small peptidesis essential to produce cost-effective and sustainable aquafeeds. Hepatopancreas (the midgut gland) is the main site for the digestion of dietary protein as well as the absorption of small peptides and AAs into the hemolymph. Besides serving as the building blocks of protein, AAs (particularly aspartate, glutamate, glutamine and alanine) are the primary metabolic fuels for the gut and extra-hepatopancreas tissues (e.g., kidneys and skeletal muscle) of crustaceans. In addition, AAs are precursors for the syntheses of glucose, lipids, H2S, and low-molecular-weight molecules (e.g., nitric oxide, glutathione, polyamines, histamine, and hormones) with enormous biological importance, such as physical barrier, immunological and antioxidant defenses. Therefore, both nutritionally essential and nonessential AAs are needed in diets to improve the growth, development, molt rate, survival, and reproduction of crustaceans. There are technical difficulties and challenges in the use of crystalline AAs for research and practical production due to the loss of free AAs during feed processing, the leaching of in-feed free AAs to the surrounding water environment, and asynchronous absorption with peptide-bounded AAs. At present, much knowledge about AA metabolism and functions in crustaceans is based on studies of mammals and fish species. Basic research in this area is necessary to lay a solid foundation for improving the balances and bioavailability of AAs in the diets for optimum growth, health and wellbeing of crustaceans, while preventing and treating their metabolic diseases. This review highlights recent advances in AA nutrition and metabolism in aquatic crustacean species at their different life stages. The new knowledge is expected to guide the development of the next generation of their improved diets.

Changes in the secondary structures and zeta potential of soybean peptide and its calcium complexes in different solution environments.

To illustrate the relationship between environment hydrophobicity and soybean peptide and its calcium complexes when they are absorbed transmembrane, different solution environments (HBS buffer, TFE hydrophobic solution and cell suspension) were used to simulate hydrophilic and hydrophobic environments. In this study, soybean peptides (10-30 kDa) with a high calcium binding capacity were prepared by enzymatic hydrolysis and ultrafiltration. The results of cell experiments showed that the peptide could transport calcium into cells for absorption. Secondary structure changes of the peptide and its calcium complexes in different solution environments showed that the secondary structure of the peptide changed during the transmembrane absorption, and the contents of α-helix and β-sheet structures increased. Besides, the β-sheet structures in the peptide-calcium complexes were further converted to an α-helix structure. This conversion may be induced by the hydrophobicity of peptide solutions. In addition, when the conformation changes, the positively charged peptides in the sample will be exposed and then interact with cells, which is beneficial for the transmembrane of peptide-calcium complexes.

Antarctic krill-derived peptides with consecutive Glu residues enhanced iron binding, solubility, and absorption.

Three peptides containing three glutamic acid (Glu) residues at different positions derived from Antarctic krill were obtained to investigate their iron-binding properties, digestive stability, and effectiveness on enhancing iron solubility and absorption. Results indicated that Fe2+ bound to the carbonyl, carboxyl, or hydroxyl groups of DELEDSLER, EEEFDATR, and DTDSEEEIR at stoichiometric ratios of 0.453, 0.466, and 0.490, respectively. DTDSEEEIR with three consecutive Glu in the middle of the sequence possessed higher iron-binding ability and iron release potential than EEEFDATR with three consecutive Glu in the N-terminal, and DELEDSLER with three discontinuous Glu showed the lowest values. Although EEEFDATR showed remarkably lower digestion stability than DTDSEEEIR, the effect of EEEFDATR-iron on iron solubility and absorption was comparable to that of DTDSEEEIR-iron, but better than that of DELEDSLER-iron and FeSO4. Thus, peptides with consecutive Glu have the potential as an effective iron carrier to improve iron absorption.

In Vitro and In Vivo Evaluation of 3D Printed Capsules with Pressure Triggered Release Mechanism for Oral Peptide Delivery

In this study a 3D printed capsule designed to break from the physiological pressures in the antropyloric region was evaluated for its ability to deliver the synthetic octapeptide octreotide in beagle dogs when co-formulated with the permeation enhancer sodium caprate. The pressure sensitive capsules were compared to traditional enteric coated hard gelatin capsules and enteric coated tablets. Paracetamol, which is completely absorbed in dogs, was included in the formulations and used as an absorption marker to give information about the in vivo performance of the dosage forms. The pressure sensitive capsules released drug in 50% of the dogs. In the cases where drug was released, there was no difference in octreotide bioavailability or Cmax compared to the enteric coated dosage forms. When comparing all dosage forms, a correlation was seen between paracetamol Cmax and octreotide bioavailability, suggesting that a high drug release rate may be beneficial for peptide absorption when delivered together with sodium caprate.

Design of a self-unfolding delivery concept for oral administration of macromolecules

Delivering macromolecular drugs, e.g. peptides, to the systemic circulation by oral administration is challenging due to their degradation in the gastrointestinal tract and low transmucosal permeation. In this study, the concept of an oral delivery device utilizing an elastomeric material is presented with the potential of increasing the absorption of peptides, e.g. insulin. Absorption enhancement in the intestine is proposed as a result of self-unfolding of a polydimethylsiloxane foil upon release from enteric coated capsules. A pH-sensitive polymer coating prevents capsule disintegration until arrival in the small intestine where complete unfolding of the elastomeric foil ensures close contact with the intestinal mucosa. Foils with close-packed hexagonal compartments for optimal drug loading are produced by casting against a deep-etched silicon master. Complete unfolding of the foil upon capsule disintegration is verified in vitro and the insulin release profile of the final delivery device confirms insulin protection at gastric pH. In vivo performance is evaluated with the outcome of quantifiable plasma insulin concentrations in all rats receiving duodenal administration of the novel delivery device. By taking advantage of elastomeric material properties for drug delivery, this approach might serve as inspiration for further development of commercially viable biocompatible devices for oral delivery of macromolecules.

Absorption, translocation, and effects of Bt Cry1Ac peptides from transgenic cotton to the intercrops and soil functional bacteria

Insecticidal proteins encoded by the truncated genes from Bacillus thuringiensis (Bt) in transgenic crops are released into soil mainly through root exudate and crop residues. In the present study, Bt Cry1Ac protein was hydrolyzed by pronase that was secreted by the soil bacterium Streptomyces griseus. Six peptides were identified as the products of enzymatic hydrolysis by nano liquid chromatography tandem mass spectrometry (LC–MS/MS). One of the six peptides was labeled with radioactive isotope iodine-125 and then purified. The 125I-peptide solution was irrigated to the rhizosphere soil of watermelon seedlings (Citrullus lanatus L.) and wheat seedlings (Triticum aestivum L.), which the two crops usually intercrop with cotton in China. Detection of radioactivity in both plant tissues within one hour proved adsorption, uptake and translocation of the peptide into watermelon and wheat seedlings. Three of the identified peptides were sprayed onto the seedling leaves of watermelon, wheat and maize (Zea mays L.) in the field or the growth chamber. No significant effects on plant growth were observed. These peptides also did not affect growth of organic phosphate-dissolving, nitrogen-fixing, and potassium-dissolving bacteria in the culture. This study provides a new view of GMO risk assessment methodology.

The Influence of Peptidases in Intestinal Brush Border Membranes on the Absorption of Oligopeptides from Whey Protein Hydrolysate: An Ex Vivo Study Using an Ussing Chamber

For many years, it was believed that only amino acids, dipeptides, and tripeptides could be absorbed and thus reach the bloodstream. Nowadays, the bioavailability of oligopeptides is also considered possible, leading to new research. This pilot study investigates the activity of brush border enzymes on undigested whey protein hydrolysate (WPH) and on simulated intestinal digested (ID) whey hydrolysate and the subsequent absorption of resultant peptides through the proximal jejunum of a 7-week old piglet setup in an Ussing chamber model. Amongst all samples taken, 884 oligopeptides were identified. The brush border peptidase activity was intense in the first 10 min of the experiment, producing several new peptides in the apical compartment. With respect to the ID substrate, 286 peptides were detected in the basolateral compartment after 120 min of enzyme activity, originating from β-lactoglobulin (60%) and β-casein (20%). Nevertheless, only 0.6 to 3.35% of any specific peptide could pass through the epithelial barrier and thus reach the basolateral compartment. This study demonstrates transepithelial jejunum absorption of whey oligopeptides in an ex vivo model. It also confirmed the proteolytic activity of brush border enzymes on these oligopeptides, giving birth to a myriad of new bioactive peptides available for absorption.

Passive Internalization of Bioactive β-Casein Peptides into Phospholipid (POPC) Bilayers. Free Energy Landscapes from Unbiased Equilibrium MD Simulations at μs-Time Scale

Absorption of bioactive peptides in the intestinal epithelium take place in the apical or the basolateral tight junctions of the cells. Depending on the peptide size and hydrophobicity, translocation mechanisms involve processes of passive diffusion, active transport by peptide-cotransporters such as members of the PepT family, and transcytosis by internalization vesicles. In this work, we investigated passive diffusion of bioactive peptides of 6, 17, and 30 amino acids into lipid bilayers of (POPC) phospholipid molecules. We initially selected these three peptides because such fragments are produced by partial hydrolysis of β-casein (BCN), and because of their physiological functions: BCN6 is an agonist of opioid receptors; BCN17 is an inhibitor of thrombin and angiotensin-converting enzymes, and BCN30 promotes secretion of the protective mucin barrier in the intestine. Our computational set up consisted of unbiased equilibrium molecular dynamics simulations, at the μs-time scale, using an all-atom force field. Each peptide was allowed to freely fold and unfold, as well as enter and exit the lipid bilayer, which allows determination of peptide affinity for the bilayer interface and hydrophobic core. Passive internalization of BCN6 (YPVEPF), BCN17 (YQEPVLGPVR GPFPIIV), and BCN30 (GVSKVKEAMA PKHKEMPFPK YPVEPFTESQ) displayed different dynamics at the bilayer interface: the BCN6 peptide attached and detached throughout the simulation trajectory; BCN17 and BCN30 attached irreversibly to the bilayer interface, respectively, with N- and C-terminus fragments in close contact with lipid molecules. Quenching of tyrosine fluorescence data suggest interfacial interactions of BCN6, BCN17 and BCN30 in POPC lipid bilayers, consistent with the proposed modeling set up. This approach gave valuable information of peptide insertion and folding at a lipid bilayer, allowing to explore the initial stages of the peptide adsorption at the interface, and providing a model for evaluation of amphipathic properties of potential biofunctional peptides.