Solution structure of nociceptin peptides.

Peptides embedded in the sequence of pre-pro-nociceptin, i.e. nociceptin, nocistatin and orphanin FQ2, have shed light on the complexity of the mechanisms involving the peptide hormones related to pain and have opened up new perspectives for the clinical treatment of pain. The design of new ligands with high selectivity and bioavailability, in particular for ORL1, is important both for the elucidation and control of the physiological role of the receptor and for their therapeutic importance. The failure to obtain agonists and antagonists when using, for nociceptin, the same substitutions that are successful for opioids, and the conformational flexibility of them all, justify systematic efforts to study the solution conformation under conditions as close as possible to their natural environment. Structural studies of linear peptides in solution are hampered by their high flexibility. A direct structural study of the complex between a peptide and its receptor would overcome this difficulty, but such a study is not easy since opioid receptors are membrane proteins. Thus, conformational studies of lead peptides in solution are still important for drug design. This review deals with conformational studies of natural pre-nociceptin peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yielded a completely reliable bioactive conformation, but the global conformation of the peptides in biomimetic environments can shed light on their interaction with receptors.     

Bioactive conformation of linear peptides in solution: An elusive goal?

Bioactive peptides of natural origin have, in general, short linear sequences, and are characterized by a large conformational flexibility. It is very difficult to study their conformation in solution since they exist, almost invariably, as a complex mixture of numerous conformers, most of which are extended. The so-called bioactive conformation may be one of them, although the solvents used in solution studies often have properties drastically different from those of the biological system in which the peptide acts. There is, however, no simple way of identifying the bioactive conformation amid the many existing conformers. It is possible to approach a solution to this problem using two distinct strategies: (a) Limiting the conformational freedom of the peptide, e.g., by increasing the viscosity of the solution and decreasing the temperature, in the assumption that the bioactive conformation is, even slightly, more stable than the others. (b) Trying to mimic in solution the physicochemical features of the more reliable receptor models. These two approaches will be illustrated with examples taken mainly from opioid peptides.     

Detection of small bioactive peptides from Atlantic herring (Clupea harengus L.)

Recent research has shown that fish residual materials contain a range of components with interesting biological activity. Therefore, there is a great potential in the marine bioprocess industry to utilize these by-products as starting material for generating more valuable products. The aim of the present study was to search for bioactive peptides (in particular small natural bioactive peptides with molecular weight lower than 10 kDa) in Atlantic herring (Clupea harengus L.) by-products such as skin and more general residual materials. By such means a range of peptides with claimed interesting biological activities was found. Herein the activity of the detected bioactive peptides and strategies for isolating peptide fragments containing the bioactive motif is discussed. Identification of bioactive peptides in crude peptide/protein sources (skin and residual materials) was performed directly using a combination of mass spectrometry (Orbitrap), bioinformatics and database search. This method was a good angle of approach in order to map the potential in new species and species that have been very little studied.     

Thrombin receptor-activating peptides (TRAPs): investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving alpha-methyl, ester psi(COO), and reduced-amide psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed 1H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, whereas SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).     

Conformational studies of antimetastatic laminin-1 derived peptides in different solvent systems, using solution NMR spectroscopy.

Due to its critical role in cancer progression, interactions between laminin-1 and the 67 kDa Laminin-Binding Protein (the 67 kDa LBP) have been the focus of a number of structural and biological studies. As laminin-1 is such a large and complex molecule, research interests have turned to the investigation of bioactive peptides derived from binding domains of laminin-1. Two peptides of interest, CDPGYIGSR (peptide 11) and YIGSR, both derived from the beta1 chain of laminin-1, have been shown to block invasion of basement membranes by tumor cells. Substituting the C-terminal arginine to lysine, a conservative substitution, results in a loss of peptide antimetastatic activity. This difference in bioactivity has been attributed, based on numerous modeling studies of free peptide conformations, to structural differences between YIGSR and YIGSK. Yet the nature of the 'active' free peptide backbone conformation has been a matter of debate and controversy. In order to test the validity of the structural modeling claims, we have undertaken detailed conformational studies of the two laminin-1 derived peptides YIGSR and CDPGYIGSR along with the biologically inactive YIGSK analog by two-dimensional solution 1H NMR spectroscopy in three different solvent systems. Herein we report that although both the active (YIGSR, CDPGYIGSR) and the inactive (YIGSK) peptides can adopt several closely related conformations in solution, the two peptides share similar conformational preferences, and there are no significant structural differences between the active and inactive peptides, contrary to previously reported modeling data. We conclude that the basis of the peptide biological activity, in contrast to published models, cannot be attributed to well-defined structural preferences of the free peptides. We infer that the difference in bioactivity observed between YIGSR and YIGSK originates primarily from the chemical nature of the arginine versus lysine sidechain substitution, rather than being due to a structural change in the free peptide conformations.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.     

Multifunctional peptides encrypted in milk proteins

Many bioactivities of milk are latent in that they are inactive within the protein sequence, requiring enzymatic proteolysis for release of bioactive peptides from milk proteins precursors. Bioactivities of peptides encrypted in major milk proteins are latent until released and activated, e.g. during gastrointestinal digestion or food processing. Bioactive peptides can be produced in vivo following intake of milk proteins, and the proteolytic system of bacterial species used in the production of fermented milk products and cheese can contribute to the liberation of bioactive peptides or precursors thereof. Activated peptides are potential modulators of various regulatory processes in the living system: immunomodulatory peptides stimulate the activities of cells of the immune system and several cytomodulatory peptides inhibit cancer cell growth, antimicrobial peptides kill sensitive microorganisms, angiotensin-I-converting enzyme (ACE)-inhibitory peptides exert an hypotensive effect, opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, mineral binding peptides may function as carriers for different minerals, especially calcium. Many milk-derived peptides reveal multifunctional properties, i.e. specific peptide sequences having two or more different biological activities have been reported. Milk protein-derived bioactive peptides are claimed to be health enhancing components that can be used to reduce the risk of disease or to enhance a certain physiological function.     

Recognized sequence and conformation in design of linear peptides as a competitive inhibitor for HMG-CoA reductase

This study is an attempt to develop a simple search method for lead peptide candidates, which include constrained structures in a recognized sequence, using the design of a competitive inhibitor for HMG-CoA reductase (HMGR). A structure-functional analysis of previously synthesized peptides proposes that a competitive inhibitory peptide can be designed by maintaining bioactive conformation in a recognized sequence. A conformational aspect of the structure-based approach was applied to the peptide design. By analysis of the projections obtained through a principle component analysis (PCA) for short linear and cyclic peptides, a head-to-tail peptide cycle is considered as a model for its linear analogy. It is proposed that activities of the linear peptides based on an identical amino acid sequence, which are obtained from a less flexible peptide cycle, would be relatively higher than those obtained from more flexible cyclic peptides. The design criterion was formulated in terms of a 'V' parameter, reflecting a relative deviation of an individual peptide cycle from an average statistical peptide cycle based on all optimized structures of the cyclic peptides in set. Twelve peptide cycles were selected for the peptide library. Comparing the calculated 'V' parameters, two cyclic peptides (GLPTGG and GFPTGG) were selected as lead cycles from the library. Based on these sequences, six linear peptides obtained by breaking the cycle at different positions were selected as lead peptide candidates. The linear GFPTGG peptide, showing the highest inhibitory activity against HMGR, increases the inhibitory potency nearly tenfold. Kinetic analysis reveals that the GFPTGG peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 6.4 +/- 0.3 microM. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease

Vascular diseases such as atherosclerosis, stroke or myocardial infarction are a significant public health problem worldwide. Attempts to prevent vascular diseases often imply modifications and improvement of causative risk factors such as high blood pressure, obesity, an unfavorable profile of blood lipids or insulin resistance. In addition to numerous preventive and therapeutic drug regimens, there has been increased focus on identifying dietary compounds that may contribute to cardiovascular health in recent years. Food-derived bioactive peptides represent one such source of health-enhancing components. They can be released during gastrointestinal digestion or food processing from a multitude of plant and animal proteins, especially milk, soy or fish proteins. Biologically active peptides are considered to promote diverse activities, including opiate-like, mineral binding, immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic and antihypertensive actions. By modulating and improving physiological functions, bioactive peptides may provide new therapeutic applications for the prevention or treatment of chronic diseases. As components of functional foods or nutraceuticals with certain health claims, bioactive peptides are of commercial interest as well. The current review centers on bioactive peptides with properties relevant to cardiovascular health.     

The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.

The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions.     

Conformations of model peptides in membrane-mimetic environments.

The influence of a membrane environment on the conformational energetics of a polypeptide chain has been investigated through studies of model peptides in a variety of membrane-mimetic media. Nuclear magnetic resonance (NMR) and circular dichroism (CD) data have been obtained for the peptides in bulk hydrophobic solvents, normal micelles, and reversed micelles. Several hydrophobic peptides which are sparingly soluble in water have been solubilized in aqueous sodium dodecyl sulfate (SDS) solution. NMR and CD data indicate that the micelle-solubilized peptides experience an environment with the conformational impact of bulk methanol, and have decreased conformational freedom. The site of residence of the peptides interacting with the micelles appears to be near the surfactant head groups, in a region permeated by water, and not in the micelle core. Strongly hydrophilic peptides have been solubilized in nonpolar solvents by reversed micelles. These peptides are located in small water pools in close association with the head groups of the surfactant. NMR and CD data show that there is a conformational impact of this interfacial water region on peptide solubilizates distinct from that of bulk water.     

Characterization and production of multifunctional cationic peptides derived from rice proteins

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.     

Investigating endogenous peptides and peptidases using peptidomics

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters, and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been a successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome--all the peptides in a cell, tissue, or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography--tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.     

Structure-lipophilicity relationships of peptides and peptidomimetics

Summary Understanding the physicochemical and structural properties of peptides are important prerequisites for the rational design of bioactive peptides and peptidomimetics. The present contribution reviews methods used for the assessment and prediction of lipophilicity (or hydrophobicity) and their correlation with structural elements of peptides and closely related peptidomimetics.     

Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part I: overview

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In this review, we discuss the exploitation of such fermentation towards the development of functional foods conferring specific health benefits to the consumer beyond basic nutrition. In particular, in Part I, we focus on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of LAB as cell factories for the de novo generation of bioactivities.     

Biological activities and potential health benefits of bioactive peptides derived from marine organisms

Marine organisms have been recognized as rich sources of bioactive compounds with valuable nutraceutical and pharmaceutical potentials. Recently, marine bioactive peptides have gained much attention because of their numerous health beneficial effects. Notably, these peptides exhibit various biological activities such as antioxidant, anti-hypertensive, anti-human immunodeficiency virus, anti-proliferative, anticoagulant, calcium-binding, anti-obesity and anti-diabetic activities. This review mainly presents biological activities of peptides from marine organisms and emphasizing their potential applications in foods as well as pharmaceutical areas.     

The pH sensitivity of histidine‐contain‐ ing lytic peptides

Many bioactive peptides are featured by their unique amino acid compositions such as argine/lysine‐rich peptides. However, histidine‐rich bioactive peptides are hardly found. In this study, histidine‐containing peptides were constructed by selectively replacing the corresponded lysine residues in a lytic peptide LL‐1 with histidines. Interestingly, all resulting peptides demonstrated pH‐dependent activities. The cell lysis activities of these peptides could be increased up to four times as the solution pHs dropped from pH = 7.4 to pH = 5.5. The pH sensitivity of a histidine‐containing peptide was determined by histidine substitution numbers. Peptide derivatives with more histidines were associated with increased pH sensitivity. Results showed that not the secondary structures but pH‐affected cell affinity changes were responsible for the pH‐dependent activities of histidine‐containing peptides. The histidine substitution approach demonstrated here may present a general strategy to construct bioactive peptides with desired pH sensitivity for various applications. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Cyclic peptides — Small and big and their conformational aspects

Cyclic peptides form an interesting class of compounds for study by conformational analysis, by virtue of their unique conformational features and biological properties. The small cyclic peptides having 3-6 peptide units in their ring, show a variety of conformational characteristics such as occurrence ofcis peptide units, flexibility of peptide dimension and variety in hydrogen bonding. The different possible conformations of cyclic tri- and hexa-peptides are given and certain specific conformational features are discussed for cyclic tetra and pentapeptides. For higher cyclic peptides, the hydrogen bonding requirement for stability of the backbone of the ring, is seen to be kept to a minimum. These various features and their significance are examined and discussed in the light of energy minimization studies and analysis of available experimental data.