Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete “families”, some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function.     

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography–mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3 h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography–tandem mass spectrometry method.     

Application of antimicrobial peptides in agriculture and food industry

Antimicrobial peptides have captured the attention of researchers in recent years because of their efficiency in fighting against pathogens. These peptides are found in nature and have been isolated from a wide range of organisms. Furthermore, analogs or synthetic derivatives have successfully been developed on the basis of natural peptide patterns. Long use of pesticides and antibiotics has led to development of resistance among pathogens and other pests as well as increase of environmental and health risks. Antimicrobial peptides are under consideration as new substitutes for conventional pesticides and antibiotics. Many plants and animals have been manipulated with antimicrobial peptide-encoding genes and several pesticides and drugs have been produced based on these peptides. Such strategies and products may still have a long way to go before being confirmed by regulatory bodies and others need to surmount technical problems before being accepted as applicable ones. In spite of these facts, several cases of successful use of antimicrobial peptides in agriculture and food industry indicate a promising future for extensive application of these peptides. In this review, we consider the developing field of antimicrobial peptide applications in various agricultural activities.     

Isolation, structure and properties of new endogenous peptides]

Large scale isolation and determination of amino acid sequences of endogenous peptides from various biological sources (bovine brain and red bone marrow, siberian ground squirrel brain) were carried out. A number of earlier unknown peptides were identified, many of them showing distinct activity in vivo and/or in vitro. Analysis of more than 170 isolated peptide structures resulted in the hypothesis suggesting functional proteins, in particular hemoglobin, to serve also as a source of the peptide "background" which has its own biological significance. Further directions of investigating the endogenous peptide material are mapped. Its potential for developing diagnostics for somatic diseases is demonstrated on patients with CNS disorders.     

Therapeutic peptides: Historical perspectives,current development trends,and future directions

Peptide therapeutics have played a notable role in medical practice since the advent of insulin therapy in the 1920s. Over 60 peptide drugs are approved in the United States and other major markets, and peptides continue to enter clinical development at a steady pace. Peptide drug discovery has diversified beyond its traditional focus on endogenous human peptides to include a broader range of structures identified from other natural sources or through medicinal chemistry efforts. We maintain a comprehensive dataset on peptides that have entered human clinical studies that includes over 150 peptides in active development today. Here we provide an overview of the peptide therapeutic landscape, including historical perspectives, molecular characteristics, regulatory benchmarks, and a therapeutic area breakdown.     

Biologically active peptides: prospects for drug development

Biologically active peptides aree typified by their unbiquity of distribution, their high receptor affinity and an almost infinite diversity of structure. For these reasons, considerable effort is now being expended to elucidate the possible role of peptides in brain function. This effort has been stimulated by the discovery of a number of new endogenous peptides, such as the enkephalins, endorphins, vasoactive intestinal peptide and neurotensin. At present, there is no clearly defined role for these peptides, although they may form an important basis for the chemical coding of various brain functions, including pain, mood and memory. At present, the potential for drug development of peptide agonists remains in fairly circumscribed areas such as analgesia, pituitary hormone control, and gastrointestinal motor and secretory control. Peptide antagonists may provide a vast field for future development, although only one area, that of antifertility drugs based on LHRH antagonists, shows any promise of immediate success. Industrial research approaches to new peptide agonists and antagonists mainly rely at present on rational drug design through structural analogies. Other fruitful approaches to be considered are the screening of natural microbial and plant products and the possible application of genetic engineering techniques.     

A way of functional classification of regulatory peptides. Parameters of divergent and convergent evolution of regulatory peptides

A mathematical method has been presented for systematization of functions of regulatory peptides (RP) and evaluation of directions of evolutionary development of RP systems. For this purpose, traditional methods of vector algebra and multi-dimensional space were used. Effects of various peptide regulators on anxiety, depression, and memory are considered by the example of the three-dimensional space. A way of the functional classification of peptides has been proposed.     

The genetics of lantibiotic biosynthesis

The lantibiotics are a rapidly expanding group of biologically active peptides produced by a variety of Gram-positive bacteria, and are so-called because of their content of the thioether amino acids lanthionine and β-methyllanthionine. These amino acids, and indeed a number of other unusual amino acids found in the lantibiotics, arise following post-translational modification of a ribosomally synthesised precursor peptide. A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide, ABC-superfamily transport proteins involved in lantibiotic translocation, regulatory proteins controlling lantibiotic biosynthesis and proteins that protect the producing strain from the action of its own lantibiotic. Analysis of these genes and their products is allowing greater understanding of the complex mechanism(s) of the biosynthesis of these unique peptides.     

SwePep, a database designed for endogenous peptides and mass spectrometry

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.     

The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction

Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were alpha-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-zeta, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.     

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.     

Administered peptides inhibit the degradation of endogenous peptides. The dilemma of distinguishing direct from indirect effects

Virtually all peptides are biologically active following central administration as a consequence of both direct and indirect cellular actions. Direct effects are mainly interactions with specific membrane receptors but may include unions with other components of the receptor/effector complex. Significant indirect biological effects of exogenous peptides, including apparent secretagogue effects on endogenous peptides largely overlooked in practice, result from extensive competition with endogenous peptides for degradative enzymes (peptidases). A consequence of this competition is enhancement of tonic or intermittent activity of endogenous peptides. The pharmacological profile of any peptide reflects or includes, therefore, the spectrum of endogenous peptides that is protected from peptidase action. It is likely that certain pharmacologically active peptides, including a large number of di-, tri- and oligo-peptides, elicit responses mainly or exclusively by competing for peptidases. Therefore, reliable estimates of the relative contributions of direct and indirect actions of exogenous peptides may be difficult, if not impossible, to obtain.     

Regulatory peptides and control of food intake in non-mammalian vertebrates

The current view of the control of food intake involves a central feeding system in the hypothalamus receiving input from peripheral systems. The presence of food in the gut stimulates the release of several regulatory peptides that control gut motility and secretion. Some of these peptides also act as feedback satiety signals, responsible for termination of a meal. Among the regulatory peptides suggested as peripheral satiety signals are cholecystokinin and gastrin releasing peptide. A more long-term peripheral regulation of food intake has also been postulated and leptin has been suggested as a regulator of food intake. Several regulatory peptides mediate orexigenic or anorexigenic effects in the central feeding system. Neuropeptide Y and galanin both act centrally and stimulate the intake of food, while corticotropin releasing factor reduces food intake. At present, most information about the regulation of food intake is gained from mammalian studies and these findings are used as a base for a discussion on the current knowledge of how regulatory peptides control appetite in non-mammalian vertebrates.     

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.     

The primary structure of a PYY-related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY.

Although the amino acid sequence of members of the pancreatic polypeptide (PP)-family of regulatory peptides has been poorly conserved during vertebrate evolution, the overall length of the peptides (36 amino acid residues) has remained constant. Nucleotide sequence analysis of cloned cDNAs and/or genomic fragments has shown the PP-related sequence immediately follows the signal peptide in the prepropeptides. A peptide tyrosine-tyrosine (PYY)-related peptide with 37 residues has been isolated from the chicken intestine, and its primary structure was established as: Ala-Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly10-Asp-Ala-Ala-Ser-P ro-Glu-Glu-Ile-Ala-Gln20-Tyr-Phe-Ser-Ala-Leu-Arg-His-Tyr-Il e-Asn30-Leu-Val-Thr-Arg-Gln-Arg-Tyr.CONH2. The presence of an additional alanine residue at the NH2-terminus of the peptide suggests that the site of cleavage of the signal peptide in chicken preproPYY is different from the site of cleavage in other PP-family prepropeptides.     

Endothelin converting enzyme-2: a processing enzyme involved in the generation of novel neuropeptides

Members of several metalloprotease families have been proposed to be involved in non-classical processing of neuroendocrine precursors. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits a neuroendocrine distribution, intracellular subcellular localization, and an acidic pH optimum. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. These results are consistent with an important role for ECE-2 in the processing of regulatory peptides at non-classical sites.     

A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR

The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.     

Functional continuum of regulatory peptides (RPs): vector model of RP-effects representation

During the past decades, bioactive (regulatory) peptides have been identified as the major players in the regulation of many important biological processes. Dozens of peptides have found their application as pharmaceutical agents, which further stimulated research in this field making it one of the most rapidly developing areas on the edge of biological science and medicine. However, the fast accumulation of enormous amounts of experimental data has revealed a great difficulty in their analysis and demanded the development of a systematic approach for generalization of the obtained information. We propose a new computer-based algorithm for studying biological activities of regulatory peptides and their groups based on their representation as vectors in n -dimensional functional space. Our method allows the rapid analysis of databases containing thousands of polyfunctional regulatory peptides with overlapping spectra of physiological activity. The described method permits to perform several types of correlations which, when applied to the large databases, could reveal new important information about the system of regulatory peptides. It can select the groups of peptides with similar physiological role (peptide constellations) and search for the optimal peptide combinations with predetermined spectrum of effects and minimal side effects for their further pharmacological application. It can also reveal the role of regulatory peptides in induction of chain physiological reactions.     

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One area of the search for hormonal signaling systems regulators is development of peptides that correspond to the cytoplasmic regions of G protein-coupled receptors (GPCR). Modification of such peptides with hydrophobic radicals increases their efficiency and selectivity. However, at present it has not been studied how the activity of the peptide depends on the localization of hydrophobic radicals, their number, and chemical nature. The aim of this work consisted in synthesis of peptide 562–572 derivatives modified by fatty-acid radicals and corresponding to the C-terminal region of the luteinizing hormone receptor (LHR) and in the study of regulatory effects of the acylated LHR peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in rat tissues. To elucidate the effects of localization of hydrophobic radicals and of their number, modifications of peptide 562–572 were carried out only at the N-or at the C-terminus or at both ends. To study the effect of hydrophobicity, residues of palmitic (Pal) and decanoic (Dec) acids were chosen. Using a solid-phase strategy synthesis was performed of the unmodified peptide NKDTKIAKK-Nle-A^562-572-KA (1) and five of its acylated analogues, N[K(Dec)]DTKIAKK-Nle-A^562-572-KA (2), NKDTKIAKK-Nle-A^562-572-[K(Dec)]A (3), N[K(Dec)]DTKIAKK-Nle-A^562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A^562-572-KA (5), and NKDTKIAKK-Nle-A^562-572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the basal AC activity and reduced the AC stimulating effect of human chorionic gonadotropin (hCG) in testes of rats; peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the unmodified peptide 1; and peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by tissue and the receptor specificity. Thus, modification of the LHR peptide 562–572 with fatty-acid radicals at the C-terminus enhances its regulatory effect on the functional activity of the adenylyl cyclase system in rat testes, which indicates a promising modification of GPCR peptides with hydrophobic radicals. These data confirm the hypothesis that the hydrophobic radical is to be localized in the locus of GPCR peptide, where a transmembrane domain is located in the receptor.