The multifunctionality of regulatory peptides and the rule of "what--where--when" as a principle of their ordered action]

Consistent study of stages and history of discovery of regulatory peptides (RP) as a new class of multifunctional endogenic regulators puts forward some actual problems. A question what new notions on the manner of control of physiological functions have been introduced by RP investigation is considered. Two historical tendencies in the RP studies are estimated. A conception on peptides as multifunctional and colocated factors of neurohumoral regulation of organism was opposed to the idea of "single" substance--a regulator of "unique" function. In this connection a conception of peptide-regulatory continuum is considered and the author postulated the principle "what--where--when" as the main rule of RP well-regulated action. The statement on tissue (regional) specificity of the biogenesis processes of different RP as molecular basis of their regulatory function is grounded.     

Search for evolutionary ancient, relict, regulatory peptides

Among numerous regulatory peptides (RP) it is possible to presumably indicate the relict, evolutionary ancient RP. They combine three features: formation from non-specialized peptides-precursors, a comparatively high resistance to action of proteases in the organism media, and maximal simplicity of their structure. The examples of them are glyprolines—a recently identified RP family, as well as tuftsin. Several other praline-containing RP in terminal sites also seem to belong to the evolutionary ancient RP. The proposed approach to studies on the RP evolution is additional to those used traditionally in this problem.     

Vestibulo-protective properties of regulatory peptides

Endogenous opioid peptides take an active part in the pathogenesis of motion sickness (MS). Some regulatory peptides (substances P, gamma and des-tyr-gamma-endorphins) manifest antiemetic properties, unlike opioid peptides and morphine. Thus, we have examined the vestibulo-protective properties of some regulatory peptides during simulation of MS in cats. Vestibulo-protective properties have been discovered in some peptides: sweet water hydra undecapeptide, substances P, gamma and des-tyr-gamma-endorphins. It has been suggested that regulatory peptides take an active part in the genesis of vestibulo-protective disorders during motion sickness.     

Regulatory peptides. A functional continuum

A hypothesis postulating the regulatory peptides continuum is presented. At certain combinations, the numerous regulatory peptides (RP) make up a continuous set of bioactivity spectra, i.e., functionally continuous integrity (continuum) which provides a way for any complex influences on organism functions. Nevertheless, each RP retains its qualitative differences as compared to other RPs. Data on reciprocal influences of RP on their release into body fluids are generalized. The experimental results testify to complex chain reactions of the whole RP continuum in response to changes in any single peptide concentration.     

Functional continuum of the regulatory peptides enhancing anxiety. Search for the optimal complexes as a basis for developing inhibitory therapeutic agents

Regulatory peptides (RP) take an active part in managing the majority of physiological processes. One of these functional applications is the monitoring of the anxiety level, of panic state. This work represents the presumptive analysis of literature data of 1960-2004 on the effects of regulatory peptides enhancing anxiety (RP-AT). This information database was used for researching the characteristics of organization and functionality of the system of anxiogenic RP. Taking into account the method of vector representation of RP effects, estimation of spectra of physiological effects that can go with each of RP-AT and their combination, was carried out. The most perspective RP complexes of anxiogenic profile for further experimental development of inhibitory therapeutic agents are proposed.     

Development and application of an expert system for analysis of the functional continuum of regulatory peptides

An expert computer system was developed to analyze the effects of regulatory peptides (RPs) and some other biologically active compounds. The database includes information on their structures and physiological and inductive effects taking into account the method of administration, doses, and types of organisms. The developed method of vector representation of RP effects and the software modules provide for the evaluation of the role of single RPs and their combinations in the regulatory system of organism. The expert system was used for solving the following tasks: (1) an analysis of the structural and functional organization of the RP continuum and a search for effective RP combinations regulating the levels of anxiety, depression, cognitive processes, etc; (2) design of a network of complicated cross inductive connections of major members of RP families; and (3) an analysis of the peculiar features of functioning of numerous internal reward factors.     

Development and application of an expert system for analysis of the functional continuum of regulatory peptides

An expert computer system was developed to analyze the effects of regulatory peptides (RPs) and some other biologically active compounds. The database includes information on their structures and physiological and inductive effects taking in account the method of administration, doses, and types of organisms. The developed method of vector representation of RP effects and the software modules provide for the estimation of the role of single RPs and their combinations in the regulatory system of organism. The expert system was used for solving the following tasks: (1) an analysis of the structural and functional organization of the RP continuum and a search for effective RP combinations regulating the levels of anxiety, depression, cognitive processes, etc; (2) design of a network of complicated cross inductive connections of major members of RP families; and (3) an analysis of the peculiar features of functioning of numerous internal reward factors. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Search for evolutionary ancient, relict regulatory peptides

Among numerous regulatory peptide (RP) it is possible to presumably indicate the relict, evolutionary ancient RP. They combine three features: formation from non-specialized proteins-precursors, a comparatively high resistance to action of proteases in the organism media, and maximal simplicity of their structure. The examples of them are glyprolines--a recently identified RP family, as well as tuftsin. Several other praline-containing RP in terminal sites also seem to belong to the evolutionary ancient RP. The proposed approach to studies on the RP evolution is additional to those used traditionally in this problem.     

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.     

Cation exchange as a single polishing step for conjugated peptides

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.     

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immunoreactivity of human tissue mast cells: nonspecific binding of primary antibodies against regulatory peptides by ionic linkage

Tissue mast cells (TMC) are known to react with antibodies against various regulatory peptides (RP). The specificity of such reactions was investigated by various methods in this study. When normal immunohistochemical staining procedures were employed. TMC in the vermiform appendix and in a cutaneous mastocytoma reacted with antibodies against ACTH, Leu-enkephalin, Met-enkephalin, and peptide histidine isoleucine (PHI). Antibody specificity was tested by absorption controls, and staining specificity by varying the concentration of the primary antibodies and the pH and sodium chloride concentration of the buffer used for rinsing and diluting. In absorption controls, staining of the TMC by anti-PHI was diminished but staining by anti-ACTH, anti-Leu-enkephalin, and anti-Met-enkephalin remained unchanged. Unlike control reactions, immunostaining of TMC with antibodies against RP exhibited marked dependence on antibody concentration and the pH and sodium chloride concentration of the buffer. Alkalization of the buffer led to an obvious increase in the reaction with antibodies against RP, and lowering the pH to 6.0 usually resulted in abolition of the reaction. These results indicate that the immunostaining of TMC with antibodies against RP, including PHI, was nonspecific. It is postulated that the granules of TMC bind certain antibodies by a cation-exchange mechanism involving ionic interactions with positively charged groups in the F(ab')2 and/or Fc segments.     

Crystallization of proton channel peptides.

Crystals have been grown of two similar peptides that form ion-conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2-trifluoroethanol. Crystals of one of the peptides have been characterized by X-ray diffraction, and X-ray data have been measured to 2.3 A resolution. Earlier it was proposed that the ion-conducting channels formed by these peptides consist of four peptides associated as a parallel alpha-helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.     

Immunogold staining procedure for the localisation of regulatory peptides

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.     

Emetic and antiemetic properties of regulatory peptides

Emetic and antiemetic properties of opioid peptides, substance "P", beta-lipotropin, and ACTH1-39 have been investigated in experiments on cats. It was shown that morphine, enkephalin, beta-endorphin and DADLE caused vomiting in animals, which was blocked by naloxone. Substance "P", gamma- and des-tyr-gamma-endorphin manifested antiemetic properties similar to those of naloxone. Selective antagonists of delta-opioid receptors ICI 154, 129 blocked emetic action of delta-agonist DADLE but did not prevent vomiting caused by mu-agonist morphine. It is suggested that the vomiting mechanisms of endogenous opioid peptides involve stimulation of mu- and delta-opioid receptors in the chemoreceptor trigger zone of the vomiting centre.     

Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides

Dipeptidyl-peptidase IV (DPP IV/CD26) has a dual function as a regulatory protease and as a binding protein. Its role in the inactivation of bioactive peptides was recognized 20 years ago due to its unique ability to liberate Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of regulatory peptides, but further examples are now emerging from in vitro and vivo experiments. Despite the minimal N-terminal truncation by DPP IV, many mammalian regulatory peptides are inactivated--either totally or only differentially--for certain receptor subtypes. Important DPP IV substrates include neuropeptides like neuropeptide Y or endomorphin, circulating peptide hormones like peptide YY, growth hormone-releasing hormone, glucagon-like peptides(GLP)-1 and -2, gastric inhibitory polypeptide as well as paracrine chemokines like RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokine. Based on these findings the potential clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, have been tested to enhance insulin secretion and to improve glucose tolerance in diabetic animals. Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones and chemokines.     

Examination of possible structural constraints of MHC-binding peptides by assessment of their native structure within their source proteins

Antigenic peptides bind to major histocompatibility complex (MHC) molecules as a prerequisite for their presentation to T cells. In this study, we investigate possible structural preferences of MHC-binding peptides by examining the conformation space defined by the structures of these peptides within their native source proteins. Comparison of the conformation space of the native structures of MHC-binding nonamers and a corresponding conformation space defined by a random set of nonamers showed no significant difference. This suggests that the environment of the MHC binding groove has evolved to bind peptides with essentially any "structural background." A slight tendency for an extended beta-conformation at positions 8 and 9 was observed for the set of native structures. We suggest that such a preference may facilitate the binding of the C-terminal anchor position of processed peptides into the corresponding specificity pocket. MHC-binding peptides represent examples of short subsequences that are present in two different structural environments: within their native protein and within the MHC binding groove. Comparison of the native and of the bound structure of the peptides showed that peptides up to 14 residues long may adopt different conformations within different protein environments. This has direct implications for structure prediction algorithms.