Use of Peptide Strategy for Study of Molecular Mechanisms of Hormonal Signal Transduction into Cell

At present the peptide strategy is used extensively to study molecular mechanisms of interaction between signal proteins, components of hormonal signal systems. The strategy is based on use of synthetic peptides as probes corresponding to functionally important sites of these proteins. This review summarizes and analyzes literature data and results of our own works on use of the peptide strategy for studying functional coupling of receptors of serpentine and tyrosine kinase types with heterotrimeric G-proteins. Alongside with peptides derived from the primary structure of cytoplasmic loops and transmembrane domains as well as from different sites of G-protein α, β, and γ-subunits, natural and synthetic peptides are considered which have no homology with receptors and G-proteins, but are able to affect effectively interaction between them.     

Peptides derived from the extracellular loop of receptors: structure, mechanisms of action and application in physiology and medicine

One of the main tasks of the peptide strategy, a new direction in modern biochemistry and physiology, is the creation of selective and effective regulators of hormonal signaling systems on the basis of the peptides corresponding to functionally important regions of signal proteins. At the last years the greatest interest is connected with peptides, derivatives of the extracellular loops of receptors of the serpentine type. With these peptides the molecular basis of interaction between receptors and their ligands are studied, the new approaches for construction and testing of highly selective agonists and antagonists are developed, the etiology and pathogenesis of diseases of human and animals induced by autoimmune reactions to the extracellular loops of receptors are investigated. It is shown that peptides corresponding to the extracellular loops of the receptors and the specific antibodies to them are capable to regulate the activity of hormonal signaling systems in vitro and in vivo and can be considered as functional probes for studying of physiological functions in the norm and pathology. In the review the data obtained during the last years concerning the structures, functions, mechanisms of action and practical application ofpeptides, derivatives of the extracellular loops of serpentine type receptors, are summarized and analyzed. The prospective of their use in fundamental biology and practical medicine are discussed.     

Receptor and tissue specificity of the effects of peptides corresponding to intracellular regions of the serpentine type receptors

Proximal regions of the third intracellular loop (ICL-3) are responsible for the interaction with heterotrimeric G proteins in most of the serpentine type receptors. The peptides corresponding to these regions are able to activate G proteins in the absence of hormone and to alter the transduction of hormonal signal via the respective homologous receptor. However, the molecular mechanisms of action of the peptides, their specificity to receptors and target tissues are currently not well understood. The goal of this work was to study the receptor and tissue specificity of peptides-derivatives of C-terminal regions of the ICL-3 of luteinizing hormone receptor (LHR), type 1 relaxin receptor (RXFP1), somatostatin receptors of types 1 and 2 (Som₁R and Som₂R), and 5-hydroxytryptamine receptors of subtype 1B and type 6 (5-HT_1BR and 5-HT₆R) on the functional activity of adenylyl cyclase (AC) and GppNHp-binding of G proteins in the brain, myocardium, and testis of rats. It was shown that the influence of peptides on AC and G proteins is well detected in tissues enriched in homologous receptors. The effects stimulating AC and GppNHp-binding were most pronounced in the testes for LHR peptide, in the brain for peptide 5-HT₆R, and in all of the tested tissues (but mainly in the myocardium) for the RXFP1 peptide. The AC-inhibiting effects of peptides Som₁R, Som₂R and 5-HT_1BR, as well as the stimulation of GppNHp binding induced by these peptides, were most pronounced in the brain. In the presence of the peptides, the AC effects of hormones acting via homologous receptors were significantly attenuated, while the AC effects of other hormones changed insignificantly. The findings suggest that biological activity of the peptides depends on their interaction with complementary regions of homologous receptors, which should be taken into account when developing highly selective regulators of hormonal signaling systems on the basis of these peptides.     

Polycationic peptides as nonhormonal regulators of chemosignal systems

This review summarizes and analyzes both literature data and results of our own studies on molecular mechanisms of action of natural and artificially created polycationic peptides on functional activity of heterotrimeric G-protein-coupled signal systems. There are considered peptide toxins from insect venom, synthetic peptides that are derivatives of cytoplasmic loops of receptors of the serpentine type as well as artificially created peptides with linear, branched, and dendrimeric structures. Action of most of these peptides on activity of G-proteins is highly selective and these themselves are able to mimic the hormone-activated receptor to be thereby non-hormonal regulators of the signal systems coupled to heterotrimeric G-proteins.     

Regulators of hormonal signalling system on the basis of peptides, derivatives of receptors of the serpentine type

In the review, own results and the data of other authors concerning synthetic peptides corresponding to intracellular and transmembrane regions of receptors of the serpentine type, which are responsible for the interaction with G-proteins and for the formation of ligand-binding site, are analyzed. These peptides affect the basal activity of hormonal signalling systems and the transduction of hormonal signal from the latter with high selectivity and efficiency. Possible molecular mechanisms of the action of peptides, the perspectives of development of a new generation of drugs for the control of endocrine function and physiological processes in the organism on the basis of receptor-based peptides, and their application as functional probes for study of structural-functional organization of hormonal signalling systems are considered.     

Biological implications of SNPs in signal peptide domains of human proteins

Proteins destined for secretion or membrane compartments possess signal peptides for insertion into the membrane. The signal peptide is therefore critical for localization and function of cell surface receptors and ligands that mediate cell-cell communication. About 4% of all human proteins listed in UniProt database have signal peptide domains in their N terminals. A comprehensive literature survey was performed to retrieve functional and disease associated genetic variants in the signal peptide domains of human proteins. In 21 human proteins we have identified 26 disease associated mutations within their signal peptide domains, 14 mutations of which have been experimentally shown to impair the signal peptide function and thus influence protein transportation. We took advantage of SignalP 3.0 predictions to characterize the signal peptide prediction score differences between the mutant and the wild-type alleles of each mutation, as well as 189 previously uncharacterized single nucleotide polymorphisms (SNPs) found to be located in the signal peptide domains of 165 human proteins. Comparisons of signal peptide prediction outcomes of mutations and SNPs, have implicated SNPs potentially impacting the signal peptide function, and thus the cellular localization of the human proteins. The majority of the top candidate proteins represented membrane and secreted proteins that are associated with molecular transport, cell signaling and cell to cell interaction processes of the cell. This is the first study that systematically characterizes genetic variation occurring in the signal peptides of all human proteins. This study represents a useful strategy for prioritization of SNPs occurring within the signal peptide domains of human proteins. Functional evaluation of candidates identified herein may reveal effects on major cellular processes including immune cell function, cell recognition and adhesion, and signal transduction.     

Development of non-hormonal regulators of the adenylyl cyclase signaling system based on the peptides,derivatives of the third intracellular loop of somatostatin receptors

In most serpentine type receptors the third intracellular loop (ICL-3) is responsible for the interaction with heterotrimeric G proteins and for transduction of a hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, consequently, may be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to the C-terminal regions 255–269 and 240–254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som₁R and Som₂R). Micromolar concentrations of these peptides activated G _i proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255–269 of Som₁R is a selective antagonist of Som₁R, and the peptide 240–254 of Som₂R is an agonist of Som₁R. The peptide 255–269 of Som₁R decreased the regulatory effects of somatostatin and the selective Som₁R agonist, CH-275, realized via the homologous receptor, while the peptide 240–254 of Som₂R, on the contrary, increased the AC inhibitory effect of CH-275. Both peptides insignificantly influenced regulatory effects of the Som₂R agonist octreotide. Thus, the peptides studied by us are selective regulators of the somatostatin-sensitive AC system. Using the peptides we have demonstrated that ICL-3 of both Som₁R and Som₂R includes the main molecular determinants that are responsible for activation of G _i proteins and regulation of the AC system by somatostatin and its analogues.     

Tribbles: a family of kinase-like proteins with potent signalling regulatory function

The recent identification of tribbles as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabetes and cancer, has generated considerable interest in these proteins. Tribbles have been reported to regulate activation of a number of intracellular signalling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. The current review summarises our current understanding of interactions between tribbles and various other proteins. Since our understanding on the molecular basis of tribbles function is far from complete, we also describe a bioinformatic analysis of various segments of tribbles proteins, which has revealed a number of highly conserved peptide motifs with potentially important functional roles.     

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Receptors of the serpentine type and heterotrimeric G-proteins of the yeasts: structural-functional organization and molecular action mechanisms

The signal systems of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, coupled to heterotrimeric G-proteins and sensitive to pheromones and alimentary molecules, are prototypes of hormonal signal systems of the higher vertebrate animals and are widely used in studies on molecular mechanisms of their functioning. This review summarizes and analyzes data on structural-functional organization of the first two components of these systems - receptors of the serpentine type and heterotrimeric G-proteins; mechanisms of functional coupling of receptors and G-proteins both between each other and to other signal proteins are discussed. It has been shown that at the early stages of evolution of signal systems, at the yeast level, various models of transduction of signals into the cell were tested; many of them differ essentially from the classic model of the three-component, G-protein-coupled signal system of the higher vertebrates.     

Fragments of Functional Proteins: Role in Endocrine Regulation

Systematic analysis of structures, localization, formation and biological activities of endogenous peptides derived from functional proteins, such as hemoglobin, myelin basic protein, immunoglobulins, etc., allowed establishing the basic features of that group of compounds. The sets of these peptides in mammalian tissues, or tissue-specific peptide pools are: (i) tissue specific; (ii) stable at normal conditions; (iii) conservative in the same tissues of different mammalian species; (iv) dependent on the general state of homeostasis of tissue or the whole organism. Formation of such peptides has features of both conformation and site specificity and also involves the action of carboxy- and amino-peptidases. As a result, the families of structurally related families of peptides are generated. The fragments of functional proteins exhibit a wide range of the biological effects, characteristic both for hormones and parahormones, from hormone-releasing to growth-regulatory activity. At the same time, the molecular mechanisms of action of the majority of such peptides are unknown. On the basis of the data obtained the components of tissue-specific peptide pools are considered to form a novel regulatory system, complementary to other peptidergic systems such as hormonal, nervous, immune, etc. The biological role of the fragments of functional proteins in vivo and the patterns of interaction with other regulatory systems are suggested.     

Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.     

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.     

Targeting of p53 peptide analogues to anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Inhibition of the interaction between the p53 tumor suppressor and its negative regulator MDM2 is of great importance to cancer therapy. The anti-apoptotic Bcl-2 family proteins are also attractive anti-cancer molecular targets, as they are key regulators of apoptotic cell death. Previously, we reported the interactions between the p53 transactivation domain (p53TAD) and diverse members of the anti-apoptotic Bcl-2 family proteins. In this study, we investigated the binding of MDM2-inhibiting p53TAD peptide analogues, p53-MDM2/MDMX inhibitor (PMI) and pDI, with anti-apoptotic Bcl-2 family proteins, Bcl-X_L and Bcl-2, by using NMR spectroscopy. The NMR chemical shift perturbation data demonstrated the direct binding of the p53 peptide analogues to Bcl-X_L and Bcl-2 and showed that the PMI and pDI peptides bind to a conserved hydrophobic groove of the anti-apoptotic Bcl-2 family proteins. Furthermore, the structural model of the Bcl-X_L/PMI peptide complex showed that the binding mode of the PMI peptide is highly similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Finally, our structural comparison provided a molecular basis for how the same PMI peptide can bind to two distinct anti-cancer target proteins Bcl-X_L and MDM2, which may have potential applications for multi-targeting cancer therapy.     

Organelle-targeted delivery of biological macromolecules using the protein transduction domain: potential applications for Peptide aptamer delivery into the nucleus

Extensive effort is currently being expended on the innovative design and engineering of new molecular carrier systems for the organelle-targeted delivery of biological cargoes (e.g., peptide aptamers or biological proteins) as tools in cell biology and for developing novel therapeutic approaches. Although cell-permeable Tat peptides are useful carriers for delivering biological molecules into the cell, much internalized Tat-fused cargo is trapped within macropinosomes and thus not delivered into organelles. Here, we devised a novel intracellular targeting technique to deliver Tat-fused cargo into the nucleus using an endosome-disruptive peptide (hemagglutinin-2 subunit) and a nuclear localization signal peptide. We show for the first time that Tat-conjugated peptide aptamers can be selectively delivered to the nucleus by using combined hemagglutinin-2 subunit and nuclear localization signal peptides. This nuclear targeting technique resulted in marked enhancement of the cytostatic activity of a Tat-fused p53-derived peptide aptamer against human MDM2 (mouse double minute 2) that inhibits p53-MDM2 binding. Thus, our technique provides a unique methodology for the development of novel therapeutic approaches based on intracellular targeting.     

Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.     

Serpentine type receptors and heterotrimeric G-proteins in yeasts: Structural-functional organization and molecular mechanisms of action

The signal systems of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, coupled to heterotrimeric G-proteins and sensitive to pheromones and alimentary molecules, are prototypes of hormonal signal systems of the higher vertebrate animals and are widely used in studies on molecular mechanisms of their functioning. This review summarizes and analyzes data on structural-functional organization of the first two components of these systems—receptors of the serpentine type and heterotrimeric G-proteins; mechanisms of functional coupling of receptors and G-proteins both between each other and to other signal proteins are discussed. It has been shown that at the early stages of evolution of signaling systems, at the yeast level, various models of transduction of signals into the cell were tested; many of them differ essentially from the classic model of the three-component, G-protein-coupled signal system of the higher vertebrates.     

Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies

In the race for supremacy, microbes are sprinting ahead. This warning by the World Health Organization clearly demonstrates that the spread of antibiotic-resistant bacteria leads to a global health problem and that antibiotics never seen before by bacteria are urgently needed. Antimicrobial peptides represent such a source for novel antibiotics due to their rapid lytic activity (within minutes) through disruption of cell membranes. However, due to the similarities between bacterial, fungal, and mammalian plasma cell membranes, a large number of antimicrobial peptides have low lytic specificities and exhibit a broad activity spectrum and/or significant toxic effect toward mammalian cells. Mutation strategies have allowed the development of analogues of existing antimicrobial peptides having greater lytic specificities, although such methods are lengthy and would be more efficient if the molecular mechanisms of action of antimicrobial peptides were clearly elucidated. Synthetic combinatorial library approaches have brought a new dimension to the design of novel biologically active compounds. Thus, a set of peptide analogues were generated based on the screening of a library built around an existing lytic peptide, and on a deconvolution strategy directed toward activity specificity. These peptide analogues also served as model systems to further study the effect of biomembrane mimetic systems on the peptides structural behavior relevant to their biological activities.     

Prediction of signal peptides using scaled window

Cells use a ZIP code system to sort newly synthesized proteins and deliver them wherever they are needed: into different internal compartments called organelles or even out of the cell altogether. One of the most essential features of the ZIP code system is the signal sequence or “address tag,” which is originally present in the N-terminal part of the protein and is trimmed away by the time it is secreted. Owing to the importance of signal peptides for understanding the molecular mechanisms of genetic diseases, reprogramming cells for gene therapy, and constructing new drugs for correcting a specific defect, it is highly desirable to develop a fast and accurate method to identify the signal peptides. In this paper, a scaled window model is proposed. Based on such a model as well as Markov chain theory, a new algorithm is formulated for predicting the signal peptides. Test results for the 1939 secretory proteins and 1440 non-secretary proteins have indicated that the new algorithm is particularly successful in the overall success rate, and hence can serve as a complementary tool to the existing algorithms for signal peptide prediction.