I. I. Tarasenko

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT1BR) and 6 (5-HT6R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT1BR-peptide ARERKATKTL307–316K-amide mimicking agonist-activated 5-HT1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT1BR-antagonists. The 5-HT6R-peptides mimicking 5-HT6R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT6R-agonists on AC and Gs proteins, but in the presence of 5-HT6R-antagonists their action was blocked. Of all the 5-HT6R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT6R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT1BR and 5-HT6R selectively activate Gi and Gs proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.

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-HT1BR 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-HT1BR, 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.

Peptide 612–627 of thyrotropin receptor and its modified analogs as regulators of adenylyl cyclase in rat thyroid gland

The specific activity of the thyroid gland is regulated by thyroid-stimulating hormone (TSH) via TSH receptor (TSHR). This receptor is coupled with various types of G proteins, including Gs proteins, through which TSH stimulates activity of enzyme adenylyl cyclase (AC). Since the use of TSH in medicine is restricted, selective regulators of TSHR with activity of agonists and antagonists are being developed. One approach to their creation is development of peptides corresponding to the functionally important TSHR regions that are located in its cytoplasmic loops and are involved in binding and activation of G proteins. We synthesized peptide corresponding to the C-terminal region 612–627 of the third cytoplasmic loop of TSHR and its derivatives modified by residues of palmitic acid (from the N- or C-termini) or by polylysine dendrimer (from the N-terminus) and we studied their effect on the basal and TSH-stimulated AC activity in membrane fractions isolated from the rat thyroid gland. The most active was peptide 612–627-K(Pal)A modified by palmitate from the C-terminus, where the hydrophobic transmembrane region is located in TSHR. At the micromolar concentrations, it increased AC activity and reduced the AC-stimulating TSH effect. The action of 612–627-K(Pal)A was directed to its homologous TSHR, which is indicated by the following facts: the ingibition of Gs proteins, the transductory component of the AC system, by treatment of the membranes with cholera toxin blocked the AC effect of peptide and this effect was not revealed in the tissues where TSHR are absent, the peptide did not influence the stimulating AC effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer had a much lower ability to activate AC in the thyroid gland than 612–627-K(Pal)A, whereas the peptide modified by palmitate from the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable with 612–627-K(Pal)A by the ability to inhibit the AC action of TSH, but it also decreased (although to the lesser degree) the AC effects of other hormones, which indicates its low receptor specificity. Thereby, the obtained data indicate the high efficiency of the peptide 612–627-K(Pal)A as a regulator of TSHR and the possibilities of creation of drugs on its basis for regulation of thyroid-gland functions in the case of pathology.

The secondary structure of peptides derived from the third intracellular loop of the serpentine-type receptors and its interrelation with their biological activity

We and other authors have shown that synthetic peptides corresponding to regions of the third cytoplasmic loop (CL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and triggering them in the absence of a hormone. To create selective regulators of hormonal signaling systems on the basis of these peptides, the relationship between their biological activity and secondary structure is studied. It is suggested that the most suitable is the helical conformation, which allows the peptide to effectively interact with signaling proteins. The goal of this study was to test the biological activity and secondary structure of linear peptides that we synthesized and their dimeric and palmitoylated analogs corresponding to the C-terminal region of CL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine (serotonin) receptor of type 6 (Ser6R). It is shown that LHR peptides at micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in plasma membranes of rat testes, while Ser6R peptides activate AC and G-proteins in synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while Ser6R peptides, the effect of Ser6R-agonist, EMD-386088, and the action of the peptides was not found in the case of nonhomologous receptors. Using circular dichroism spectroscopy, it is shown that in the neutral (pH 7) and acidic (pH 2) medium, all the peptides exist predominantly in the antiparallel β-sheet (37–42%) and disordered conformations (33–35%). In the alkaline medium (pH 10) in the case of palmitoylated peptides the increase of the contribution of the helical conformation to 12–27% was observed. In the presence of trifluoroethanol (10–80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs by 14%); however, in this case, the antiparallel β-sheet and disordered conformation prevailed. The conclusion was drawn that the lack of a clearly expressed ability to form helices in peptides derived from CL-3 of receptors did not significantly affect their activity. This is consistent with the proposed mechanism of peptide action, whereby peptide interacts with the complementary regions of homologous receptor that does not require helix formation.

Investigating the structural characteristics of monomeric layers of polymers by means of Fourier spectroscopy

This paper discusses the possibilities of using IR Fourier spectroscopy to investigate the orientational and conformational characteristics of single layers of polymers attached by one end to an impermeable surface (polymeric brushes). The method is used to determine the structural parameters of the polymeric branches of brushes attached to a cylindrical or flat surface.

Amphiphilic polypeptides with prolonged enzymatic stability for the preparation of self-assembled nanobiomaterials

Due to their excellent biocompatibility and biodegradability, polypeptides have emerged as versatile bio-inspired scaffolds for the preparation of artificial biomaterials. In order to create self-assembled polypeptide nanoparticles with enhanced stability towards enzymatic degradation, we synthesized a series of random and block polypeptides based on lysine and α-aminoisobutyric acid (Aib) by the ring-opening polymerization of N-carboxyanhydrides (ROP NCA) of the corresponding amino acids. A conformational analysis carried out by means of FT-IR absorption and CD spectroscopies revealed a noticeable difference between random and block copolymers. In turn, the spatial organization of the polypeptide chains induced the formation of nanostructures of different types. The block copolymers self-assembled in vesicle-like structures, whereas polypeptides with randomly distributed monomers formed micelles. In contrast with the polymers with only natural amino acids, all nanoparticles based on Aib/Lys polypeptides showed strong resistance to proteolytic cleavage. The cytotoxicity and the kinetics of the cellular uptake of the prepared nanostructures were also studied. The results obtained could not only contribute to the understanding of long Aib polypeptide folding and self-assembling, but also pave the way to the design of nanomaterials with finely tuned properties in the fields of drug delivery and tissue engineering.

Proinsulin C-peptide and its C-terminal fragments stimulate Gi/o-proteins but do not influence adenylyl cyclase activity

from q/11-family G-proteins. In 2013, it was demonstrated that GPR146 receptor can function as a specific C-peptide receptor, as evidenced by the following facts [3]. The GPR146 gene is expressed in all cells that are sensitive to C-peptide. C-peptide co-localizes with GPR146 receptor and induces its internalization, while this effect is highly C-peptide-specific. GPR146 gene knockout suppresses the stimulatory effect of Cpeptide on expression of c-Fos factor, a main target of its action [3]. We conducted a comparative analysis of the GPR146 receptor primary structure and found that it is homologous to Gi/oand Gq/11protein-coupled receptors of peptide hormones. The enzyme adenylyl cyclase (AC) is known to be one of the main targets of Gi/o-coupled receptors. Based on this fact, we suggested that C-peptide and its terminal fragments can inhibit AC and suppress cAMP-dependent signaling cascades in cells. It is worthwhile to note that pentaand hexapeptides corresponding to the C-peptide terminal segment exhibit the same spectrum of signaling properties as a molecule of the full-size C-peptide and their regulatory effects are also blocked by PT. The aim SHORT COMMUNICATIONS

Peptides Derived from the Third Cytoplasmic Loop of the Serotonin Subtype 1B Receptor Selectively Inhibit Transmission of Serotoninergic Signals via Their Homologous Receptors

A leading role in the interactions of most serotonin-type hormone receptors with heterotrimeric G proteins is played by their third cytoplasmic loops. Studies in recent years have shown that synthetic peptides corresponding to the membrane-proximal parts of these loops may have selective influences on the transmission of hormone signals via their homologous receptors and trigger the signal cascade in the absence of hormone. We report the first synthesis of peptides derived from the C-terminal region of the third cytoplasmic loop of type 1B serotonin receptors, along with studies of their influences on the serotonin sensitivity of the adenylate cyclase system in the rat brain. Peptides 300–316 and 306–316 (numbered from amino acid positions in the rat serotonin subtype 1B receptor molecule) at micromolar concentrations, in the absence of hormone, stimulated GTP-binding Gi-proteins coupled with subtype 1B serotonin receptors and inhibited forskolin-stimulated adenylate cyclase activity. Studies using selective serotonin receptor agonists and antagonists showed that peptides 300–316 and 306–316 inhibit the transmission of the serotonin signal via the homologous receptor but have weak effects on other types of serotonin receptor. Peptide 300–316 were markedly more active than its shortened analog 306–316 in terms of the selectivity and efficacy of actions on the adenylate cyclase signaling system, which is regulated via subtype 1B serotonin receptors. These data provide evidence that region 300–316 of subtype 1B serotonin receptors is involved in the interaction with Gi proteins and includes the main molecular determinants responsible for transmission of the serotonin signal to adenylate cyclase.