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Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(N α Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH 2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(N α Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH 2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Objective: To examine cardioprotective effects of Ρ-terminal fragment of adipokine apelin-12 (A12), its novel structural analogue [MeArg(1), NLe(10)]-A12 (I), and [d-Ala(12)]-A12 (II), a putative antagonist of APJ receptor, employing in vivo model of ischemia/reperfusion (I/R) injury., Materials and Methods: Peptides were synthesized by the automatic solid phase method using Fmoc technology. Anesthetized open-chest male Wistar rats were subjected to left anterior descending (LAD) coronary artery occlusion and coronary reperfusion. Hemodynamic variables and electrocardiogram (ECG) were monitored throughout the experiment. Myocardial injury was assessed by infarct size (IS), activity of necrosis markers in plasma, and metabolic state of the area at risk (AAR)., Results: Intravenous injection of A12, I, or II at the onset of reperfusion led to a transient reduction of the mean arterial pressure. A12 or I administration decreased the percent ratio of IS/AAR by 40% and 30%, respectively, compared with control animals which received saline. Both peptides improved preservation of high-energy phosphates, reduced lactate accumulation in the AAR, and lowered CK-MB and LDH activities in plasma at the end of reperfusion compared with these indices in control. Treatment with II did not significantly affect either the IS/AAR, % ratio, or activities of both markers of necrosis compared with control. The overall metabolic protection of the AAR in the treated groups increased in the following rank: II < A12 < I., Conclusions: The structural analogue of apelin-12 [MeArg(1), NLe(10)]-A12 may be a promising basis to create a new drug for the treatment of acute coronary syndrome.

Two methionine-containing peptides, endothelin 1 and the 1-16 fragment of the receptor of the plasminogen activator 1 for human urokinase, were synthesized and cyclized by hydrogen peroxide. Endothelin 1 was obtained by using regioselective and random schemes of disulfide bond formation. The conditions of cyclization that provided the target products in high purity were found. The general potential of disulfide bond formation by means of hydrogen peroxide was demonstrated for methionine-containing peptides. The method resulted in target products containing insignificant quantities of the corresponding Met-sulfoxide derivatives.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

The peptide H-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-NHEt corresponding to the 593-603 sequence of gp41 protein of the HIV-2 was used to evaluate different methods for the removal of Acm-protection and subsequent disulfide bond formation. The studied methods involved the treatment by salts of heavy metals (silver and mercury) and subsequent cyclization by oxygen, potassium ferricyanide or hydrogen peroxide. The direct oxidative conversion of Acm-peptide to the corresponding cyclic disulfide by iodine under acidic and neutral conditions was investigated, and the structure of by-products was also studied. The best results were obtained using mercuric acetate followed by oxidation with hydrogen peroxide.

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.

We reported earlier that peptides containing glycine as the third amino acid from the amino end undergo sequence rearrangement of the first two amino acid residues. In the course of this experimental verification of the suggested reaction mechanism, we found extensive racemization of the amino acid residue in position 1. Racemization is preferred over rearrangement in peptides containing amino acids different from glycine in position 3. We demonstrate that this reaction can be used for the selective labeling of peptides. Using model peptides, we suggest a mechanism that explains both the rearrangement and racemization of these peptides in aqueous solution. This mechanism is based on formation of a diketopiperazine-like (DKP-like) structure by attack of the N-terminal amino group on the amide carbonyl group of the second residue in the peptide chain. This tetrahedral intermediate, which contains a secondary amino group derived from the amide bond between the second and third amino acid residue, can (i) decompose with the formation of diketopiperazine and a shortened peptide sequence; (ii) form a bicyclic structure by transanular attack on the first amino acid carbonyl group in the DKP-like ring by the newly formed amino group, leading to the rearranged product; and (iii) form a bicyclic structure by transanular attack of the newly formed hydroxyl group on the carbonyl group in the DKP-like ring, leading to the race-mixed product.

Hydrolysis of Bz-Gly-Ser-Phe-Arg, C-terminal fragment of atriopeptin 2, by human cardiac angiotensin-converting enzyme has been studied. The KM for the reaction was 10(-4) M. The effect of concentration of NaCl on activity of cardiac angiotensin-converting enzyme has been determined, which allowed to regard Bz-Gly-Ser-Phe-Arg as bradykinin-like substrates. It was demonstrated that cardiac, but not pulmonary isozyme of angiotensin-converting enzyme specifically hydrolyses atriopeptin 2.

It was shown that opioid peptides stimulate nervous tissue growth in culture in the rat, which manifests itself in augmented outgrowth of neurites from explants and in an increase in the number of glial and fibroblast-like cells in the growth zone. The effects of opioid peptides ([Leu]- and [Met]-enkephalins, beta- and gamma-endorphins and some synthetic analogues of [Leu]-enkephalin) on the growth of organotypic cultures of rat sympathetic and dorsal root ganglia and spinal cord were investigated. Neurite outgrowth, cell composition, and size of the growth zone as well as the dynamics of its formation were estimated. Changes in the survival of neurons in dorsal root ganglion cultures were determined. The experiments were performed with living cultures as well as with fixed preparations. In experiments with sympathetic ganglia, it was demonstrated that a significant growth-promoting effect is exerted by peptides taken at concentrations of 10(-8) M to 10(-14) M. Naloxone does not eliminate the effects of peptides, but stimulates the growth at 10(-5) M to 10(-7) M. Studies with spinal cord revealed that naloxone (10(-6) M) enhances the response to [Leu]-enkephalin (10(-9) M). The survival of dorsal root ganglion neurons under the influence of a [leu]-enkephalin analog (10(-9) M) exceeds control values by approximately two to four times. Thus, opioid peptides were shown to exert a strong growth-promoting effect on nervous tissue in culture. This effect is dual: in neurons the peptides stimulate the outgrowth of neurites and their survival, while in glial cells they change the rate of their migration and, probably, their proliferation. It is suggested that opioid peptides, besides their already established functions, may play a role in the development and regeneration of nervous tissue.