Іванов Вадим Тихонович

The development of the peptide-based vectors for the intracellular delivery of biologically active macromolecules has opened new prospects of their application in research and therapy. Earlier the amphipathic cell-penetrating peptide (CPP) Pep-1 was reported to mediate cellular uptake of proteins without covalent binding to them. In this work we studied the ability of a series of membrane-active amphipathic peptides, based on the gramicidin A sequence, to transport a model protein across the eukaryotic cell membrane. Among them the positively charged Cys-containing peptide P10C demonstrated the most effective beta-galactosidase intracellular delivery. Besides, this peptide was shown to form noncovalent associates with beta-galactosidase as judged from electrophoresis and enzymatic activity assays. In addition, a series of new gramicidin analogues were prepared and the effect of N-terminus modification of gramicidin on the protein transduction efficiency was studied.

Using reverse-phase (MB-HIC 8 and HB-HIC 18) weak cation exchange (MB-WCX) and metal affinity ClinProt magnetoc beads peptides and protein factions were obtained from human sera for their profiling by MALDI-TOF mass spectrometry. Proteome profiling of sera from I-IV stage ovarian cancer patients (47 women, average age 51) and from healthy women (47 subjects, average age 49) using MB-WCX beads allowed calculation of the best diagnostic models based on the Genetic Algorithm and Supervised Neural Network classifiers; these model generated 100% sensitivity and specificity when the test set of subjects was analyzed. Introduction of additional sera from patients with colorectal cancer (19) and ulcerous colitis (5) to the statistical model confirmed 100% ovarian cancer recognition. Statistical mass-spectrometry analysis of mass-spectrometry peak areas included to the diagnostic classifiers showed 3 peaks distinctive for ovarian cancer and 4 peaks distinctive for ovarian and colorectal cancer.

Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA ). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2 + L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2 + chelator 1,2-bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2 + / calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.

Синтетичний пептид, відповідний амінокислотноїпослідовності 37-55 неструктурного білка NS1 вірусу кліщового енцефаліту здатний захищати 60% тварин при зараженні високопатогенним штамом вірусу. Протективна роль протівопептідних антитіл показана в експериментах по адоптівная переносу.

Rapid progress of separation techniques as well as methods of structural analysis provided conditions in the past decade for total screening of complex biologic mixtures for any given class of biomolecules. The present review updates the reader with the modern state of peptidomics, a chapter of chemical biology that deals with structure and biologic properties of sets of peptides present in biologic tissues, cells or fluids. Scope and limitations of currently employed experimental techniques are considered and the main results are outlined. Considerable attention will be afforded to the biologic role of peptides formed in vivo by proteolysis of nonspecialized precursor proteins with other well-defined functions. In conclusion, the connection is discussed between peptidomics and the much more mature and still closely related field of proteomics.

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease (s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides can not play the role of precursors of hemoglobin fragments present in tissue extracts.

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative beta-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 microM valorphin and 1 microM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 microM (but not 1 microM) epirubicin added 24 h prior to 1 microM valorphin; 1 microM valorphin added 48 h prior to 0.1 microM epirubicin, or 0.1 microM vincristine, or 0.05 microM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg / m (2) epirubicin and 1 mg / kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu -Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Нова пептидная конструкція Palm135-158-GGA-170-188 (Acm) містить специфічний T-хелперний епітоп последовательносчті 170-188 білка VP1і основний антигенний район 135-158 білка VP1 вірусу ящура. Конструкція проявляє вищу протективногороль, антигенну, імуногенну активність, а також здатність до Т-клітинної проліферації, ніж раніше описаний пептид Palm (2) 135-159.

Сінетезірован пептид Palm2 135-159, який представляє собою діпальмітоільное похідне фрагмента 135-159 білка VP1 вірусу ящура штаму A22. Показано, що пептид Palm2 135-159 проявляє противірусну активність на мишах, морських свинках і вівцях. Одноразове введення синтетичної пептидного вакцини забезпечує захист овець від захворювання на ящур протягом 1 год. Пептидная вакцина дозволена до застосування на території Росії.

he C '= O and Cα signals in the 13C nuclear magnetic resonance (NMR) spectra of valinomycin have been assigned and the vicinal 1H ... 13C coupling constants have been determined by double and triple heteronuclear resonance. In conjunction with the vicinal H-NCα-H and H-CαCβ-H proton-proton constants, the results led to unequivocal determination of the torsion angles φ and of the population distribution of the CαCβ rotational states. The Φ torsion angles for the hydroxy acid residues were estimated from the vicinal 1H-CαC'-15N constants. The combined data permitted refinement of the conformational states of valinomycin in different solvents. For the KS + complex of valinomycin the observed couplings are in complete accord with the conformations we had earlier proposed for solutions and that had also been established by X-ray analysis. In the 13C spectra of the valinomycin-Tl + complex 13C ... 203,205Tl + spin-spin couplings were observed for the l and d -valine carbonyls, unequivocal proof of the donor-acceptor interaction with the cation. In media of weak polarity (cyclohexane, chloroform) the conformation of the valinomycin molecule is similar to that of the K + complex. In such a 'bracelet' structure formed by six fused β-turns of type II and II ', the amino acid carbonyls are axial with certain inclination towards the symmetry axis. On formation of a 1: 1 complex with a cation the carbonyl orientation changes, now bending towards the center of the molecular cavity. In the 'propeller' conformation, predominant in solvents of medium polarity (for instance CCl4 / (C2H3) 2SO, 3/1) the three β-turns are of type II. The 1H and 13C chemical shifts are interpreted in terms of conformational changes in the valinomycin molecule, intermolecular and intramolecular hydrogen bonds and interaction with the metal cation.

The ability of the enniatin cyclodepsipeptides (CDP) (fig. 1) to form complexes with alkali metal ions (M +) and induce ionic permeability in artificial and biological membranes has been described in a number of papers [1,2]. The complexes were found to be equimolar in both solutions and in the crystalline state; by analogy with valinomycin and the nactins the role of the M + carriers across the membrane was ascribed to them [3,49. In the present paper evidence is produced showing that an important part in the functioning of this group of ionophores is played by complexes with 2: 1 and 3: 2 macrocycle: cation ratios.

Синтезовані аналоги природних пептидів для топохимической досліджень, які показали, якою мірою біологічно активна пептидная система повинна бути стереоелектронно комплементарна відповідного рецептора. Для депсіпептідних антибіотиків і їх топохимической аналогів показана придатність такого підходу з метою створення фізико-хімічних основ вивчення біологічних мембран. Топохимічеськие принципи можуть бути застосовані також до дослідження специфічних конкурентних інгібіторів протеолітичних ферментів.

Вивчено хімія валиномицина, енніатінов і родинних мембраноактівних депсіпептідних антибіотиків, які здійснюють проникнення іонів лужних металів через біологічні мембрани, кореляція їх антимікробної активності і катіон-зв'язуючої здатності і конформаційні властивості депсіпептідов, що визначають зв'язування їх з катіонами і мембранну активність. Передбачається, що принципи конформационно-залежних іон-дипольних взаємодій можуть становити основу функціонування систем, осущетсвляется іонну провідність біологічних мембран.