Department of Peptide and Protein Technologies

All publications (show selected)

Vadim Ivanov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 51, office. 454
  • Phone: +7(495)330-56-92
  • E-mail: ivavt@ibch.ru

Researchers from the Laboratory of biocatalysis, Laboratory of proteolytic enzyme chemistry, Laboratory of bioinformatics approaches in combinatorial chemistry and biology, Laboratory of hormonal regulation proteins IBCh RAS together with their Russian and foreign colleagues showed that amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Publications

  1. Terekhov SS, Eliseev IE, Ovchinnikova LA, Kabilov MR, Prjibelski AD, Tupikin AE, Smirnov IV, Belogurov AA, Severinov KV, Lomakin YA, Altman S, Gabibov AG (2020). Liquid drop of DNA libraries reveals total genome information. Proc Natl Acad Sci U S A 117 (44), 27300–27306

Protective allele for multiple sclerosis HLA-DRB1*0101 provides kinetic discrimination of myelin and exogenous antigenic peptides

Laboratory of biocatalysis

Risk of the development of multiple sclerosis (MS) is known to be increased in individuals bearing distinct class II human leukocyte antigen (HLA) variants, whereas some of them may have a protective effect. Here we analyzed distribution of a highly polymorphous HLA-DRB1 locus in more than one thousand relapsing-remitting MS patients and healthy individuals of Russian ethnicity. Carriage of HLA-DRB1*15 and HLA-DRB1*03 alleles was associated with MS risk, whereas carriage of HLA-DRB1*01 and HLA-DRB1*11 was found to be protective. Analysis of genotypes revealed the compensatory effect of risk and resistance alleles in trans. We have identified previously unknown MBP153-161 peptide located at the C-terminus of MBP protein and MBP90-98 peptide that bound to recombinant HLA-DRB1*01:01 protein with affinity comparable to that of classical antigenic peptide 306–318 from the hemagglutinin (HA) of the influenza virus demonstrating the ability of HLA-DRB1*01:01 to present newly identified MBP153-161 and MBP90-98 peptides. Measurements of kinetic parameters of MBP and HA peptides binding to HLA-DRB1*01:01 catalyzed by HLA-DM revealed a significantly lower rate of CLIP exchange for MBP153-161 and MBP90-98 peptides as opposed to HA peptide. Analysis of the binding of chimeric MBP-HA peptides demonstrated that the observed difference between MBP153-161, MBP90-98 and HA peptide epitopes is caused by the lack of anchor residues in the C-terminal part of the MBP peptides resulting in a moderate occupation of P6/7 and P9 pockets of HLA-DRB1*01:01 by MBP153-161 and MBP90-98 peptides in contrast to HA308-316 peptide. This leads to the P1 and P4 docking failure and rapid peptide dissociation and release of empty HLA-DM–HLA-DR complex. We would like to propose that protective properties of the HLA-DRB1*01 allele could be directly linked to the ability of HLA-DRB1*01:01 to kinetically discriminate between antigenic exogenous peptides and endogenous MBP derived peptides.

A personalised approach for treating T-cell malignancies

Laboratory of biocatalysis

Efficient and specific removal of malignant cells is the ultimate goal of cancer therapy. The current rapid development of chimeric antigen receptor T cell (CAR-T cell or CART) therapy potentially provides high efficiency and allows long-term surveillance, which have greatly extended the frontier of leukemia treatment. We validated the idea of using CARTs as targeting agents and observed excellent continuous efficacy as well as specificity. Combining CDR3 targeting with the CART approach provides a solution for a substantial portion of patients with T cell leukemia and lymphoma, with supposedly minimized side effects. After validation of this strategy to eliminate pathological T cells ex vivo and in vivo, we envisage this approach as a generally useful alternative and supplement to the popular approach of common antigen targeting to treat T cell malignancies, especially considering its safety.

Publications

  1. Huang J, Stepanov A, Li J, Jones T, Grande G, Douthit L, Xie J, Chen D, Wu X, Michael M, Xiao C, Zhao J, Xie X, Xie J, Chen X, Fu G, Gabibov A, Tzeng CM (2019). Unique CDR3 epitope targeting by CAR-T cells is a viable approach for treating T-cell malignancies. Leukemia 33 (9), 2315–2319

Discovery of novel class of ubiquitin-independent proteasomal degrons

Laboratory of hormonal regulation proteins

Majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin, representing universal degradation signal operated by ubiquitination system. Ubiquitin-independent proteasome targeting is rationalized by existence of two types of direct proteasome signals (DPS) – specific amino acid sequence or posttranslational modification that are recognized by proteasome regulatory subunits. Historically, the first one was shown to be existed in ornithine decarboxylase, whereas acetylation of core histones recently was reported as second type of DPS. Here we declare the third one, representing charge-mediated DPS. Discovered DPS may be classified as monopartite composition- but not sequence-dependent element of approximately 70 Å in length enriched in basic and flexible amino acids. This type of degradation signal that may be either provided by cationic chemicals is most efficiently engaged by REGa or REGg-capped proteasomes in ATP-independent manner. Taken together, our findings suggest novel modality of proteasome-substrate interrelation bypassing ubiquitination.

Publications

  1. Kudriaeva A, Kuzina ES, Zubenko O, Smirnov IV, Belogurov A (2019). Charge-mediated proteasome targeting. FASEB J 33 (6), fj201802237R

Preparation of active pharmaceutical ingridient based on Nα-acetyl C-amide terminal form of HLDF-6 peptide using optimized synthetic method

Laboratory of hormonal regulation proteins,  Group of Peptide Chemistry

Standard solid phase methods could not be applied for the preparation of the API samples due to the fact that current SPPS methodologies are still imperfect. Therfore, fragment condensation in solution method was developed, optimized and used for the preparation of the amide form of HLDF-6 peptide. That material could be purified and acetylated under the conditions where peptide salts of weak acids are acylated with variuos weak electrophilic derivatives of acetic acid including active esters and azolides. Appliaction of the acetylation reaction conditions described provides API samples in high yield and purity

Publications

  1. Zolotarev YuA, Dadayan AK, Kozik VS, Shram SI, Nagaev IYu, Azev VN, Bogachouk AP, Lipkin VM, Myasoedov NF (2019). Proteolytic Hydrolysis of the Antitumor Peptide HLDF-6-AA in Blood Plasma. Russ. J. Bioorganic Chem. 45 (5), 347–352

Development of a monocomponent polyvalent vaccine for the prevention of diseases caused by bacteria producing IgA1 protease

Laboratory of proteolytic enzyme chemistry

For the first time the ability of recombinant IgA1 protease N. meningitidis serogroup B and chimeric recombinant proteins created by us, consisting of two (I) or three  (II) interconnected fragments from different sites of the primary structure of the enzyme, to protect the animals immunized with fatal doses of meningococcal and some pneumococcal infections was shown. The decrease in the level of bacteremia (number of CFU, %) depended on the compound structure and the strain of the pathogen (Fig. 1).

For the first time in the sera of rabbits immunized with inactivated S. pneumonia microbial cells of different serotypes, antibodies to the N. meningitidis IgA1 protease and to the proteins I and II were detected (Fig.2). On the model of passive protection of animals, it was shown that the sera of these rabbits are able to protect animals from infection with N. meningitidis serogroup B, that indicates the possibility of cross immunity in pneumococcal and meningococcal infections (Fig.3).

The data obtained allow to conclude that a monocomponent polyvalent vaccine can be created based on the proteins of proteins I and II for the prevention of diseases caused by bacteria whose common virulence factor is the IgA1 protease: N. meningitidis, N. gonorrheoeae, H. influenzae, S. pneumoniae, S. sanguis, S. oralis, etc.

In the practice of world health, analogs of such a vaccine does not exist.

Profile of Gene Expression in the Kidneys of Mice with the insrr Gene Knockout

Laboratory of Receptor Cell Biology,  Group of Molecular Physiology

The insulin receptor-related receptor (IRR) is a receptor tyrosine kinase and a close homologue of the insulin receptor and the insulin-like growth factor receptor; however, peptide and protein ligands for this receptor have not been identified. IRR is encoded by the insrr gene and is found in some cell populations of kidneys, stomach, pancreas, as well as in some sympathetic and cholinergic neurons. In the present study, a comparative analysis of the transcriptomes of kidneys from wild-type mice and IRR-knockout mice has been performed by the method of deep sequencing and by the analysis of gene expression on microarrays and in real-time PCR. A significant change (a more than 1.5 fold increase in gene expression in knockout animals) was found for the transcripts of two genes: hsd3b2 and igf2. The results suggest that the line of IRR-knockout mice can find application as an animal model in studies of the role of these genes in the kidney.

Publications

  1. Deyev IE, Shayahmetova DM, Zhenilo SV, Radionov NV, Petrenko AG (2018). Profile of Gene Expression in the Kidneys of Mice with the insrr Gene Knockout. Russ. J. Bioorganic Chem. 44 (2), 256–260

Oral microbiome of Siberian bear as a source of new antibiotics c

Laboratory of biocatalysis

A new pipeline for antibiotic discovery using microfluidic screening technologies has been proposed. The biosynthetic pathways of the new antibiotic amicoumacin has been discovered. Specific enzymes: kinase and phosphatase, responsible for the inactivation and activation of the antibiotic were found

Publications

  1. Terekhov SS, Smirnov IV, Malakhova MV, Samoilov AE, Manolo AI, Nazarov AS, Danilov DV, Dubiley SA, Osterman IA, Rubtsova MP, Kostryukova ES, Ziganshin RH, Kornienko MA, Vanyushkina AA, Bukato ON, Ilina EN, Vlasov VV, Severinov KV, Gabibov AG, Altmani S (2018). Ultrahigh-throughput functional profiling of microbiota communities. Proc Natl Acad Sci U S A 115 (38), 9551–9556

Personalized therapy of B-cell lymphomas

Laboratory of biocatalysis

We report the development of a novel platform to significantly enhance the efficacy and safety of Follicular lymphoma treatment. Since lymphoma is a clonal malignancy of a diversity system every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a CAR-T format for redirection of human CTLs. Science Advances 2018. Stepanov AV,...,Gabibov AG, Lerner RA

Publications

  1. Stepanov AV, Markov OV, Chernikov IV, Gladkikh DV, Zhang H, Jones T, Senkova AV, Chernolovskaya EL, Zenkova MA, Kalinin RS, Rubtsova MP, Meleshko AN, Genkin DD, Belogurov AA, Xie J, Gabibov AG, Lerner RA (2018). Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma. Sci Adv 4 (11), eaau4580

Effect of hyperosmotic stress on post-translational modifications of the Kaiso transcription factor

Group of Molecular Physiology

This joint study with the laboratory of Professor Egor Prokhortchouk shed light on the role of post-translational modifications of the methyl-sensitive Kaiso transcription factor. We found that Kaiso exists mainly as a monoSUMOylated protein, that is, it is linked to one SUMO molecule. However, if cells expressing this protein are hyperosmotic stressed, then we suddenly find Kaiso quickly losing this modification (deSUMOylation). We have shown that the loss of the SUMO modification is reversible, since the removal of the osmotic pressure returns Kaiso almost completely the SUMO modified form. Then, using point mutagenesis, we were able to map the site of modification in this protein, and this turned out to be lysine in 42 positions.

Publications

  1. Zhenilo S, Deyev I, Litvinova E, Zhigalova N, Kaplun D, Sokolov A, Mazur A, Prokhortchouk E (2018). DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress. Cell Death Differ 25 (11), 1938–1951

Orphan Receptor Tyrosine Kinase of the Insulin Receptor Family Takes Shape: Structural study by Small-Angle X-Ray Scattering and AFM

Group of Molecular Physiology,  Laboratory of Receptor Cell Biology

Activation of the IRR can be achieved by increasing the extracellular pH value. The activation by alkaline media is specific, dose-dependent and reversible, which resembles typical features of ligand-receptor interaction. It was also revealed that the pH sensitivity of IRR is defined by its extracellular region, that is similar to other members of the IR minifamily. Since the activation of the IRR is determined by its extracellular part (ectodomain), the isolation and study of the structure of the ectodomain IRR (ectoIRR) is of particular interest for understanding the fundamental basis of the mechanism of alkaline sensitivity. This work is devoted to the determination of possible conformational rearrangements in the ectodomain of IRR induced by pH changing using small-angle X-ray scattering (SAXS). SAXS is a particularly useful tool for investigation of the non-crystallizable proteins, the structural organization of multidomain proteins and it allows for the rigid body modeling of the quaternary structure from subunits for which crystal structures or other models of the constituent domains are available.

From the data obtained from SAXS experiments it can be concluded that the protein (soluble IRR ectodomain) in solution exists as a dimer having a molecular mass close to that calculated from an amino acid sequence with the contribution of glycosylation, but we can’t find significant differences in X-ray scattering between conditions of pH 7.0 and pH 9.0.

To obtain detailed structural organization of the ectoIRR in solution, hybrid modeling was performed using CORAL software. Available high-resolution X-ray crystal structure of insulin receptor ectodomain as the closest IRR homologue was split into individual subdomains. In total, two polypeptide chains (ectoIRR dimer) were modeled in CORAL, followed by the search for optimal positions and orientation of the rigid subdomains and feasible conformation of the linkers to achieve the best fit to the experimental data. The modeling yields good fit with χ2 = 1.3 confirming that the chosen modeling scheme with two domains per chain was adequate for the data fitting.

The data obtained by atomic force microscopy also demonstrates good agreement with the results of structural analysis by SAXS. AFM experiments of the IRR ectodomain adsorbed on the surface of atomically flat mica revealed the structure similar to the one observed by SAXS, with no significant differences between conditions of pH 7.0 and pH 9.0.

Mechanism of the nonopioid β-endorphin action on the activity of the hypothalamic–pituitary–adrenal axis

Laboratory of hormonal regulation proteins,  Laboratory of toxicology in vitro

The binding of β-endorphin to a nonopioid receptor on the anterior pituitary cells has been found to increase the expression of inducible NO-synthase, which leads to a decrease in the secretion of ACTH and corticosterone. Thus, it has been shown that the nonopioid β-endorphin receptor and its agonists are involved in the regulation of the activity of the hypothalamic–pituitary–adrenal axis at the level of the pituitary and adrenal glands.

Neuroprotective activity of HLDF-6 peptide was shown on double- transgene model of Alzheimer’s disease

Laboratory of hormonal regulation proteins

Neuroprotective and nootropic activities of amide form (AF) of HLDF-6 peptide (TGENHR-NH2) were studied on transgene mice of B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line – animal model of inherited Alzheimer’s disease. Animals of this line express the mutant human presenilin and chimeric mouse/human amyloid protein. A typical feature of this line is early development of an Alzheimer-like pathology caused by accelerated βA deposition and cognitive impairment in the brain, which is evaluated using the spatial learning tests. The cognitive abilities of B6C3-Tg-  have not been fully characterized.

Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line

Group of Molecular Physiology,  Laboratory of Receptor Cell Biology

Secretion of mildly alkaline (pH 8.0-8.5) juice to intestines is one of the key functions of the pancreas. Recent reports indicate that the pancreatic duct system containing the alkaline juice may adjoin the endocrine cells of pancreatic islets. We have previously identified the insulin receptor-related receptor (IRR) that is expressed in islets as a sensor of mildly alkaline extracellular media.  In this study, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. We further analyzed the effects of alkaline media on pancreatic beta cell line MIN6. Activation of endogenous IRR but not of the insulin receptor was detected that could be inhibited with linsitinib. The IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of insulin receptor substrate 1 (IRS-1), the primary adaptor in the insulin signaling pathway. However, in contrast with insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of alkali treatment. We observed overexpression of several early response genes (EGR2, IER2, FOSB, EGR1 and NPAS4) upon alkali treatment of MIN6 cells but those were IRR-independent. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali might be part of a local loop of signaling between the exocrine and endocrine parts of the pancreas where alkalinization of the juice facilitate  insulin release which, in turn, might induce further duct secretion.

Site-directed mutagenesis of the fibronectin domains in insulin receptor-related receptor

Laboratory of Receptor Cell Biology,  Group of Molecular Physiology

We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains and show positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR, we now address the role of the FnIII repeats in the IRR pH-sensing. We found that the first activation site includes the intrinsically disordered subdomain ID (646-716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

Fluorogenic marker for instant live-cell membrane staining and imaging

Group of growth factors expression and differentiation,  Group of Molecular Physiology,  Laboratory of molecular theranostics

A new organic-compound-based fluorogenic marker has been created for live-cell membrane staining. Unlike current commercial cell markers, the obtained fluorogenic marker does not fluoresce in the aquatic environment, but acquires fluorescence immediately after being placed in a nonpolar medium, for example, in the cell membrane. This property allows one to instantly stain cells without further washing out the unbound dye. This marker can be applied in fluorescence microscopy for live-cell imaging and flow cytometry.

Publications

  1. Pakhomov AA, Deyev IE, Ratnikova NM, Chumakov SP, Mironiuk VB, Kononevich YN, Muzafarov AM, Martynov VI (2017). BODIPY-based dye for no-wash live-cell staining and imaging. Biotechniques 63 (2), 77–79

Molecular mechanism of the non-opioid beta-endorphin action

Laboratory of hormonal regulation proteins,  Laboratory of pharmacokinetics,  Laboratory of toxicology in vitro

Using a synthetic peptide octarphin (TPLVTLFK), a selective agonist of non-opioid beta-endorphin receptor, data on the molecular mechanism of the non-opioid action of the hormone have been obtained. It was found that the non-opioid effect of beta- endorphin on the target cell is realized by the following way: increase in the inducible NO synthase expression → increase in NO production → increase in the activity of soluble guanylate cyclase → increase in the intracellular level of cGMP.

Multiple sclerosis is a chronic autoimmune disease with neurological pathology. The dominant role of immunological processes in the development of the disease dictates the need for medications that specifically minimize the activity of immune processes. The peptides A8AMS and mA8AMS, homologous to the fragment of the human IgG VH domain, have been shown to act in vitro and in vivo, effectively reducing the symptoms of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. The results provide new opportunities for the development of peptide drugs for the treatment of multiple sclerosis.

Development of ultrahigh-throughput method for screening of catalytic activity. Validation.

Laboratory of biocatalysis,  Group of combinatorial methods for constructing biocatalysts

The research is aimed at the search and creation of catalytic biological antidotes. To find new catalytic antidotes based on butyrylcholinesterase, a panel of libraries of the active center of BuChE was created. The library of 284-288 loop was successfully used to validate the method of ultrahigh-throughput screening of biocatalytic activity in droplets of a microfluidic emulsion. The obtained results, together with the developed quantum-mechanical algorithm for calculating the dephosphorylation step of covalent BChE-paraoxon complex, made it possible to create an optimized library for the BChE RKH-4 yeast display, representing 1 * 10 ^ 6 variants.

Publications

  1. Terekhov SS, Smirnov IV, Stepanova AV, Bobik TV, Mokrushina YA, Ponomarenko NA, Belogurov AA, Rubtsova MP, Kartseva OV, Gomzikova MO, Moskovtsev AA, Bukatin AS, Dubina MV, Kostryukova ES, Babenko VV, Vakhitova MT, Manolov AI, Malakhova MV, Kornienko MA, Tyakht AV, Vanyushkina AA, Ilina EN, Masson P, Gabibov AG, Altman S (2017). Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity. Proc Natl Acad Sci U S A 114 (10), 2550–2555

Generation of enantioselective bioscavengers based on antibodies.

Laboratory of biocatalysis,  Group of combinatorial methods for constructing biocatalysts

In the framework of the study of catalytic antidotes based on antibodies, an algorithm for predicting mutants capable of selectively interacting with (R) and (S) isomers of PCT was proposed.

Publications

  1. Golovin AV, Smirnov IV, Stepanova AV, Zalevskiy AO, Zlobin AS, Ponomarenko NA, Belogurov AA, Knorre VD, Hurs EN, Chatziefthimiou SD, Wilmanns M, Blackburn GM, Khomutov RM, Gabibov AG (2017). Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer. Dokl Biochem Biophys 475 (1), 245–249