Laboratory of hormonal regulation proteins

Department of Peptide and Protein Technologies

Head: Valery Lipkin, corresponding member of the academy of sciences

Proteins, peptides, differentiation, apoptosis, Alzheimer’s disease, ischemia, myopia

The Laboratory researches the proteins including in transmembrane signaling processes, differentiation and cell apoptosis, and also new proteins and peptides associated with the pathogenesis of social diseases. As a result researchers develop new approaches for the treatment and diagnosis of these diseases. In addition, the Laboratory develops methods of genotyping used in agriculture.

The Laboratory have discovered and studied a new factor of differentiation of blood cells (HLDF) which suppress the proliferation of promyelocytic leukemia cell lines. In six-membered HLDF-peptide 6 (TGENHR) factor was identified composition having nootropic and neuroprotective activity. During successful preclinical testing in animal models of clinical disease (Alzheimer's disease and ischemic stroke) Anne Bogachuk’s Group showed that protected by amidation at the C-terminal carboxyl group of the peptide form (HLDF-6-NH2) considerably exceeds the activity of the peptide original shape almost completely restores impaired cognitive function and does not cause some toxic effects and can be recommended for further clinical studies.

The attention of researchers was drawn by another neurotrophic factor – PEDF (Pigment Epithelium Derived Factor), which accumulates with myopia and promotes the formation of amyloid fibrillar structures. This knowledge opens new approaches to the treatment of eye diseases widespread.

Researchers conduct studies laboratory EIF1AD protein (gaponina) which was opened in the Laboratory and regulates the processes in the body related to oxidative stress (OS). EIF1AD localized in the nucleus and controls the transcriptional activity of p53 protein, caused by reactive oxygen species. The suppression of EIF1AD expression via shRNK inhibits apoptosis induced by the OS and promotes cell survival, such as ultraviolet, gamma irradiation or during inflammation.

Tatiana Shuvaeva’s Group develops methods for genotyping of cattle for identification of genetic diseases and the further improvement of breed herds.

Laboratory cooperates with laboratories of the Institute, as well as with The Anokhin Institute of Normal Physiology RAS, The Zakusov Institute of Pharmacology, The Institute of Cell Biophysics RAS, The Institute of Molecular Genetics RAS, The Helmholtz Moscow Research Institute of Eye Diseases of the Russian Ministry of Health, People’s Friendship University of Russia, The Kuban State Agrarian University, Department of Medical Biochemistry and Biophysics of Umeå University (Sweden) etc.

The Laboratory was formed in 1986 on the basis of the group existing from 1975 in the Department of protein chemistry, heading by academician Yu.A. Ovchinnikov.

  1. Physico-chemical biology;
  2. Investigations of structure and functioning mechanism of proteins and peptides, participating in transmembrane signal transduction, cells differentiation and apoptosis;
  3. Elaboration of new preparations for cancer and neurodegenerative diseases therapy, regeneration of human damaged tissues and organs.

The Laboratory was formed in 1986 on the basis of the group existing from 1975 in the Department of protein chemistry, heading by academician Yu.A. Ovchinnikov.

1986—1997. Primary structure of all subunits of bovine and human cGMP phosphodiesterase was estimated. Using immunochemical methods and site-directed mutagenesis, in photoreceptor cGMP phosphodiesterase, functional amino acid residues were localized and mechanisms of photoactivation and inhibition of the enzyme were elucidated. In 1997, the work was awarded by Yu.A. Ovchinnikov prize.

1996—2008. Two new secretory proteins with molecular weights 28 and 45 kDa were isolated from the rat’s olfactory epithelium. Their complete amino acid sequences were determined. 28 kDa protein has been identified as peroxyredoxin VI. It was shown to act as antioxidant and might be used as a novel remedy, promoting regeneration of damaged human tissues and organs (in cooperation with the Institute of the Cell Biophysics RAS). In the structure of 45 kDa protein, a lipid-binding domain was identified. The protein was ascertained to belong to lipid-transferring class, and phosphatydilinositol-(3,4,5)-triphosphate is its specific ligand.

2006—2008. In the HL-60 cell line of human promyelocytic leukemia, a new protein was discovered with molecular mass 19,2 kDa, named haponin (HLDF-alike protein), immunoreactive with polyclonal antibodies to one of HLDF analogs (HLDFβ). Full-sized cDNA of haponin was cloned, and its structure was determined. We suppose participation short form of haponin (molecular mass. kDa) in normal apoptosis of lymphoid tissue.

2006—2014. Else one line of investigation in the Laboratory is connected with study of functional role of neurotrophic factor from pigment epithelium PEDF (Pigment Epithelium Derived Factor) on myopia progression. We have recently shown, that normal PEDF factor, secreted by sclera phibroblasts, undergoes partial proteolysis by 382Leu-383Тhr bond that results in removal of C-terminal fragment. At myopia this process is disturbed that gives rise to accumulation in pericellular space of full-size PEDF, able to form amyloid—like fibrillary structures, decreasing biomechanical stability of sclera tissues. We suppose that using of the substances preventing fibrils formation would permit to stop myopia progression.

20122015. Preclinical studies of HLDF-peptide 6 were conducted to developing a medicament for the prevention and treatment of neurodegenerative (Alzheimer's disease) or cerebrovascular (ischemic stroke) diseases. The most active form of HLDF-6 peptide having neuroprotective and nootropic activity was identified. In the comparative study of the activity of the parent peptide and the peptide protected from protease action with the C-terminus amide group was found that the peptide protected form in all investigated animal models of ischemic stroke and Alzheimer's disease almost completely restores impaired cognitive function, efficiency exceeding the parent peptide. The study of acute, sub-chronic and chronic toxicity, allergenicity, mutagenicity and mechanism of the peptide amide form action was conducted.

The conclusion was made on the basis of the study that the drug, based on the peptide HLDF-6 amide form with repeated intranasal administration to male and female rats does not cause toxic effects and is safe. The drug based on the peptide HLDF-6 amide form at a dose of 5000 mg / kg does not cause toxic effects, it complies with the requirements of the low hazard class 4 "low-hazard substances" and could be recommended for further research. The advantages of our product over other existing drugs determined by the aggregate of such medicinal properties as originality, expression of therapeutic effect, a high level and wide range of combined nootropic and neuroprotective activity (effective influence on both diseases - asthma and ischemic stroke), high efficiency in small and ultralow doses, no undesirable side effects.

Valery Lipkin, corresponding member of the academy of sciencesdepart.
Tat'jana Shuvaeva, D.Scl. r.
Anna Bogachuk, Ph.D.s. r.
Tat'jana Rakitina, Ph.D.s. r.
Dmitrij Kakuevr.
Vitaly Radchenko, Ph.D.r.
Elena Surinar.
Evgenia Smirnova, Ph.D.r.
Alexey Garkovenkoj. r.
Natal'ja Minkevich, Ph.D.j. r.
Elena Il'nitskaya, Ph.D.j. r.
Elena Vorob'evaPhD
Anastasia Frolovat. q. - lab. as.
Irina Smirnovares.

Former members:

Igor' Artamonov, Ph.D.s. r. f.
Irina Kostanjan, Ph.D.s. r. f.

Selected publications

  1. Bogachouk A.P., Storozheva Z.I., Telegin G.B., Chernov A.S., Proshin A.T., Sherstnev V.V., Zolotarev Y.A., Lipkin V.M. (2017). Studying the Specific Activity of the Amide Form of HLDF-6 Peptide using the Transgenic Model of Alzheimer's Disease. Acta Naturae 9 (3), 64–70 [+]

    The neuroprotective and nootropic activities of the amide form (AF) of the HLDF-6 peptide (TGENHR-NH2) were studied in transgenic mice of the B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line (the animal model of familial Alzheimer's disease (AD)). The study was performed in 4 mouse groups: group 1 (study group): Tg+ mice intranasally injected with the peptide at a dose of 250 μg/kg; group 2 (active control): Tg+ mice intranasally injected with normal saline; group 3 (control 1): Tg- mice; and group 4 (control 2): C57Bl/6 mice. The cognitive functions were evaluated using three tests: the novel object recognition test, the conditioned passive avoidance task, and the Morris water maze. The results testify to the fact that the pharmaceutical substance (PhS) based on the AF of HLDF-6 peptide at a dose of 250 μg/kg administered intranasally efficiently restores the disturbed cognitive functions in transgenic mice. These results are fully consistent with the data obtained in animal models of Alzheimer's disease induced by the injection of the beta-amyloid (βA) fragment 25-35 into the giant-cell nucleus basalis of Meynert or by co-injection of the βA fragment 25-35 and ibotenic acid into the hippocampus, and the model of ischemia stroke (chronic bilateral occlusion of carotids, 2VO). According to the overall results, PhS based on AF HLDF-6 was chosen as an object for further investigation; the dose of 250 μg/kg was used as an effective therapeutic dose. Intranasal administration was the route for delivery.

  2. Ilnitskaya E.V., Radchenko V.V., Rodionova A.S., Kosyreva A.M., Shuvaeva T.M., Lipkin V.M. (2013). Cloning and sequence analysis of cDNA encoding a new short form of rat acid chitinase. Dokl. Biochem. Biophys. 452 (1), 241–4 ID:1052
  3. Minkevich N.I., Rakitina T.V., Bogachuk A.P., Radchenko V.V., Surina E.A., MorozovaRoche L.A., Yanamandra K., Iomdina E.N., Babichenko I.I., Kostanian I.A., Lipkin V.M. (2012). [Amyloid-like fibrils forming and fibroblasts destruction in Tenon's capsule in progressive myopia as a result of pigment epithelium derived factor resistance to restricted proteolysis]. Bioorg. Khim. 38 (6), 683–90 [+]

    We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

  4. Smirnova E.V., Rakitina T.V., Bogatova O.V., Ivanova D.L., Vorobyeva E.E., Lipkin A.V., Kostanyan I.A., Lipkin V.M. (2011). Novel protein haponin regulates cellular response to oxidative stress. Dokl. Biochem. Biophys. 440, 225–7 ID:1054
  5. Kostanyan I.A., Storozheva Z.I., Semenova N.A., Lipkin V.M. (2009). Postischemic administration of HLDF-6 peptide ameliorates cognitive dysfunction and brain damage induced by chronic cerebral ischemia in rats. Dokl. Biol. Sci. 428, 418–22 ID:1056
  6. Merkulova M., Huynh H., Radchenko V., Saito K., Lipkin V., Shuvaeva T., Mustelin T. (2005). Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein. FEBS J. 272 (21), 5595–605 [+]

    Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P3 and far weaker to less phosphorylated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P3. Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phospholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium.

  7. Rzhevsky D.I., Zhokhov S.S., Babichenko I.I., Goleva A.V., Goncharenko E.N., Baizhumanov A.A., Murashev A.N., Lipkin V.M., Kostanyan I.A. (2005). HLDF-6 peptide affects behavioral reactions and organism functions dependent on androgen hormones in normal and castrated male mice. Regul. Pept. 127 (1-3), 111–21 [+]

    The hexapeptide Thr-Gly-Glu-Asn-His-Arg (HLDF-6), which was first identified as an active fragment of the human leukemia differentiation factor (HLDF) molecule, displays differentiation-inducing, neuroprotective and anti-drug abuse activities. Most of its in vivo effects were revealed only on male animals. We have studied HLDF-6 effects on a variety of organism functions and behavioral reactions, which are known to be dependent on androgen steroid hormones, both on castrated and normal (sham-operated) animals. Male NMRI mice were castrated or sham-operated at the age of 55 days (after puberty). After that, HLDF-6 peptide was injected daily during 3 weeks, followed by behavioral, morphological and biochemical testing. HLDF-6 increased testosterone level (1.5- to 2-fold) both in sham-operated and castrated animals. Sexual activity and pain sensitivity, which are strongly reduced in castrates, were completely or partially recovered by HLDF-6. At the same time, the peptide caused some effects similar to castration in sham-operated animals: aggression and locomotor activity were decreased; oral grooming was prolonged. Morphological studies of accessory sex glands showed that HLDF-6 partially normalizes the morphology and functional activity of seminal vesicles in castrates, but it does not prevent castration-induced apoptosis of prostate epithelial cells. Based on these observations, we can assume that HLDF-6 peptide displays at least two effects on androgen hormones metabolism in males: it stimulates testosterone biosynthesis by both testes and adrenals and simultaneously inhibits its conversion to dihydrotestosterone (DHT), most probably by diminution of 5alpha-reductase isoform 1 mRNA expression.

  8. Merkulova M.I., Andreeva S.G., Shuvaeva T.M., Novoselov S.V., Peshenko I.V., Bystrova M.F., Novoselov V.I., Fesenko E.E., Lipkin V.M. (1999). A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation. FEBS Lett. 450 (1-2), 126–30 [+]

    cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.

  9. Kostanyan I.A., Astapova M.V., Starovoytova E.V., Dranitsina S.M., Lipkin V.M. (1994). A new human leukemia cell 8.2 kDa differentiation factor: isolation and primary structure. FEBS Lett. 356 (2-3), 327–9 [+]

    A new 8.2 kDa differentiation factor has been purified to homogeneity from the cultural media of human myelogenous HL-60 leukemia cells induced by retinoic acid. cDNA clones encoding this factor were isolated from a cDNA library prepared from HL-60 differentiated cells and their nucleotide sequence has been determined. The deduced amino acid sequence of the differentiation factor molecule consists of 54 amino acid residues. The protein is shown to be glycosylated. It was shown by Northern blot experiments that the level of poly(A)+ RNA with a length of 450 nucleotides was higher in differentiated cells than in non-differentiated cells.

  10. Khramtsov N.V., Feshchenko E.A., Suslova V.A., Shmukler B.E., Terpugov B.E., Rakitina T.V., Atabekova N.V., Lipkin V.M. (1993). The human rod photoreceptor cGMP phosphodiesterase beta-subunit. Structural studies of its cDNA and gene. FEBS Lett. 327 (3), 275–8 [+]

    cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed alpha-, beta- and alpha'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4-22 of the PDE beta-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the beta-subunit gene was observed between exons 20 and 21 which led to the formation of the beta'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE beta'-subunit in human retina.

  11. Lipkin V.M., Khramtsov N.V., Vasilevskaya I.A., Atabekova N.V., Muradov K.G., Gubanov V.V., Li T., Johnston J.P., Volpp K.J., Applebury M.L. (1990). Beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase. Comparison with the phosphodiesterase family. J. Biol. Chem. 265 (22), 12955–9 [+]

    A group of cDNA clones encoding the beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase were isolated for structural analysis. The encoded polypeptide has 853 residues with a calculated molecular mass of 98 kDa. The beta-subunit is 72% identical to the rod cGMP phosphodiesterase alpha-subunit. Like the alpha-subunit and the cone alpha'-subunit, the beta-subunit belongs to the family of phosphodiesterase genes. The beta- and alpha-subunits are more similar to each other than either is to the cone alpha'-subunit, suggesting either that the beta- and alpha-subunits diverged more recently or that their divergence was restrained by the rod functional environment.


Valery Lipkin

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Neuroprotective activity of HLDF-6 peptide was shown on double- transgene model of Alzheimer’s disease (2017-12-15)

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.