Laboratory of hormonal regulation proteins

Department of Peptide and Protein Technologies

Head: Alexey Belogurov, D.Sc
belogurov@mx.ibch.ru+7(495)7273860

Proteostasis, 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.

NamePositionContacts
Valery Lipkin, corresponding member of the academy of sciencesheadvmlipkin@ibch.ru+7(495)336-61-66
Alexey Belogurov, D.Scheadbelogurov@mx.ibch.ru+7(495)7273860
Tat'jana Shuvaeva, D.Scl. r. f.shuvaeva@mx.ibch.ru+7(495)336-51-11
Anna Bogachuk, Ph.D.s. r. f.apbogachouk@gmail.com+7(495)336-51-11
Tat'jana Rakitina, Ph.D.s. r. f.taniarakitina@yahoo.com+7(495)330-63-47
Dmitrij Kakuevr. f.dkakuev@mail.ru+7(495)330-63-47
Vitaly Radchenko, Ph.D.r. f.vitalyradchenko@yandex.ru+7(495)336-55-11
Elena Surinar. f.surinae@mx.ibch.ru+7(495)336-55-11
Elena Il'nitskaya, Ph.D.r. f.nevrilemma@yandex.ru+7(495)335-26-00
Evgenia Smirnova, Ph.D.r. f.smirnova.evgeniya@gmail.com+7(495)330-63-47
Natal'ja Minkevich, Ph.D.j. r. f.ni-minkevich@gmail.com+7(495)336-55-11
Elena Vorob'evaPhD stud.e-elena777@yandex.ru
Irina Smirnovares. eng.smirnova@mx.ibch.ru

Former members:

Igor' Artamonov, Ph.D.s. r. f.
Irina Kostanjan, Ph.D.s. r. f.
Alexey Garkovenkoj. r. f.garkovenko@gmail.com

All publications (show selected)

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Alexey Belogurov

Discovery of novel class of ubiquitin-independent proteasomal degrons

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

In collaboration with 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

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

In collaboration with 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

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.

Molecular mechanism of the non-opioid beta-endorphin action

In collaboration with 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.

Development of peptide drugs for the treatment of multiple sclerosis

In collaboration with Group of Peptide Chemistry,  Laboratory of Biological Testing,  Laboratory of pharmacokinetics

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.