Laboratory of bioengineering of neuromodulators and neuroreceptors

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Ekaterina Lyukmanova

Human Three-finger Proteins Inhibit the Growth of Carcinoma Cells

In collaboration with Laboratory of structural biology of ion channels

Nicotinic acetylcholine receptors (nAChR) play an important role in the physiology of epithelial cells, and their activation contributes to the development of carcinomas. Natural modulators of nicotinic acetylcholine receptors may become promising prototypes of new antitumor agents.

Recombinant analogs of human three-finger proteins ws-Lynx1 and rSLURP-1 were shown to inhibit the growth of lung carcinoma and melanoma cells. Ws-Lynx1 in A549 cells stimulates antiproliferative and proapoptotic signaling cascades associated with activation of α7-nAChR. rSLURP-1 inhibits nicotine-induced lung carcinoma cell growth, and also abolishes nicotine-induced increase in the α7-nAChR expression and decrease in the PTEN tumor suppressor gene expression. In addition, rSLURP-1 inhibits the growth of multicellular spheroids from cells of various carcinomas. The combined use of rSLURP-1 with other antitumor drugs (gefitinib, bortezomib, doxorubicin) leads to a complete stop in the growth of spheroids.

Thus, ws-Lynx1 and rSLURP-1 are promising prototypes for the development of new drugs for the cancer treatment.

Combinatorial selective incorporation of stable13C and 15N isotopes facilitates NMR spectra analysis and allows mapping of the binding interfaces between membrane receptors and their ligands

In collaboration with Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Combinatorial incorporation of stable 13C and 15N isotopes into protein molecules can significantly simplify the analysis of NMR spectra. For the first time, the problem was solved and the CombLabel algorithm was developed for calculating combinatorial 13C and 15N  labeling schemes with a minimum price. The application of the program allowed to assign 50% of the NMR signals of the backbone of the second voltage-sensing domain of human sodium channel Nav1.4 (VSD-II). Leak currents through mutant variants of Nav1.4 containing Arg675Gly mutation in VSD-II lead to the development of a hereditary disease – normokalemic periodic paralysis. Hm-3 toxin from the venom of spider Heriaeus melloteei is able to block leak currents in VSD-II. By the means of NMR spectroscopy the interaction interface between VSD-II and Hm-3 toxin was determined. According to the model of the VSD-II/Hm-3 complex, based on the NMR data, the toxin binds to the extracellular S1-S2 loop, destabilizing the state of the domain, at which leak currents are observed. Using the example of the complexes of Hm-3 toxin with VSD-I and VSD-II of the Nav1.4 channel, it has been shown that arachnid toxins can interact differently with different domains within the same sodium channel.

The physiological effect of two bisbenzylisoquinoline alkaloids having activity on ASIC1a

In collaboration with Laboratory of Biological Testing,  Laboratory of neuroreceptors and neuroregulators

The ASIC1a is the most sensitive subtype of acid-sensing ion channel in the cell membrane, and it plays an important role in the excitation of neurons of CNS. Long time the ligands to this ASIC subtype are under intense attention for the development of drugs for pain relief, as well as protectors from strokes and neurodegenerative diseases. In in vitro experiments on heterologically expressed ASIC1a channels, the action of two bisbenzylisoquinoline alkaloids from plants was studied by electrophysiological method of two-electrode potential fixing on oocyte cells.

The alkaloid lindoldhamine extracted from the leaves of Laurus nobilis L. significantly inhibited the ASIC1a channel’s response to physiologically-relevant stimuli of pH 6.5–6.85 with IC50 range 150–9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund’s adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid. Thus, it was shown not only a prospective of plant alkaloids using for a pain relief, but was indirectly confirmed the involvement of the ASIC1a channels of the peripheral nervous system in the generation of a pain response to mild acidification.

The structural analogue named daurisoline, unlike lindoldamine, did not inhibit the activation of the ASIC1a channel by protons, but produced the second peak component of the ASIC1a current. This second peak manifested with a 2.5 seconds delay after the first fast respond followed by completely desensitization with the same kinetics as the main peak. The presence of second current components was specific characteristic of ASIC2 and ASIC3 subtypes early, but this component is sustained, that last all time while the acid stimulus presented. The discovery of the second component of ASIC1a current allows us to declare the common mechanism of opening and desensitization for all ASICs, which will be interesting to determine in further experiments.

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells.

The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action.

Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Mammalian three-finger proteins protect against cancer

In collaboration with Laboratory of structural biology of ion channels,  Laboratory of optical microscopy and spectroscopy of biomolecules

Efremenko A.V., Sharonov G.V., Feofanov A.V. (Laboratory of optical microscopy and spectroscopy of biomolecules), Lyukmanova E.N., Bychkov M.L., Shulepko M.A., Kulbatskii D.S., Dolgikh D.A., Kirpichnikov M.P. (Group of bioengineering of neuromodulators and neuroreceptors), Shenkarev Z.O. (Group of structural biology of ion channels).

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells. The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action. Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Spider toxin inhibits aberrant currents in mutant ion channels

In collaboration with Laboratory of structural biology of ion channels,  Laboratory of Molecular Instruments for Neurobiology

Toxin from the venom of the crab spider Heriaeus melloteei may serve as a hit in drug discovery for hypokalemic periodic paralysis type 2; there is no reliable medication for all cases of this disease. It is caused by mutations in the gene encoding voltage-gated sodium channels NaV1.4, characteristic of skeletal muscles. As a result of the mutations, these channels conduct aberrant currents, the muscles are unable to respond to the signals of the nervous system, and weakness develops followed by paralysis. Hm-3 toxin was found to be able to selectively inhibit such currents through voltage-sensing domain I of mutant channels. Read more in the press release on the IBCh website.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Interaction of gating modifier toxin Hm-3 with voltage-sensing domains of Nav1.4 sodium channel: structural view on the membrane-mediated binding

In collaboration with Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Voltage-gated Na+ channels (Nav) are essential for the functioning of cardiovascular, muscular, and nervous systems. Certain mutations trigger a leak current through voltage-sensing domains (VSDs) of Nav leading to various diseases. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the S4 segments of VSDs in the human skeletal muscle channel NaV1.4. The gating modifier toxin Hm-3 (crab spider Heriaeus melloteei) inhibits leak currents through such mutant channels. To investigate molecular basis of Hm-3 interaction with NaV1.4 channel, we studied isolated VSD-I by NMR spectroscopy in membrane mimicking environment. Hm-3/VSD-I complex was modeled using protein-protein docking guided by NMR restrains. The toxin initially anchors onto the membrane surface and then forms the complex with the S3b-S4 loop of the VSD-I. The Hm-3 binding blocks movement of the voltage-sensor helix S4 and induces some allosteric changes that prevent development of gating-pore currents. Our report is the first NMR study of structural interactions between gating modifier toxins and Nav channels.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Secondary structure and dynamics of the voltage-sensing domain of second pseudosubunit of human skeletal muscle sodium channel Nav1.4

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Laboratory of biomolecular modeling,  Laboratory of structural biology of ion channels

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues. This complexity significantly impedes structural studies of full-sized Na+ channels. The isolated voltage-sensing domain (VSD-II) of human skeletal muscle Nav1.4 channel was studied by NMR in membrane mimicking environment. Secondary structure of VSD-II showed similarity with the bacterial Na+ channels. Fragment of S4 helix between the first and second conserved Arg residues probably adopts 3/10-helical conformation. 15N-relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels.