Publications by authors named "Vasiliy I Vladimirov"

6 Publications

  • Page 1 of 1

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Int J Mol Sci 2021 Nov 22;22(22). Epub 2021 Nov 22.

Belozersky Institute of Physico Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.
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http://dx.doi.org/10.3390/ijms222212602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8623652PMC
November 2021

Polyamines Counteract Carbonate-Driven Proteasome Stalling in Alkaline Conditions.

Biomolecules 2020 11 24;10(12). Epub 2020 Nov 24.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

Cancer cells tend to increase intracellular pH and, at the same time, are known to intensively produce and uptake polyamines such as spermine. Here, we show that various amines, including biogenic polyamines, boost the activity of proteasomes in a dose-dependent manner. Proteasome activity in the classical amine-containing buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES), Tris, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycylglycine, bis-Tris propane, and bicine, has a skewed distribution with a maximum at pH of 7.0-8.0. The activity of proteasomes in buffers containing imidazole and bis-Tris is maintained almost on the same level, in the pH range of 6.5-8.5. The third type of activation is observed in buffers based on the amino acids arginine and ornithine, as well as the natural polyamines spermine and spermidine. Proteasome activity in these buffers is dramatically increased at pH values greater than 7.5. Anionic buffers such as phosphate or carbonate, in contrast, inhibit proteasome activity during alkalization. Importantly, supplementation of a carbonate-phosphate buffer with spermine counteracts carbonate-driven proteasome stalling in alkaline conditions, predicting an additional physiological role of polyamines in maintaining the metabolism and survival of cancer cells.
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http://dx.doi.org/10.3390/biom10121597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760842PMC
November 2020

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins.

Biomolecules 2020 07 10;10(7). Epub 2020 Jul 10.

Laboratory of pharmacokinetics, Department of Biological Testing, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences in Puschino, Pushchino, Moscow region 142290, Russia.

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.
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http://dx.doi.org/10.3390/biom10071025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407513PMC
July 2020

Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization.

Front Mol Neurosci 2018 7;11:474. Epub 2019 Jan 7.

Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Russia.

The excessive light illumination of mammalian retina is known to induce oxidative stress and photoreceptor cell death linked to progression of age-related macular degeneration. The photochemical damage of photoreceptors is suggested to occur via two apoptotic pathways that involve either excessive rhodopsin activation or constitutive phototransduction, depending on the light intensity. Both pathways are dramatically activated in the absence of rhodopsin desensitization by GRK1. Previously, we have shown that moderate illumination (halogen lamp, 1,500 lx, 1-5 h) of mammalian eyes provokes disulfide dimerization of recoverin, a calcium-dependent regulator of GRK1. Here, we demonstrate under conditions that both moderate long-term (metal halide lamp, 2,500 lx, 14 h, rat model) and intense short-term (halogen lamp, 30,000 lx for 3 h, rabbit model) illumination of the mammalian retina are accompanied by accumulation of disulfide dimer of recoverin. Furthermore, in the second case we reveal alternatively oxidized derivatives of the protein, apparently including its monomer with sulfinic group. Histological data indicate that thiol oxidation of recoverin precedes apoptosis of photoreceptors. Both disulfide dimer and oxidized monomer (or oxidation mimicking C39D mutant) of recoverin exhibit lowered α-helical content and thermal stability of their apo-forms, as well as increased Ca affinity. Meanwhile, the oxidized monomer and C39D mutant of recoverin demonstrate impaired ability to bind photoreceptor membranes and regulate GRK1, whereas disulfide dimer exhibits notably improved membrane binding and GRK1 inhibition in absence of Ca. The latter effect is expected to slow down rhodopsin desensitization in the light, thereby favoring support of the light-induced oxidative stress, ultimately leading to photoreceptor apoptosis. Overall, the intensity and duration of illumination of the retina affect thiol oxidation of recoverin likely contributing to propagation of the oxidative stress and photoreceptor damage.
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http://dx.doi.org/10.3389/fnmol.2018.00474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330308PMC
January 2019

Functional Status of Neuronal Calcium Sensor-1 Is Modulated by Zinc Binding.

Front Mol Neurosci 2018 14;11:459. Epub 2018 Dec 14.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 10 M) and one low-affinity (2 × 10 M) Zn-binding sites, whereas Mg-loaded and Ca-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn-binding sites of apo- and Mg-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn concentrations displace Mg from the EF-hands and decrease the stoichiometry of Ca binding. Meanwhile, one of the EF-hands of Zn-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn binding to Ca-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca-free NCS-1 and favours the interaction of its Ca-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.
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http://dx.doi.org/10.3389/fnmol.2018.00459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302015PMC
December 2018

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1.

Cell Calcium 2018 07 14;73:55-69. Epub 2018 Apr 14.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca-signals thereby maintaining efficient phototransduction recovery and light adaptation.
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http://dx.doi.org/10.1016/j.ceca.2018.04.003DOI Listing
July 2018
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