Vladimir Polshakov | Moscow State University (original) (raw)

Papers by Vladimir Polshakov

Alzheimer's & Dementia, 2014

ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes ... more ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes the metal-binding domain of human Aβ, and amino acids 6-14 form the minimal zinc-binding site. Zinc-dependent Aβ dimerization is mediated by a tetrapeptide stretch 11-14. Isomerization of Asp7, the most abundant aging-associated spontaneous chemical modification of Aβ, makes the resulting isoAsp7-containing Aβ (isoAβ) much more susceptible to Zn 2+ -bound dimerization with dimers serving as aggregation seeds. Indeed, it has been shown in experiments on transgenic mice that intravenous injections of isoAβ cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. In the present study we show the influence of Ser-8 phosphorilation and H6R mutation on the formation of potentially pathogenic zinc-induced Aβ dimers. Methods: We have used synthetic peptide analogs of the metal-binding domains of corresponding Aβ isoforms in order to study their zinc-induced complexes. Their thermodynamic, kinetic and structural properties were probed by isothermal titration calorimetry, SPR biosensing, mass-spectrometry and NMR spectroscopy. Results: It has been shown that the Ser-8 phosphorylation as well as H6R mutation result in higher capability of the modified metal-binding domains to form stable zinc-bound dimers. Conclusions: The obtained data show that the Aβ species in which the 11-14 stretch is more accessible for intermolecular interactions due to specific chemical modifications or mutations in the region 6-10 can act as triggers for zinc-induced aggregation of Aβ. These results suggest that the information on three-dimensional structure of the Zn 2+ -binding interface of the dimers can be used for rational drug design, targeting Alzheimer's disease.

Alzheimer's & Dementia, 2014

ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes ... more ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes the metal-binding domain of human Aβ, and amino acids 6-14 form the minimal zinc-binding site. Zinc-dependent Aβ dimerization is mediated by a tetrapeptide stretch 11-14. Isomerization of Asp7, the most abundant aging-associated spontaneous chemical modification of Aβ, makes the resulting isoAsp7-containing Aβ (isoAβ) much more susceptible to Zn 2+ -bound dimerization with dimers serving as aggregation seeds. Indeed, it has been shown in experiments on transgenic mice that intravenous injections of isoAβ cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. In the present study we show the influence of Ser-8 phosphorilation and H6R mutation on the formation of potentially pathogenic zinc-induced Aβ dimers. Methods: We have used synthetic peptide analogs of the metal-binding domains of corresponding Aβ isoforms in order to study their zinc-induced complexes. Their thermodynamic, kinetic and structural properties were probed by isothermal titration calorimetry, SPR biosensing, mass-spectrometry and NMR spectroscopy. Results: It has been shown that the Ser-8 phosphorylation as well as H6R mutation result in higher capability of the modified metal-binding domains to form stable zinc-bound dimers. Conclusions: The obtained data show that the Aβ species in which the 11-14 stretch is more accessible for intermolecular interactions due to specific chemical modifications or mutations in the region 6-10 can act as triggers for zinc-induced aggregation of Aβ. These results suggest that the information on three-dimensional structure of the Zn 2+ -binding interface of the dimers can be used for rational drug design, targeting Alzheimer's disease.

Conformational changes of Aβ peptide result in its transformation from native monomeric state to ... more Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer's disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ 1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6 HDSGYEVHH 14. The residue H6 and segment 11 EVHH 14 , which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation. According to the amyloid hypothesis, which has been the predominant framework for Alzheimer disease (AD) studies, Aβ aggregation has a unique and critical role as an initiator of AD pathology 1,2. What triggers Aβ aggre-gation still remains unclear, however, some genetically and/or post-translationally modified Aβ species accumulated in the amyloid plaques appear to act as the pathogenic aggregation seeds 3. It has been shown in animal models of AD that zinc ions might play a crucial role in the Aβ plaque formation in vivo 4–7. Indeed, at concentration as high as that detected in the synapse, zinc ions specifically bind Aβ and are able to facilitate Aβ aggre-gation 8 , which could explain abnormally high levels of zinc ions within amyloid plaques of AD patients 9,10. It is assumed that zinc ions promote Aβ aggregation via population shift of polymorphic states 11 with a mechanism similar to that observed for larger zinc-binding proteins 12. When zinc-induced Aβ amyloidogenesis is observed in vitro, conformational changes in Aβ are also identified 13. However, precise structural details of these changes were elusive since three-dimensional structures of Aβ oligomers complexed with zinc ions were unavailable 14. Interaction of Zn 2+ with monomeric Aβ species is mediated by the metal binding domain which comprises the N-terminal region 1–16 of Aβ 15–17. Aβ 1–16 exists in health and disease as a separate entity 18 , suggesting its possible role as the structural and functional unit of the full-length Aβ. Indeed, the interaction of N-terminal region 1–16 with the β-strand hydrophobic region 17-42 is negligible in the model amyloid aggregates 19,20 , and also synthetic peptides Aβ 1–16 exhaustively simulate the metal binding properties of Aβ 15,16,21. Previous NMR studies of the N-terminus of Aβ showed that the first 9 residues are poorly structured, whereas residues beyond 10 form a distinct local conformation 17,22–30. Structure of the tethered N-terminus of the Alzheimer's disease amyloid-β peptide obtained using X-ray crystallography 31 showed that Aβ region 10–16 is relatively rigid and adopts a mixture of the local polyproline II-helix (PPII) and turn type conformations. The fragment Aβ 1–16 includes several charged residues with their location typical of ionic self-complementary peptides 32. These residues participate in the formation of electrostatic contacts, which can stabilize both intra and intermolecular interactions. The region 10–16 of Aβ appeared to be an effective metal ion trapping unit 33. The fragment 6–14 of Aβ has been determined as the minimal Zn 2+ binding site wherein the ion is coordinated by H6, E11, H13, and H14 34. Under

Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed... more Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of β-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z/E isomerism of β-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of β-proline oligopeptides. Preferences in their structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied β-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the β-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating β-proline pentapeptide, the presence of an E configuration for at least of one β-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of β-peptide bonds forming a wave-like extended structure.

The elongation of single-stranded DNA repeats at the 3-ends of chromosomes by telomerase is a key... more The elongation of single-stranded DNA repeats at the 3-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids––telomerase RNA, telom-eric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the ther-motolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Protoplasma, 2016

Vacuole is a multifunctional compartment central to a large number of functions (storage, catabol... more Vacuole is a multifunctional compartment central to a large number of functions (storage, catabolism, maintenance of the cell homeostasis) in oxygenic phototrophs including microalgae. Still, microalgal cell vacuole is much less studied than that of higher plants although knowledge of the vacuolar structure and function is essential for understanding physiology of nutrition and stress tolerance of microalgae. Here, we combined the advanced analytical and conventional transmission electron microscopy methods to obtain semi-quantitative, spatially resolved at the subcellular level information on elemental composition of the cell vacuoles in several free-living and symbiotic chlorophytes. We obtained a detailed record of the changes in cell and vacuolar ultrastructure in response to environmental stimuli under diverse conditions. We suggested that the vacuolar inclusions could be divided into responsible for storage of phosphorus (mainly in form of polyphosphate) and those accommodating non-protein nitrogen (presumably polyamine) reserves, respectively.The ultrastructural findings, together with the data on elemental composition of different cell compartments, allowed us to speculate on the role of the vacuolar membrane in the biosynthesis and sequestration of polyphosphate. We also describe the ultrastructural evidence of possible involvement of the tonoplast in the membrane lipid turnover and exchange of energy and metabolites between chloroplasts and mitochondria. These processes might play a significant role in acclimation in different stresses including nitrogen starvation and extremely high level of CO2 and might also be of importance for microalgal biotechnology. Advantages and limitations of application of analytical electron microscopy to biosamples such as microalgal cells are discussed.

FEBS Journal, 2007

The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the... more The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring alpha-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest alpha-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other.

Molecular Biology, 2008

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2,... more Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α 1 of the M domain. Some residues adjacent to α 1 and belonging to strand β 5 and short helices α 2 and α 3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α 1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight proteinribosome contact.

Journal of Biomolecular NMR, 2006

Molecular Biology, 2008

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2,... more Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α 1 of the M domain. Some residues adjacent to α 1 and belonging to strand β 5 and short helices α 2 and α 3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α 1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight proteinribosome contact.

ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

PLoS genetics, 2015

The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in num... more The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in numerous interacting genes. In our mouse model system, the whole-genome quantitative trait loci (QTLs) scan revealed three QTLs involved in TB control on chromosomes 3, 9, and in the vicinity of the H2 complex on chromosome 17. For the present study, we have established a panel of new congenic, MHC-recombinant mouse strains bearing differential small segments of chromosome 17 transferred from the TB-susceptible I/St (H2j) strain onto the genetic background of TB-resistant C57BL/6 (B6) mice (H2b). This allowed narrowing the QTL interval to 17Ch: 33, 77-34, 34 Mb, containing 36 protein-encoding genes. Cloning and sequencing of the H2j allelic variants of these genes demonstrated profound polymorphic variations compare to the H2b haplotype. In two recombinant strains, B6.I-249.1.15.100 and B6.I-249.1.15.139, recombination breakpoints occurred in different sites of the H2-Aβ 1 gene (beta-chain ...

Heterocyclic Communications, 2000

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Biochemistry, 1991

heteronuclear N M R techniques have been used to make sequential 'H and I5N resonance assignments... more heteronuclear N M R techniques have been used to make sequential 'H and I5N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18 300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D lsN/'H nuclear Overhauserheteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 'H-IH through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows N O E cross peaks to be detected between N H protons even when their 'H chemical shifts are degenerate as long as the amide ISN chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+l NOES define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR-methotrexateeNADPH complex J . Biol. G e m . 257, 13650-136621, while the NMRdetermined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-1 10 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of @-sheet structure were characterized in the binary complex by strong aCHi-NHi+, NOES and by slowly exchanging amide protons. In addition, several long-range NOES were identified linking together these stretches to form a 6-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR-methotrexate-NADPH complex, which contains an eight-stranded 6-sheet, indicating that the main body of the @-sheet is preserved in the binary complex in solution.

Chemistry, an Asian journal, 2015

Functionalized oligomeric organic compounds with well-defined β-proline scaffold have been synthe... more Functionalized oligomeric organic compounds with well-defined β-proline scaffold have been synthesized by a cycloadditive oligomerization approach in racemic and enantiopure forms. The structure of the novel β-peptides was investigated by NMR spectroscopic and X-ray methods determining the conformational shapes of the β-proline oligomers in solution and solid states. The main structural elements subject to conformational switches are β-peptide bonds between 5-arylpyrrolidine-2-carboxylic acid units existing in Z/E configurations. The whole library of short β-peptides and intermediate acrylamides has been tested on antiproliferative activity towards the hormone-refractory prostate cancer cell line PC-3 revealing several oligomeric compounds with low micromolar and submicromolar activities. Bromine-substituted dimeric and trimeric acrylamides induced caspase-dependent apoptosis of PC-3 cells through cell-cycle arrest and mitochondrial damage.

Biochemistry, 1994

Two-and three-dimensional (2D and 3D) N M R techniques have been used to assign the signals from ... more Two-and three-dimensional (2D and 3D) N M R techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (Mr 18 300) in its 1:l complex with the antibacterial drug trimethoprim. A sample of uniformly ISN-labeled protein was examined using 3D 15N/IH experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOES between trimethoprim and protein protons and four intramolecular NOES in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15Nand 13C-labeled trimethoprim molecules ([ 1 ,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound tounlabeled protein. The ligand-

Journal of Biomolecular NMR, 1999

A new method is proposed for docking ligands into proteins in cases where an NMR-determined solut... more A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein-ligand, ligand-ligand, and protein-protein restraints for residues in or near to the binding site, combined with a set of protein-protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in 1 H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase.trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase.methotrexate reference structure.

Biochemistry, 1995

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlati... more Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1: 1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-0 position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of lo3 more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 1 1 intramolecular NOES were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tl = -153" f 4" (C4-C5-C7-C1') and t2 = 53" k 4" (C5-C7-C1'-C2'):

Journal of Biomolecular NMR, 2005

Alzheimer's & Dementia, 2014

ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes ... more ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes the metal-binding domain of human Aβ, and amino acids 6-14 form the minimal zinc-binding site. Zinc-dependent Aβ dimerization is mediated by a tetrapeptide stretch 11-14. Isomerization of Asp7, the most abundant aging-associated spontaneous chemical modification of Aβ, makes the resulting isoAsp7-containing Aβ (isoAβ) much more susceptible to Zn 2+ -bound dimerization with dimers serving as aggregation seeds. Indeed, it has been shown in experiments on transgenic mice that intravenous injections of isoAβ cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. In the present study we show the influence of Ser-8 phosphorilation and H6R mutation on the formation of potentially pathogenic zinc-induced Aβ dimers. Methods: We have used synthetic peptide analogs of the metal-binding domains of corresponding Aβ isoforms in order to study their zinc-induced complexes. Their thermodynamic, kinetic and structural properties were probed by isothermal titration calorimetry, SPR biosensing, mass-spectrometry and NMR spectroscopy. Results: It has been shown that the Ser-8 phosphorylation as well as H6R mutation result in higher capability of the modified metal-binding domains to form stable zinc-bound dimers. Conclusions: The obtained data show that the Aβ species in which the 11-14 stretch is more accessible for intermolecular interactions due to specific chemical modifications or mutations in the region 6-10 can act as triggers for zinc-induced aggregation of Aβ. These results suggest that the information on three-dimensional structure of the Zn 2+ -binding interface of the dimers can be used for rational drug design, targeting Alzheimer's disease.

Alzheimer's & Dementia, 2014

ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes ... more ABSTRACT Background: Zinc ions are necessary for Aβ aggregation in vivo. Region 1-16 constitutes the metal-binding domain of human Aβ, and amino acids 6-14 form the minimal zinc-binding site. Zinc-dependent Aβ dimerization is mediated by a tetrapeptide stretch 11-14. Isomerization of Asp7, the most abundant aging-associated spontaneous chemical modification of Aβ, makes the resulting isoAsp7-containing Aβ (isoAβ) much more susceptible to Zn 2+ -bound dimerization with dimers serving as aggregation seeds. Indeed, it has been shown in experiments on transgenic mice that intravenous injections of isoAβ cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. In the present study we show the influence of Ser-8 phosphorilation and H6R mutation on the formation of potentially pathogenic zinc-induced Aβ dimers. Methods: We have used synthetic peptide analogs of the metal-binding domains of corresponding Aβ isoforms in order to study their zinc-induced complexes. Their thermodynamic, kinetic and structural properties were probed by isothermal titration calorimetry, SPR biosensing, mass-spectrometry and NMR spectroscopy. Results: It has been shown that the Ser-8 phosphorylation as well as H6R mutation result in higher capability of the modified metal-binding domains to form stable zinc-bound dimers. Conclusions: The obtained data show that the Aβ species in which the 11-14 stretch is more accessible for intermolecular interactions due to specific chemical modifications or mutations in the region 6-10 can act as triggers for zinc-induced aggregation of Aβ. These results suggest that the information on three-dimensional structure of the Zn 2+ -binding interface of the dimers can be used for rational drug design, targeting Alzheimer's disease.

Conformational changes of Aβ peptide result in its transformation from native monomeric state to ... more Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer's disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ 1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6 HDSGYEVHH 14. The residue H6 and segment 11 EVHH 14 , which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation. According to the amyloid hypothesis, which has been the predominant framework for Alzheimer disease (AD) studies, Aβ aggregation has a unique and critical role as an initiator of AD pathology 1,2. What triggers Aβ aggre-gation still remains unclear, however, some genetically and/or post-translationally modified Aβ species accumulated in the amyloid plaques appear to act as the pathogenic aggregation seeds 3. It has been shown in animal models of AD that zinc ions might play a crucial role in the Aβ plaque formation in vivo 4–7. Indeed, at concentration as high as that detected in the synapse, zinc ions specifically bind Aβ and are able to facilitate Aβ aggre-gation 8 , which could explain abnormally high levels of zinc ions within amyloid plaques of AD patients 9,10. It is assumed that zinc ions promote Aβ aggregation via population shift of polymorphic states 11 with a mechanism similar to that observed for larger zinc-binding proteins 12. When zinc-induced Aβ amyloidogenesis is observed in vitro, conformational changes in Aβ are also identified 13. However, precise structural details of these changes were elusive since three-dimensional structures of Aβ oligomers complexed with zinc ions were unavailable 14. Interaction of Zn 2+ with monomeric Aβ species is mediated by the metal binding domain which comprises the N-terminal region 1–16 of Aβ 15–17. Aβ 1–16 exists in health and disease as a separate entity 18 , suggesting its possible role as the structural and functional unit of the full-length Aβ. Indeed, the interaction of N-terminal region 1–16 with the β-strand hydrophobic region 17-42 is negligible in the model amyloid aggregates 19,20 , and also synthetic peptides Aβ 1–16 exhaustively simulate the metal binding properties of Aβ 15,16,21. Previous NMR studies of the N-terminus of Aβ showed that the first 9 residues are poorly structured, whereas residues beyond 10 form a distinct local conformation 17,22–30. Structure of the tethered N-terminus of the Alzheimer's disease amyloid-β peptide obtained using X-ray crystallography 31 showed that Aβ region 10–16 is relatively rigid and adopts a mixture of the local polyproline II-helix (PPII) and turn type conformations. The fragment Aβ 1–16 includes several charged residues with their location typical of ionic self-complementary peptides 32. These residues participate in the formation of electrostatic contacts, which can stabilize both intra and intermolecular interactions. The region 10–16 of Aβ appeared to be an effective metal ion trapping unit 33. The fragment 6–14 of Aβ has been determined as the minimal Zn 2+ binding site wherein the ion is coordinated by H6, E11, H13, and H14 34. Under

Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed... more Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of β-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z/E isomerism of β-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of β-proline oligopeptides. Preferences in their structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied β-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the β-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating β-proline pentapeptide, the presence of an E configuration for at least of one β-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of β-peptide bonds forming a wave-like extended structure.

The elongation of single-stranded DNA repeats at the 3-ends of chromosomes by telomerase is a key... more The elongation of single-stranded DNA repeats at the 3-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids––telomerase RNA, telom-eric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the ther-motolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Protoplasma, 2016

Vacuole is a multifunctional compartment central to a large number of functions (storage, catabol... more Vacuole is a multifunctional compartment central to a large number of functions (storage, catabolism, maintenance of the cell homeostasis) in oxygenic phototrophs including microalgae. Still, microalgal cell vacuole is much less studied than that of higher plants although knowledge of the vacuolar structure and function is essential for understanding physiology of nutrition and stress tolerance of microalgae. Here, we combined the advanced analytical and conventional transmission electron microscopy methods to obtain semi-quantitative, spatially resolved at the subcellular level information on elemental composition of the cell vacuoles in several free-living and symbiotic chlorophytes. We obtained a detailed record of the changes in cell and vacuolar ultrastructure in response to environmental stimuli under diverse conditions. We suggested that the vacuolar inclusions could be divided into responsible for storage of phosphorus (mainly in form of polyphosphate) and those accommodating non-protein nitrogen (presumably polyamine) reserves, respectively.The ultrastructural findings, together with the data on elemental composition of different cell compartments, allowed us to speculate on the role of the vacuolar membrane in the biosynthesis and sequestration of polyphosphate. We also describe the ultrastructural evidence of possible involvement of the tonoplast in the membrane lipid turnover and exchange of energy and metabolites between chloroplasts and mitochondria. These processes might play a significant role in acclimation in different stresses including nitrogen starvation and extremely high level of CO2 and might also be of importance for microalgal biotechnology. Advantages and limitations of application of analytical electron microscopy to biosamples such as microalgal cells are discussed.

FEBS Journal, 2007

The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the... more The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring alpha-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest alpha-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other.

Molecular Biology, 2008

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2,... more Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α 1 of the M domain. Some residues adjacent to α 1 and belonging to strand β 5 and short helices α 2 and α 3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α 1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight proteinribosome contact.

Journal of Biomolecular NMR, 2006

Molecular Biology, 2008

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2,... more Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α 1 of the M domain. Some residues adjacent to α 1 and belonging to strand β 5 and short helices α 2 and α 3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α 1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight proteinribosome contact.

ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

PLoS genetics, 2015

The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in num... more The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in numerous interacting genes. In our mouse model system, the whole-genome quantitative trait loci (QTLs) scan revealed three QTLs involved in TB control on chromosomes 3, 9, and in the vicinity of the H2 complex on chromosome 17. For the present study, we have established a panel of new congenic, MHC-recombinant mouse strains bearing differential small segments of chromosome 17 transferred from the TB-susceptible I/St (H2j) strain onto the genetic background of TB-resistant C57BL/6 (B6) mice (H2b). This allowed narrowing the QTL interval to 17Ch: 33, 77-34, 34 Mb, containing 36 protein-encoding genes. Cloning and sequencing of the H2j allelic variants of these genes demonstrated profound polymorphic variations compare to the H2b haplotype. In two recombinant strains, B6.I-249.1.15.100 and B6.I-249.1.15.139, recombination breakpoints occurred in different sites of the H2-Aβ 1 gene (beta-chain ...

Heterocyclic Communications, 2000

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Biochemistry, 1991

heteronuclear N M R techniques have been used to make sequential 'H and I5N resonance assignments... more heteronuclear N M R techniques have been used to make sequential 'H and I5N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18 300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D lsN/'H nuclear Overhauserheteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 'H-IH through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows N O E cross peaks to be detected between N H protons even when their 'H chemical shifts are degenerate as long as the amide ISN chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+l NOES define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR-methotrexateeNADPH complex J . Biol. G e m . 257, 13650-136621, while the NMRdetermined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-1 10 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of @-sheet structure were characterized in the binary complex by strong aCHi-NHi+, NOES and by slowly exchanging amide protons. In addition, several long-range NOES were identified linking together these stretches to form a 6-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR-methotrexate-NADPH complex, which contains an eight-stranded 6-sheet, indicating that the main body of the @-sheet is preserved in the binary complex in solution.

Chemistry, an Asian journal, 2015

Functionalized oligomeric organic compounds with well-defined β-proline scaffold have been synthe... more Functionalized oligomeric organic compounds with well-defined β-proline scaffold have been synthesized by a cycloadditive oligomerization approach in racemic and enantiopure forms. The structure of the novel β-peptides was investigated by NMR spectroscopic and X-ray methods determining the conformational shapes of the β-proline oligomers in solution and solid states. The main structural elements subject to conformational switches are β-peptide bonds between 5-arylpyrrolidine-2-carboxylic acid units existing in Z/E configurations. The whole library of short β-peptides and intermediate acrylamides has been tested on antiproliferative activity towards the hormone-refractory prostate cancer cell line PC-3 revealing several oligomeric compounds with low micromolar and submicromolar activities. Bromine-substituted dimeric and trimeric acrylamides induced caspase-dependent apoptosis of PC-3 cells through cell-cycle arrest and mitochondrial damage.

Biochemistry, 1994

Two-and three-dimensional (2D and 3D) N M R techniques have been used to assign the signals from ... more Two-and three-dimensional (2D and 3D) N M R techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (Mr 18 300) in its 1:l complex with the antibacterial drug trimethoprim. A sample of uniformly ISN-labeled protein was examined using 3D 15N/IH experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOES between trimethoprim and protein protons and four intramolecular NOES in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15Nand 13C-labeled trimethoprim molecules ([ 1 ,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound tounlabeled protein. The ligand-

Journal of Biomolecular NMR, 1999

A new method is proposed for docking ligands into proteins in cases where an NMR-determined solut... more A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein-ligand, ligand-ligand, and protein-protein restraints for residues in or near to the binding site, combined with a set of protein-protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in 1 H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase.trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase.methotrexate reference structure.

Biochemistry, 1995

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlati... more Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1: 1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-0 position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of lo3 more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 1 1 intramolecular NOES were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tl = -153" f 4" (C4-C5-C7-C1') and t2 = 53" k 4" (C5-C7-C1'-C2'):

Journal of Biomolecular NMR, 2005