Biochemistry (Moscow) (v.81, #13)

Investigation of ribosomes using molecular dynamics simulation methods by G. I. Makarov; T. M. Makarova; N. V. Sumbatyan; A. A. Bogdanov (1579-1588).
The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.
Keywords: ribosome; molecular dynamics simulations; allosteric signal transmission

Investigation of structure of the ribosomal L12/P stalk by I. V. Mitroshin; M. B. Garber; A. G. Gabdulkhakov (1589-1601).
This review contains recent data on the structure of the functionally important ribosomal domain, L12/P stalk, of the large ribosomal subunit. It is the most mobile site of the ribosome; it has been found in ribosomes of all living cells, and it is involved in the interaction between ribosomes and translation factors. The difference between the structures of the ribosomal proteins forming this protuberance (despite their general resemblance) determines the specificity of interaction between eukaryotic and prokaryotic ribosomes and the respective protein factors of translation. In this review, works on the structures of ribosomal proteins forming the L12/P-stalk in bacteria, archaea, and eukaryotes and data on structural aspects of interactions between these proteins and rRNA are described in detail.
Keywords: ribosome; stalk; ribosomal proteins

G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding–unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA–RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.
Keywords: G-quadruplex thermodynamics and kinetics; multiquadruplexes; G-quadruplex-specific ligands and proteins; RNA G-quadruplexes; hybrid DNA–RNA G-quadruplexes; telomeric G-quadruplexes; G-quadruplexes in oncogene promoters

Genomics and biochemistry of Saccharomyces cerevisiae wine yeast strains by M. A. Eldarov; S. A. Kishkovskaia; T. N. Tanaschuk; A. V. Mardanov (1650-1668).
Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term “unconscious” selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using “classical” and modern techniques for improving wine-making technology.
Keywords: wine; yeast strains; diversity; artificial selection; stress response; evolution; comparative genomics; metagenomics; microbial interactions; flor yeast; adhesion; aromatic compounds; gene regulation; strain improvement

ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA–DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia.
Keywords: ATM; Ataxia Telangiectasia mutated; DNA damage; DNA single-strand breaks; DNA double-strand breaks; R-loops

This review discusses and summarizes the results of molecular and cellular investigations of myosin light chain kinase (MLCK, MYLK1), the key regulator of cell motility. The structure and regulation of a complex mylk1 gene and the domain organization of its products is presented. The interactions of the mylk1 gene protein products with other proteins and posttranslational modifications of the mylk1 gene protein products are reviewed, which altogether might determine the role and place of MLCK in physiological and pathological reactions of cells and entire organisms. Translational potential of MLCK as a drug target is evaluated.
Keywords: myosin light chain kinase; KRP/telokin; mylk1 gene organization and regulation; posttranslational modifications; protein–protein interactions; MLCK-dependent cellular reactions; MLCK in pathology

Control of myofibroblast differentiation and function by cytoskeletal signaling by N. Sandbo; L. V. Smolyaninova; S. N. Orlov; N. O. Dulin (1698-1708).
The cytoskeleton consists of three distinct types of protein polymer structures–microfilaments, intermediate filaments, and microtubules; each serves distinct roles in controlling cell shape, division, contraction, migration, and other processes. In addition to mechanical functions, the cytoskeleton accepts signals from outside the cell and triggers additional signals to extracellular matrix, thus playing a key role in signal transduction from extracellular stimuli through dynamic recruitment of diverse intermediates of the intracellular signaling machinery. This review summarizes current knowledge about the role of cytoskeleton in the signaling mechanism of fibroblast-to-myofibroblast differentiation–a process characterized by accumulation of contractile proteins and secretion of extracellular matrix proteins, and being critical for normal wound healing in response to tissue injury as well as for aberrant tissue remodeling in fibrotic disorders. Specifically, we discuss control of serum response factor and Hippo signaling pathways by actin and microtubule dynamics as well as regulation of collagen synthesis by intermediate filaments.
Keywords: myofibroblast; actin stress fibers; transcription; microtubules; differentiation; intermediate filaments; collagen synthesis

Role of proteolytic enzymes in the interaction of phytopathogenic microorganisms with plants by T. A. Valueva; B. Ts. Zaichik; N. N. Kudryavtseva (1709-1718).
Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.
Keywords: phytopathogenic microorganisms; proteolytic enzymes; pathogenesis-related proteins

Rpf proteins are the factors of reactivation of the dormant forms of actinobacteria by V. D. Nikitushkin; G. R. Demina; A. S. Kaprelyants (1719-1734).
As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its par-ticipation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis – an infectious disease that affects one third of the World’s population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine–in diagnostics and in creation of antituberculosis subunit vaccines.
Keywords: Resuscitation promoting factor (Rpf); actinobacteria; dormancy; tuberculosis; lytic enzymes; bacterial cell wall; muropeptides; MALDI; vaccines; diagnostics

In addition to reversible O2 binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO2 and MbO2 are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E 0, 200-600 mV) and high stability constants, such as [Cu(phen)2]2+, [Cu(dmphen)2]2+, and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu2+, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E 0≤ 100-150 mV) react with HbO2 and MbO2 through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO2 and MbO2 by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PPi proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO2 with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO2 and cytochrome b 5 via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO2 and MbO2 transformation into met-forms in the presence of small amounts of metal ions or complexes (catalysis), which, until recently, had been demonstrated only for copper compounds with intermediate redox potentials. The main contribution to the reaction rate comes from copper binding to the “inner” histidines, His97 (0.66 nm from the heme) that forms a hydrogen bond with the heme propionate COO group, and the distal His64. The affinity of both histidines for copper is much lower than that of the surface histidines residues, and they are inaccessible for modification with chemical reagents. However, it was found recently that the high-potential Fe(3) complex, potassium ferricyanide (400 mV), at a 5 to 20% of molar protein concentration can be an efficient catalyst of MbO2 oxidation into metMb. The catalytic process includes binding of ferrocyanide anion in the region of the His119 residue due to the presence there of a large positive local electrostatic potential and existence of a “pocket” formed by Lys16, Ala19, Asp20, and Arg118 that is sufficient to accommodate [Fe(CN)6]4–. Fast, proton-assisted reoxidation of the bound ferrocyanide by oxygen (which is required for completion of the catalytic cycle), unlike slow [Fe(CN)6]4– oxidation in solution, is provided by the optimal location of neighboring protonated His113 and His116, as it occurs in the enzyme active site.
Keywords: hemoglobin; myoglobin; leghemoglobin; copper and iron salts and complexes; redox reactions; electrostatic potential

Immuno-PCR: achievements and perspectives by D. Y. Ryazantsev; D. V. Voronina; S. K. Zavriev (1754-1770).
The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of “visualization” of antigen–antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.
Keywords: immuno-PCR; quantitative PCR; antibody; antigen; DNA–antibody conjugate

Biomedical applications of multifunctional gold-based nanocomposites by L. A. Dykman; N. G. Khlebtsov (1771-1789).
Active application of gold nanoparticles for various diagnostic and therapeutic purposes started in recent decades due to the emergence of new data on their unique optical and physicochemical properties. In addition to colloidal gold conjugates, growth in the number of publications devoted to the synthesis and application of multifunctional nanocomposites has occurred in recent years. This review considers the application in biomedicine of multifunctional nanoparticles that can be produced in three different ways. The first method involves design of composite nanostructures with various components intended for either diagnostic or therapeutic functions. The second approach uses new bioconjugation techniques that allow functionalization of gold nanoparticles with various molecules, thus combining diagnostic and therapeutic functions in one medical procedure. Finally, the third method for production of multifunctional nanoparticles combines the first two approaches, in which a composite nanoparticle is additionally functionalized by molecules having different properties.
Keywords: biomedicine; colloidal gold; nanocomposites; multifunctionality

Erratum: “Regulation of zygotic genome and cellular pluripotency” by D. V. Onichtchouk; A. S. Voronina (1790-1790).