Biochemistry (Moscow) (v.83, #3)

The Role of Transposable Elements in Emergence of Metazoa by R. N. Mustafin; E. K. Khusnutdinova (185-199).
Systems initially emerged for protecting genomes against insertions of transposable elements and represented by mechanisms of splicing regulation, RNA–interference, and epigenetic factors have played a key role in the evolution of animals. Many studies have shown inherited transpositions of mobile elements in embryogenesis and preservation of their activities in certain tissues of adult organisms. It was supposed that on the emergence of Metazoa the self–regulation mechanisms of transposons related with the gene networks controlling their activity could be involved in intercellular cell coordination in the cascade of successive divisions with differentiated gene expression for generation of tissues and organs. It was supposed that during evolution species–specific features of transposons in the genomes of eukaryotes could form the basis for creation of dynamically related complexes of systems for epigenetic regulation of gene expression. These complexes could be produced due to the influence of noncoding transposon–derived RNAs on DNA methylation, histone modifications, and processing of alternative splicing variants, whereas the mobile elements themselves could be directly involved in the regulation of gene expression in cis and in trans. Transposons are widely distributed in the genomes of eukaryotes; therefore, their activation can change the expression of specific genes. In turn, this can play an important role in cell differentiation during ontogenesis. It is supposed that transposons can form a species–specific pattern for control of gene expression, and that some variants of this pattern can be favorable for adaptation. The presented data indicate the possible influence of transposons in karyotype formation. It is supposed that transposon localization relative to one another and to protein–coding genes can influence the species–specific epigenetic regulation of ontogenesis.
Keywords: alternative splicing; differentiation; noncoding RNAs; ontogenesis; RNA interference; transposons; evolution

Plasticity of Human THP–1 Cell Phagocytic Activity during Macrophagic Differentiation by A. V. Kurynina; M. V. Erokhina; O. A. Makarevich; V. Yu. Sysoeva; L. N. Lepekha; S. A. Kuznetsov; G. E. Onishchenko (200-214).
Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism’s defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP–1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)–induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5–2.0–fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin-and Fc–covered beads were high; however, the intensity of ingestion of mannan–conjugated beads via mannose receptors increased 2.5–3.0–fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.
Keywords: proinflammatory macrophages; antiinflammatory macrophages; macrophage plasticity; phagocytosis; cell differentiation; functionally mature phenotype

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O–glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.
Keywords: protein glycosylation; glycoengineering; pharmaceutical proteins; plant expression systems; glycosyltransferases

Accumulation of mutations in mitochondrial DNA leads to the development of severe, currently untreatable diseases. The contribution of these mutations to aging and progress of neurodegenerative diseases is actively studied. Elucidation of DNA repair mechanisms in mitochondria is necessary for both developing approaches to the therapy of diseases caused by mitochondrial mutations and understanding specific features of mitochondrial genome functioning. Mitochondrial DNA repair systems have become a subject of extensive studies only in the last decade due to development of molecular biology methods. DNA repair systems of mammalian mitochondria appear to be more diverse and effective than it had been thought earlier. Even now, one may speak about the existence of mitochondrial mechanisms for the repair of single–and double–stranded DNA lesions. Homologous recombination also takes place in mammalian mitochondria, although its functional significance and molecular mechanisms remain obscure. In this review, I describe DNA repair systems in mammalian mitochondria, such as base excision repair (BER) and microhomology–mediated end joining (MMEJ) and discuss a possibility of existence of mitochondrial DNA repair mechanisms otherwise typical for the nuclear DNA, e.g., nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination, and classical non–homologous end joining (NHEJ). I also present data on the mechanisms for coordination of the nuclear and mitochondrial DNA repair systems that have been actively studied recently.
Keywords: mitochondria; mitochondrial DNA; mutations; DNA repair; BER; MMEJ; homologous recombination; PARP1

Identification of Single Amino Acid Substitutions in Proteogenomics by S. A. Moshkovskii; M. V. Ivanov; K. G. Kuznetsova; M. V. Gorshkov (250-258).
An important aim of proteogenomics, which combines data of high throughput nucleic acid and protein analysis, is to reliably identify single amino acid substitutions representing a main type of coding genome variants. Exact knowledge of deviations from the consensus genome can be utilized in several biomedical fields, such as studies of expression of mutated proteins in cancer, deciphering heterozygosity mechanisms, identification of neoantigens in anticancer vaccine production, search for RNA editing sites at the level of the proteome, etc. Generation of this new knowledge requires processing of large data arrays from high–resolution mass spectrometry, where information on single–point protein variation is often difficult to extract. Accordingly, a significant problem in proteogenomic analysis is the presence of high levels of false positive results for variant–containing peptides in the produced results. Here we review recently suggested approaches of high quality proteomics data processing that may provide more reliable identification of single amino acid substitutions, especially contrary to residue modifications occurring in vitro and in vivo. Optimized methods for assessment of false discovery rate save instrumental and computational time spent for validation of interesting findings of amino acid polymorphism by orthogonal methods.
Keywords: proteogenomics; proteomics; mass spectrometry; single amino acid polymorphism; single nucleotide polymorphism

Expression of Soluble Active Interferon αA in Escherichia coli Periplasm by Fusion with Thermostable Lichenase Using the Domain Insertion Approach by A. A. Tyurin; K. V. Kabardaeva; O. N. Mustafaev; O. S. Pavlenko; N. S. Sadovskaya; V. S. Fadeev; E. A. Zvonova; I. V. Goldenkova-Pavlova (259-269).
A recombinant DNA in which the interferon αA (IFN–αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN–αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN–αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.
Keywords: interferon αA; thermostable lichenase; domain insertion; expression; solubility; biological activity

Possible Role of Escherichia coli Protein YbgI by O. V. Sergeeva; D. O. Bredikhin; M. V. Nesterchuk; M. V. Serebryakova; P. V. Sergiev; O. A. Dontsova (270-280).
Proteins containing the NIF3 domain are highly conserved and are found in bacteria, eukaryotes, and archaea. YbgI is an Escherichia coli protein whose gene is conserved among bacteria. The structure of YbgI is known; however, the function of this protein in cells remains obscure. Our studies of E. coli cells with deleted ybgI gene suggest that YbgI is involved in formation of the bacterial cell wall.
Keywords: Escherichia coli ; cell stress; bacterial cell wall

Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System by M. V. Monakhova; A. I. Penkina; A. V. Pavlova; A. M. Lyaschuk; V. V. Kucherenko; A. V. Alexeevski; V. G. Lunin; P. Friedhoff; G. Klug; T. S. Oretskaya; E. A. Kubareva (281-293).
We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.
Keywords: DNA repair; mismatch; MutL protein

Glutamic Acid Signal Synchronizes Protein Synthesis Kinetics in Hepatocytes from Old Rats for the Following Several Days. Cell Metabolism Memory by V. Y. Brodsky; L. A. Malchenko; D. S. Lazarev; N. N. Butorina; T. K. Dubovaya; N. D. Zvezdina (294-298).
The kinetics of protein synthesis was investigated in primary cultures of hepatocytes from old rats in serum–free medium. The rats were fed mixed fodder supplemented with glutamic acid and then transferred to a regular mixed fodder. The amplitude of protein synthesis rhythm in hepatocytes isolated from these rats increased on average 2–fold in comparison with the rats not receiving glutamic acid supplement. Based on this indicator reflecting the degree of cell–cell interactions, the cells from old rats were not different from those of young rats. The effect was preserved for 3–4 days. These results are discussed in connection with our previous data on preservation of the effect of single administration of gangliosides, noradrenaline, serotonin, and other synchronizers on various cell populations. In contrast to the other investigated factors, glutamic acid is capable of penetrating the blood–brain barrier, which makes its effect possible not only in the case of hepatocytes and other non–brain cells, but also in neurons.
Keywords: protein synthesis kinetics; protein synthesis rhythm; glutamic acid; aging; biochemistry of direct cell–cell interactions; cell memory