Biochemistry (Moscow) (v.82, #11)

Modern approaches for identification of modified nucleotides in RNA by J. A. Filippova; D. V. Semenov; E. S. Juravlev; A. B. Komissarov; V. A. Richter; G. A. Stepanov (1217-1233).
This review considers approaches for detection of modified monomers in the RNA structure of living organisms. Recently, some data on dynamic alterations in the pool of modifications of the key RNA species that depend on external factors affecting the cells and physiological conditions of the whole organism have been accumulated. The recent studies have presented experimental data on relationship between the mechanisms of formation of modified/minor nucleotides of RNA in mammalian cells and the development of various pathologies. The development of novel methods for detection of chemical modifications of RNA nucleotides in the cells of living organisms and accumulation of knowledge on the contribution of modified monomers to metabolism and functioning of individual RNA species establish the basis for creation of novel diagnostic and therapeutic approaches. This review includes a short description of routine methods for determination of modified nucleotides in RNA and considers in detail modern approaches that enable not only detection but also quantitative assessment of the modification level of various nucleotides in individual RNA species.
Keywords: post-transcriptional RNA modifications; chemical modifications of nucleotides; minor nucleotides; RNA editing; reverse transcription; mass spectrometry; transcriptome-wide high-throughput sequencing

The power and limitations of influenza virus hemagglutinin assays by N. B. Ustinov; E. G. Zavyalova; I. G. Smirnova; A. M. Kopylov (1234-1248).
Influenza virus hemagglutinins (HAs) are surface proteins that bind to sialic acid residues at the host cell surface and ensure further virus internalization. Development of methods for the inhibition of these processes drives progress in the design of new antiviral drugs. The state of the isolated HA (i.e. combining tertiary structure and extent of oligomerization) is defined by multiple factors, like the HA source and purification method, posttranslational modifications, pH, etc. The HA state affects HA functional activity and significantly impacts the results of numerous HA assays. In this review, we analyze the power and limitations of currently used HA assays regarding the state of HA.
Keywords: influenza virus; surface antigens; influenza hemagglutinin; inhibitors of hemagglutination; monoclonal antibodies; ELISA

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen by D. A. Cherepanov; G. E. Milanovsky; A. A. Petrova; A. N. Tikhonov; A. Yu. Semenov (1249-1268).
This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A1A/A1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters FX, FA, and FB have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.
Keywords: photosystem I; electron transport; kinetic modeling of electron transfer; exogenous electron acceptors; interaction with oxygen

Temperature dependence of tryptophan fluorescence lifetime in aqueous glycerol and trehalose solutions by V. V. Gorokhov; P. P. Knox; B. N. Korvatovskiy; N. Kh. Seifullina; S. N. Goryachev; V. Z. Paschenko (1269-1275).
The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5–417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from –60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wave-length B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.
Keywords: tryptophans; rotamers; fluorescence; temperature dependence; fluorescence decay kinetics

Antiinflammatory effect of rosiglitazone via modulation of mRNA stability of interleukin 10 and cyclooxygenase 2 in astrocytes by E. V. Pankevich; A. A. Astakhova; D. V. Chistyakov; M. G. Sergeeva (1276-1284).
Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E2, PGE2) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.
Keywords: rosiglitazone; inflammatory response; astrocytes; mRNA stability

Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system by T. M. Grunina; A. V. Demidenko; A. M. Lyaschuk; M. S. Poponova; Z. M. Galushkina; L. A. Soboleva; S. A. Cherepushkin; N. B. Polyakov; D. A. Grumov; A. I. Solovyev; V. G. Zhukhovitsky; I. S. Boksha; M. E. Subbotina; A. V. Gromov; V. G. Lunin; A. S. Karyagina (1285-1294).
Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.
Keywords: erythropoietin; heterologous expression; Escherichia coli

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.
Keywords: hydroxypyruvate reductase; methanotrophs; serine cycle of carbon assimilation

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments by E. V. Sidorin; V. A. Khomenko; N. Yu. Kim; P. S. Dmitrenok; A. M. Stenkova; O. D. Novikova; T. F. Solov’eva (1304-1313).
Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.
Keywords: Yersinia pseudotuberculosis ; self-organization and aggregation of outer membrane protein; recombinant porin OmpF; dynamic light scattering

Salusin-α attenuates inflammatory responses in vascular endothelial cells by Maryam Esfahani; Masoud Saidijam; Mohammad Taghi Goodarzi; Ahmad Movahedian; Rezvan Najafi (1314-1323).
Atherosclerosis accounts for numerous cardiovascular diseases, and cytokines have a critical role in acceleration or suppression of disease. Salusin-α presents a new class of bioactive peptides that can have anti-atherogenic properties. Therefore, the effects of salusin-α on the expression of some pro- and anti-inflammatory cytokines and on TNF-α-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) were examined. The involvement of the NF-κB pathway in effects of salusin-α in HUVECs was checked using Bay 11-7082 as an NF-κB inhibitor. The mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, and IL-18 and anti-inflammatory cytokine IL-1Ra was assessed by real-time PCR. The protein levels of cytokines were measured by the ELISA method. Salusin-α suppressed both mRNA and protein expression of pro-inflammatory cytokines and induced mRNA and protein expression of IL-1Ra in HUVECs. Salusin-α suppressed TNF-α-induced inflammatory responses in HUVECs. The down-regulatory or up-regulatory effects of salusin-α on expression of cytokines could not be influenced by Bay 11-7082 pretreatment. Our findings indicate anti-inflammatory effects of salusin-α and suggest a novel peptide-based therapeutic strategy for atherosclerosis.
Keywords: atherosclerosis; salusin-α; inflammation; endothelial cells; cytokines

Factors beyond enolase 2 and mitochondrial lysyl-tRNA synthetase precursor are required for tRNA import into yeast mitochondria by M. V. Baleva; M. Meyer; N. Entelis; I. Tarassov; P. Kamenski; B. Masquida (1324-1335).
In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.
Keywords: RNA mitochondrial import; RNA chaperone; tRNA–enolase interaction; enolase; moonlighting protein

Cathelicidin LL37 promotes epithelial and smooth-muscle-like differentiation of adipose-derived stem cells through the Wnt/β-catenin and NF-κB pathways by Yongwei Li; Zhengfei Shan; Bin Yang; Diandong Yang; Changping Men; Yuanshan Cui; Jitao Wu (1336-1345).
Ureter reconstruction is a difficult procedure in urology. Adipose-derived stem cells (ADSCs), along with multipotency and self-renewal capacity, are a preferred choice for tissue engineering-based ureteral reconstruction. We explored the synergic role of cathelicidin LL37 (LL37) in epithelial and smooth-muscle-like differentiation. ADSCs were separated from adipose tissues of mouse and characterized by flow cytometry. The ADSCs were then stably transfected with pGC-FU-GFP (pGC) or pGC containing full-length LL37 (pGC-LL37), respectively. Cell viability and apoptosis were respectively estimated in the stably transfected cells and non-transfected cells. Then, qRT-PCR and Western blot analysis were used for determinations of epithelial marker expressions after induction by all-trans retinoic acid as well as smooth-muscle-like marker expressions after induction by transforming growth factor-β1. Then, possibly involved signaling pathways and extracellular expression of LL37 were detected. Cell viability and apoptosis were not changed after LL37 overexpression. Expression levels of epithelial and smooth-muscle-like markers were significantly upregulated by LL37 overexpression. Moreover, expressions of key kinases involved in the Wnt/β-catenin pathway as well as epithelial marker were upregulated by the LL37 overexpression, while it was reversed by Wnt/β-catenin inhibitor. Likewise, expressions of key kinases involved in the nuclear factor κB (NF-κB) pathway as well as smooth-muscle-like markers were upregulated by LL37 overexpression, which was reversed by NF-κB inhibitor. LL37 was found in the culture medium. LL37, which could be released into the medium, had no impact on cell proliferation and apoptosis of ADSCs. However, LL37 promoted epithelial and smooth-muscle-like differentiation through activating the Wnt/β-catenin and NF-κB pathways, respectively.
Keywords: tissue engineering-based ureteral reconstruction; ADSCs; cathelicidin LL37; epithelial and smooth-muscle-like differentiation; Wnt/β-catenin and NF-κB pathways

Cell-free expression, purification, and characterization of the functional β2-adrenergic receptor for multianalyte detection of β-agonists by Jian Wang; Yuan Liu; Junhua Zhang; Zhengzheng Han; Wei Wang; Yang Liu; Dong Wei; Wei Huang (1346-1353).
Large-scale expression of β2-adrenergic receptor (β2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have many critical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β2-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β2-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β2-AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC50 values were 45.99, 60.38, and 78.02 μg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.
Keywords: cell-free expression; β2-adrenergic receptor; codon optimization; purification; β-agonist; receptor-based assay

Spectral-kinetic analysis of recombination reaction of heme centers of bd-type quinol oxidase from Escherichia coli with carbon monoxide by S. A. Siletsky; A. V. Dyuba; D. A. Elkina; M. V. Monakhova; V. B. Borisov (1354-1366).
Recombination of the isolated, fully reduced bd-type quinol oxidase from Escherichia coli with carbon monoxide was studied by pulsed absorption spectrophotometry with microsecond time resolution. Analysis of the kinetic phases of recombination was carried out using the global analysis of multiwavelength kinetic data (“Global fitting”). It was found that the unresolved photodissociation of CO is followed by a stepwise (with four phases) recombination with characteristic times (τ) of about 20 μs, 250 μs, 1.1 ms, and 24 ms. The 20-μs phase most likely reflects bimolecular recombination of CO with heme d. Two subsequent kinetic transitions, with τ ~ 250 μs and 1.1 ms, were resolved for the first time. It is assumed that the 250-μs phase is heterogeneous and includes two different processes: recombination of CO with ~7% of heme b 595 and transition of heme d from a pentacoordinate to a transient hexacoordinate state in this enzyme population. The 24-ms transition probably reflects a return of heme d to the pentacoordinate state in the same protein fraction. The 1.1-ms phase can be explained by recombination of CO with ~15% of heme b 558. Possible models of interaction of CO with different heme centers are discussed.
Keywords: axial ligand; heme; cytochrome; respiratory chain; terminal oxidase; laser spectroscopy; flash photolysis; recombination; carbon monoxide

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine and N-docosahexaenoyl dopamine examined in neuronal precursors derived from human pluripotent stem cells by E. V. Novosadova; E. L. Arsenyeva; E. S. Manuilova; L. G. Khaspekov; M. Yu. Bobrov; V. V. Bezuglov; S. N. Illarioshkin; I. A. Grivennikov (1367-1372).
Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1–10 μM range. However, both agents at 10 μM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.
Keywords: endocannabinoids; N-arachidonoyl dopamine; N-docosahexaenoyl dopamine; neuroprotection; induced pluripotent stem cells; neural precursors; oxidative stress

The mitochondrial genome of the moss Brachythecium rivulare (Hypnales, Brachytheciaceae) by D. V. Goryunov; M. D. Logacheva; M. S. Ignatov; I. A. Milyutina; A. V. Fedorova; A. V. Troitsky (1373-1379).
The mitochondrial genome of the pleurocarpous moss Brachythecium rivulare has been sequenced and annotated. The genome consists of 104,460 base pairs and has approximately the same gene set and organization as other bryophyte mitogenomes. Whole mitochondrial genome comparison between B. rivulare and Physcomitrella patens, Tetraphis pellucida, Anomodon rugelii, and Anomodon attenuatus was performed. The primary cause of bryophyte mitochondrial gene length variation was found to be numerous indels in the introns. Bryophyte mitochondrial gene conservation level was estimated, and it was in a good congruence with the overall phylogeny of bryophytes with the percentage of mitogenome similarity being proportional to the age estimated by phylochronologic analysis. Annotation discrepancies in the analyzed mitogenome sequences were identified. The simple sequence repeat (SSR) content was evaluated, and candidate sites of RNA editing were predicted in the B. rivulare mitochondrial genome.
Keywords: mitochondrial genome; Brachythecium rivulare ; Bryophyta; mosses

Role of microRNA in development of instability of atherosclerotic plaques by I. A. Koroleva; M. S. Nazarenko; A. N. Kucher (1380-1390).
MicroRNAs are small noncoding single-stranded RNAs that regulate gene expression. Today, we see an increasing number of studies highlighting the important role of microRNAs in the development and progression of cardiovascular diseases caused by atherosclerotic lesions of arteries. We review the available scientific data on association of the expression of these biomolecules with instability of atherosclerotic plaques in animal models and humans. We made special emphasis on miR-21, -100, -127, -133, -143/145, -221/222, and -494 because they were analyzed in more than one study. We discuss the possibility of microRNAs using in the diagnosis and therapy of atherosclerosis and its complications.
Keywords: atherosclerosis; instability of human atherosclerotic plaques; microRNA

It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared.
Keywords: DNA damage; poly(ADP-ribosyl)ation; poly(ADP-ribose) polymerase; genome stability; stationary phase aging; replicative aging; cell proliferation