Biochemistry (Moscow) (v.78, #1)

Nucleic acids in exosomes: Disease markers and intercellular communication molecules by O. N. Gusachenko; M. A. Zenkova; V. V. Vlassov (1-7).
The term “exosomes” is currently used to describe specific vesicular structures of endosomal origin produced by the majority of eukaryotic cells. These natural vesicles have been under study for more than two decades. Nevertheless, a real splash of scientific interest in studies on exosomes took place only during recent years, when the concept of the role and functions of exosomes in multicellular organisms was essentially reconsidered. The major role in this was played by the discovery of exosomal mRNA and miRNA in 2007, which stimulated the idea of regulatory and communicative role of exosomes in the organism and also encouraged considering exosomes and other vesicles as potential biomarkers. The present review summarizes the up to date knowledge on the composition and probable physiological functions of nucleic acids released by different cells as components of exosomes. We also touch upon the problem of using these data in clinical diagnosis.
Keywords: exosomes; microvesicles; extracellular nucleic acids; biomarkers; intercellular communication; miRNA; mRNA

Insulin resistance and adipogenesis: Role of transcription and secreted factors by D. N. Penkov; A. D. Egorov; M. N. Mozgovaya; V. A. Tkachuk (8-18).
Insulin stimulates carbohydrate uptake by cells and induces their conversion into lipids as a more efficient form of energy storage. Insulin resistance is associated with a decrease in glucose uptake by muscle and adipose cells and also with a decrease in glycogen synthesis on retention of glucose synthesis by liver cells. Disorders in the insulin signaling cascade on development of insulin resistance can be caused by both changes in functioning of transcriptional factors and in the secretion profile of hormone-like substances. Diacylglycerols and ceramides responsible for activation of some kinases and phosphatases can directly trigger these changes in muscle and liver cells. In adipose tissue, insulin mainly stimulates adipogenesis (adipocyte differentiation) and lipogenesis (lipid accumulation in the cells). Thus, studies on the action mechanisms of factors influencing adipogenesis can be of help for understanding the molecular mechanisms of insulin resistance.
Keywords: insulin resistance; insulin; adipogenesis; adipose tissue; Prep1

Keap1/Nrf2/ARE redox-sensitive signaling system as a pharmacological target by N. K. Zenkov; E. B. Menshchikova; V. O. Tkachev (19-36).
The redox-sensitive signaling system Keap1/Nrf2/ARE plays a key role in maintenance of cellular homeostasis under stress, inflammatory, carcinogenic, and proapoptotic conditions, which allows us to consider it as a pharmacological target. Here we review the basic regulatory mechanisms of the Keap1/Nrf2/ARE system, key targets for pharmacological intervention, and interconnection of this system with other redox-sensitive transcriptional factors. We also discuss the range of currently available pharmaceuticals. Finally, we promote “indirect” antioxidants as a promising strategy for prevention and treatment of wide range of diseases associated with oxidative stress.
Keywords: Nrf2; antioxidant-responsive element (ARE); transcription factors; pharmaceuticals

Regulatory functions of microtubules by J. M. Vasiliev; V. I. Samoylov (37-40).
This mini-review summarizes literature and original data about the role of microtubules in interphase animal cells. Recent data have shown that functioning of microtubules is essential for such diverse phenomena as directional cell movements, distribution of organelles in the cytoplasm, and neuronal memory in the central nervous system. It is suggested that microtubules can act as an important regulatory system in eukaryotic cells. Possible mechanisms of these functions are discussed.
Keywords: microtubules; cell movements; Golgi apparatus; neurons; memory; memory fading

Hydrolysis of DNA catalyzed by wheat endonucleases WEN1 and WEN2 is pronouncedly processive. A correlation has been revealed between appearance of new products of DNA hydrolysis with different length and conformational changes in the enzymes. The first conformational conversion of the endonucleases is associated with appearance of large fragments of DNA hydrolysis with length longer than 500 bp, and the second conversion is associated with formation of oligonucleotides with length of 120–140 bp, and the third conversion is associated with formation of short oligonucleotides and mononucleotides. Formation of the DNA-enzyme complex is accompanied by appearance of fluorescence at λ = 410–440 nm. The intensity, positions, and numbers of maximums of the fluorescence spectra of DNA-WEN1 and DNA-WEN2 complexes are different and depend on the methylation status of the DNA and on the presence of Mg2+. The endonucleases hydrolyze DNA by two mechanisms: one is metal-independent, and the other depends on one or two Mg2+ ions. One Mg2+ ion is located inside the catalytic center of WEN1, whereas the WEN2 center contains two Mg2+ ions. The first (sitespecific) stage of DNA hydrolysis does not depend on Mg2+. Mg2+ ions evoke changes in the site specificity of the endonuclease action (WEN1) and abolish their ability to recognize the methylation status of DNA. Products of DNA hydrolysis by endonucleases WEN1 and WEN2 in the presence of Mg2+ are similar in length (120–140 bp). The endonucleases have at least two centers (domains) — catalytic and substrate-binding. Two histidine and apparently two lysine plus two dicarboxylic amino acid residues are present inside the catalytic center of WEN1. The catalytic center of WEN2 contains at least one histidine residue and apparently two residues of aspartic or glutamic acid, which are involved in coordination of the metal ions. The catalytic centers of WEN1 and WEN2 seem to be formed, respectively, by HD/E(D/EK)KH and HD/ED/E amino acid residues. The site-specificity of the endonuclease action is due to the DNA-binding domain. This domain contains dicarboxylic amino acid residues, which seem to be responsible for sensitivity of the enzymes to the methylation status of DNA. The hydroxyl groups of tyrosine residues in the enzymes also seem to contribute to recognizing methylated bases in DNA.
Keywords: endonucleases; DNA hydrolysis kinetics; catalytic mechanism; DNA methylation; wheat; plants

Initiation of translation of genomic RNA (gRNA) of hepatitis C virus (HCV) is provided by a highly structured fragment in its 5′-untranslated region, the so-called Internal Ribosome Entry Site (IRES). In this work, the exposed NH2-groups of proteins in the 40S subunit of the human ribosome and in its binary complexes with RNA transcripts corresponding to the full-size HCV IRES or its fragments were probed using the N-hydroxysuccinimide derivative of the fluorescent dye Cy3. Comparison of efficiencies of modification of ribosomal proteins in free subunits and in their binary complexes with the RNA transcripts revealed ribosomal proteins involved in the HCV IRES binding. It was found that binding of the 40S subunits with the RNA transcript corresponding to full-size HCV IRES results in a decrease in modification levels of ribosomal protein (rp) S27 and, to a lesser extent of rpS10; also, a noticeable decrease in the efficiency of labeling of proteins RACK1/S2/S3a was observed. When a fragment of HCV IRES containing the initial part of the open reading frame (ORF) of the viral gRNA was deleted, the level of rpS10 modification became the same as in free subunits, whereas the levels of modification of rpS27 and the RACK1/S2/S3a group remained virtually unchanged compared to those observed in the complex of 40S subunit with the full-size HCV IRES. Binding of 40S subunits to a fragment of the HCV IRES lacking an ORF and domain II increased the modification level of the RACK1/S2/S3a proteins, while the efficiencies of labeling of rpS10 and rpS27 remained the same as upon the deletion of the ORF fragment. Comparison of these results with known structural and biochemical data on the organization of 40S subunit and the location of the HCV IRES on it revealed structural elements of the IRES contacting exposed lysine residues of the above-mentioned ribosomal proteins. Thus, it was found that the majority of exposed lysine residues of rpS27 are involved in the binding of the HCV IRES region formed by the junction of subdomains IIIa, IIIb, and IIIc with the central stalk of domain III, and that several lysine residues of rpS10 participate in the binding of the HCV IRES region corresponding to the initial part of the ORF of the viral gRNA. In addition, we concluded that lysine residues of rpS3a are involved in the binding of domains II and III of HCV IRES.
Keywords: HCV IRES; human ribosome; fluorescent labeling; ribosomal protein S3a; ribosomal protein S10; ribosomal protein S27

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor by A. Yu. Khmelnitskiy; R. A. Khatypov; A. M. Khristin; M. M. Leonova; L. G. Vasilieva; V. A. Shuvalov (60-67).
Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll BA at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P+H A state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P+B A HA and P+BAH A states. The data give grounds for assuming that the value of the energy difference between the states P*, P+B A HA, and P+BAH A at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll BA is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P+B A state with respect to P*.
Keywords: photosynthesis; charge separation; reaction center; femtosecond spectroscopy; electron transfer

Plastoquinone or its methylated form covalently bound to the membrane-penetrating decyltriphenylphosphonium cation (SkQ1 and SkQ3) retarded the senescence of Arabidopsis thaliana rosette leaves and their death. Dodecyltriphenylphosphonium (C12TPP+) had a similar effect. Much like SkQ1, C12TPP+ prevented production of reactive oxygen species (ROS) measured by the fluorescence of 2′,7′-dichlorofluorescein in mitochondria of the plant cells. SkQ1 augmented the length of the vegetation period and the common and productive tillering, improved the crop structure and the productivity of the wheat Triticum aestivum. These results indicate that the tested compounds act as antioxidants, that ROS participate in aging and death of A. thaliana leaves, and wheat tillering is increased and the crop structure is improved by SkQ1.
Keywords: mitochondria-addressed compounds; reactive oxygen species; programmed cell death; Arabidopsis thaliana ; veg-etation period; crop structure; Triticum aestivum

The mechanism of the effect of tert-butyl hydroperoxide (tBHP) on the kinetics of decrease in liver mitochondrial ΔΨ (transmembrane electric potential) in response to successive additions of tBHP in low concentrations has been studied. FeSO4 was found to increase significantly the damaging effect of tBHP; this effect was shown to increase in the presence of low concentrations of Ca2+ starting from 2 μM CaCl2. Cyclosporin A prevents these effects. The data show that the damaging effect of low concentrations of tBHP in the course of pyruvate oxidation in isolated liver mitochondria is caused by the opening of the nonspecific Ca2+-dependent cyclosporin A-sensitive pore in the inner mitochondrial membrane. Application of a method of studying oxidative stress regulators, developed in this work, is illustrated by an example of the prooxidant action of ascorbate. This method is proposed for studying mitochondria in hemochromatosis, a pathology caused by excessive accumulation of iron.
Keywords: oxidative stress; liver mitochondria; tert-butyl hydroperoxide; pyruvate; cyclosporin A-sensitive pore; hemochromatosis

Effect of potential-dependent potassium uptake on calcium accumulation in rat brain mitochondria by O. V. Akopova; L. I. Kolchinskaya; V. I. Nosar; V. A. Bouryi; I. N. Mankovskaya; V. F. Sagach (80-90).
The effect of potential-dependent potassium uptake at 0–120 mM K+ on matrix Ca2+ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K+ content in the medium was observed due to K+ uptake into the mitochondria. The accumulation of Ca2+ was shown to depend on K+ concentration in the medium. At K+ concentration −30 mM, Ca2+ uptake is decreased due to K+-induced membrane depolarization, whereas at higher K+ concentrations, up to 120 mM K+, Ca2+ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K+ uptake. Mitochondrial K ATP + -channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K+ uptake as well as K+-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca2+ uptake, i.e. increase in Ca2+ uptake at low K+ content in the medium due to the smaller membrane depolarization and decrease in Ca2+ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K ATP + channel, in the regulation of calcium accumulation in rat brain mitochondria.
Keywords: brain; mitochondria; Ca2+ uptake; K+ uptake; K ATP + channel

Localization of non-native D-glyceraldehyde-3-phosphate dehydrogenase in growing and apoptotic HeLa cells by E. I. Arutyunova; L. V. Domnina; A. A. Chudinova; O. N. Makshakova; D. Y. Arutyunov; V. I. Muronetz (91-95).
Monoclonal antibodies that could not bind native tetramers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) but could bind to dimeric, monomeric, or denatured forms of GAPDH were used to investigate its intracellular localization. These antibodies distinctly stained the nucleus in growing HeLa cells. In the cytoplasm, non-native GAPDH was colocalized with actin filaments. Incubation of HeLa cells with tumor necrosis factor α (TNF-α) and the protein synthesis inhibitor emetine led to a drastic increase in the amount of the non-native GAPDH in the nuclei. Overproduction of Bcl-2 protein did not change the non-native GAPDH localization in the growing HeLa cells but prevented the development of apoptosis and the increase in the amount of non-native GAPDH in the nuclei upon incubation with TNF-α.
Keywords: GAPDH; actin; HeLa; localization; apoptosis; TNF-α

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants by G. S. Shatskaya; V. L. Drutsa; O. N. Koroleva; I. A. Osterman; T. M. Dmitrieva (96-101).
The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryp-tophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3′-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.
Keywords: RNA-dependent RNA polymerase; mengovirus; IMPACT system

Gastrin-releasing peptide (GRP) is a kind of neural peptide that plays an important role in the growth of various human cancer cells. However, very little is known about the relationship between GRP and apoptosis in human hepatocellular carcinoma cells. This study investigated the influences of GRP on apoptosis, as well as the mechanism that triggers HepG2 growth. The effects of GRP on cell proliferation were examined by analysis of lactate dehydrogenase. The GRP, caspase 12, and CHOP protein were detected in HepG2 and HL-7702 cells by Western blot, and endoplasmic reticulum (ER) stress-related mRNA transcription was detected by reverse transcription polymerase chain reaction. To explore the specific pathway by which GRP induces the cell growth, we investigated the apoptosis-related pathway. The expression of GRP in HL-7702 cells inhibited tunicamycin triggered ER stress-associated XBP1, ATF4, and TRAF2 mRNA transcription. Three main ER stress-unfolded protein response pathways proteins, including spliced XBP1, cleaved ATF6, IRE1-α, PERK, and eIF2-α, were increased significantly. Furthermore, the cleaved caspase 12 activation was blocked and CHOP expression was inhibited when GRP was expressed either in HepG2 or HL-7702 cells. In conclusion, GRP triggers the growth of HepG2 cells through blocking the ER stress-mediated pathway.
Keywords: gastrin-releasing peptide; growth; endoplasmic reticulum stress; apoptosis Published in Russian in Biokhimiya, 2013, Vol. 78, No. 1, pp. 131–142.