Biochemistry (Moscow) (v.78, #4)

Role of microRNAs in mechanisms of glioblastoma resistance to radio- and chemotherapy by Ph. A. Koshkin; D. A. Chistiakov; V. P. Chekhonin (325-334).
Low-grade gliomas and multiform glioblastoma are characterized by highly pronounced anaplasia, malignization, proliferation, and invasiveness; moreover, they are highly resistant to chemo- and radiotherapy. The very low efficiency of traditional approaches in the treatment of patients with glioblastomas is due to the intensive invasive growth of the tumor resulting in deep infiltration of adjacent normal perivascular and nervous tissue and formation of areas of perineural infiltration differently remote from the tumor epicenter. MicroRNAs are key posttranscriptional regulators of gene activities, and their expression is markedly increased in tumors, in particular in gliomas. MicroRNAs have been shown to promote the growth, proliferation, migration, and survival of tumor stem and non-stem cells. However, a population of microRNA possessing antitumor effects is also detected in gliomas. As a rule, the expression of antitumor microRNAs is suppressed in tumors. In this review, we consider microRNAs, their influence on radio- and chemoresistance of gliomas, and prospects for their use as specific agents in targeted therapy of gliomas. The pool of these microRNAs has distinct therapeutic value, because on use in combined therapy it can decrease the resistance of glioma tumor stem cells to existing pharmaceuticals and improve the efficiency of radio- and chemotherapy.
Keywords: microRNA; brain tumors; glioma; chemotherapy; radiotherapy; targeted delivery

Bidirectional promoters in the transcription of mammalian genomes by A. S. Orekhova; P. M. Rubtsov (335-341).
In the genomes of humans and other mammals a large number of closely spaced pairs of genes that are transcribed in opposite directions were revealed. Their transcription is directed by so-called bidirectional promoters. This review is devoted to the characteristics of bidirectional promoters and features of their structure. The composition of “core” promoter elements in conventional unidirectional and bidirectional promoters is compared. Data on binding sites of transcription factors that are primarily specific for bidirectional promoters are discussed. The examples of promoters that share protein-coding genes transcribed by RNA polymerase II and the non-coding RNA genes transcribed by RNA polymerase III are described. Data obtained from global transcriptome analysis about the existence of short noncoding antisense RNA associated with the promoters in the context of the hypothesis of bidirectional transcription initiation as an inherent property of eukaryotic promoters are discussed.
Keywords: genome structure; bidirectional promoters; divergent transcription; transcriptome; promoter core-elements; transcription initiation; CpG islands; transcription factors; short noncoding RNAs

Molecular switch for cold acclimation — anatomy of the cold-inducible promoter in plants by Yue Jiang; Dan Peng; Li-Ping Bai; Hui Ma; Li-Jing Chen; Ming-Hui Zhao; Zheng-Jin Xu; Zhi-Fu Guo (342-354).
In plants, a promoter is essential to drive the transcription and expression of genes under stress conditions. The cold-regulated promoter is an important molecular switch involved in transcriptional regulation of a dynamic network of genes associated with cold acclimation processes. However, the structure and functions of the cold-regulated promoter are ambiguous. In this review, we first describe the common type and structures of the cold-regulated promoter, such as the core promoter and transcription factor binding sites, and then discuss the synergistic actions of promoter elements and cold-regulated genes. We also describe the transcriptional responses and cross-talk among cold-regulated genes in the ICE-CBF-COR cold-response pathway. Many stress-inducible genes are known to be regulated by endogenous abscisic acid (ABA), which accumulates during osmotic and cold stress. We discuss the regulation of promoters of cold-inducible genes in ABA-dependent and ABA-independent regulatory systems. We also describe the cross-talk among gene networks regulated by different cis-acting regulatory elements. Finally, we propose potential further research on, and practical applications of, the cold-regulated promoter.
Keywords: promoter; cold regulation; transcription; cis-elements; molecular mechanism

Autophagy: Mechanisms, regulation, and its role in tumorigenesis by A. A. Parkhitko; O. O. Favorova; E. P. Henske (355-367).
Autophagy (from Greek “auto” — self, “phagos” — to eat) is the major catabolic process involved in the delivery and lysosomal degradation of long-lived intracellular components: proteins, lipids, nucleic acids, and organelles. Since the discovery of genes involved in regulation of autophagy in the 1990s, there has been a significant increase in studies of autophagy as a process involved in maintaining cellular homeostasis, as well as its role in the development of different pathologies. This review focuses on the basics of autophagy and its regulatory mechanisms. The role of autophagy in the maintenance of cellular homeostasis and tumorigenesis is also discussed.
Keywords: autophagy; metabolism; mitophagy; mTORC1

Intact Bence-Jones proteins TIM and LUS under simulated physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 37°C) did not display amyloidogenic properties. However, their isolated variable domains exhibit these qualities in full measure. Therefore, both intact proteins and their variable domains were studied using a complex of physical methods (scanning microcalorimetry, analytical centrifugation, optics) that allowed us to assess the stability of their tertiary and quaternary structures. The experimentally obtained thermodynamic functions indicated that the stability of iso-lated variable domains of TIM and LUS was comparable to the stability of similar domains in amyloidogenic proteins described earlier. However, inside the whole protein their stability was comparable to the stability of VL domains of ordinary Bence-Jones proteins. The decreased stability of the isolated variable domains of TIM and LUS was shown to be due both to weak interactions between a pair of variable domains (trans -interaction) and to a natural lack of interaction with the con-stant domains (cis-interaction).
Keywords: Bence-Jones proteins; amyloid fibrils; variable domains; stabilization free energy; interdomain interactions

Chemical modification of photosystem II core complex pigments with sodium borohydride by M. I. Vishnev; A. A. Zabelin; V. A. Shkuropatova; M. F. Yanyushin; V. A. Shuvalov; A. Ya. Shkuropatov (377-384).
The reaction of the irreversible chemical reduction of the 131-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ∼25% of Pheo a molecules are modified to 131-deoxo-131-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 131-deoxo-131-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH4-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheo-phytin PheoD2 is subjected to the borohydride treatment; (iv) the Qx optical transition of the PheoD2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Qy spectral region, PheoD2 probably absorbs at ∼680 nm.
Keywords: photosystem II core complex; pheophytin a ; chemical modification; sodium borohydride

Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants by K. A. Novototskaya-Vlasova; L. E. Petrovskaya; E. M. Rivkina; D. A. Dolgikh; M. P. Kirpichnikov (385-394).
A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5T isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30–35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.
Keywords: cryopeg; permafrost; Psychrobacter cryohalolentis ; cold-active lipase; thermostability; deletion mutants

Vectorial charge transfer reactions on the donor side of manganese-depleted and reconstituted photosystem 2 core complexes by I. O. Petrova; V. N. Kurashov; A. A. Zaspa; A. Yu. Semenov; M. D. Mamedov (395-402).
The light-induced functioning of photosystem 2 (PS 2) is directly linked to the translocation of both electrons and protons across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). Generation of ΔΨ due to S-state transitions of the water oxidation complex was demonstrated for the first time in Mn-depleted and reconstituted PS 2 core complexes incorporated into liposomes. The kinetics and relative amplitudes of the electrogenic reactions in dark-adapted samples during S1→S2, S2→S3, and S4→S0 transitions in response to the first, second and third laser flashes were comparable to those obtained in the intact PS 2 core particles. These results expand current understanding of the nature and mechanisms of electrogenic (vectorial) reactions due to a charge transfer on the donor side of PS 2.
Keywords: photosystem 2; apo-WOC-PS 2; photoactivation; S-state transitions; proteoliposomes; direct electrometric

The structural dynamics of three different ligand-activated G-protein coupled receptors (GPCRs) and the photoreactive receptor rhodopsin from mammals were comparatively studied. As a result, diagrams demonstrating the main structural differences between the studied membrane receptors were obtained. These diagrams represent the projection of the crystal structures of rhodopsin photointermediates and ligand-activated receptors onto the plane defined by the principal components. Thus, we were able to associate the activation process of the receptors with large-scale movements of their individual transmembrane (TM) domains. In addition, the dynamics of extracellular loops of ligand-activated receptors responsible for recognition and initial binding of ligands was studied. Based on these results, two parameters of functionally significant structural dynamics of membrane receptors can be thoroughly analyzed simultaneously — movements of individual TM helices and of extracellular loops.
Keywords: dynamics; conformation; membrane proteins; X-ray crystallography; principal component analysis; structural bioinformatics

Long-chain saturated monocarboxylic fatty acids can induce nonspecific permeability of the inner membrane (open pores) of liver mitochondria loaded with Ca2+ or Sr2+ by the mechanism insensitive to cyclosporin A. In this work we investigated the effect of their metabolites — α,ω-dioic (dicarboxylic) acids — as potential inducers of pore opening by a similar mechanism. It was established that the addition of α,ω-hexadecanedioic acid (HDA) at a concentration of 10–30 μM to liver mitochondria loaded with Ca2+ or Sr2+ leads to swelling of the organelles and release of these ions from the matrix. The maximum effect of HDA is observed at 50 μM Ca2+ concentration. Cyclosporin A at a concentration of 1 μM, previously added to the mitochondria, did not inhibit the observed processes. The calcium uniporter inhibitor ruthenium red, which blocks influx of Ca2+ and Sr2+ to the matrix of mitochondria, prevented HDA-induced swelling. The effect of HDA as inducer of swelling of mitochondria was compared with similar effects of α,ω-tetradecanedioic and α,ω-dodecanedioic acids whose acyl chains are two and four carbon atoms shorter than HDA, respectively. It was found that the efficiency of these α,ω-dioic acids decreases with reducing number of carbon atoms in their acyl chains. It was concluded that in the presence of Ca2+ or Sr2+ long-chain saturated α,ω-dioic acids can induce a cyclosporin A-insensitive permeability of the inner membrane (open pores) of liver mitochondria as well as their monocarboxylic analogs.
Keywords: liver mitochondria; α,ω-dioic acid; cyclosporin A-insensitive pore; calcium ions

A conjugate of the ligand of FS2 venom dihydropyridine receptors with a cationic arginine-containing oligopeptide was synthesized. It was found that the conjugate provides siRNA delivery to murine myotubes differentiated in vitro. The effect of RNA interference with the use of siRNA complexes with the conjugate was observed when siRNA concentrations were an order of magnitude lower than those used in the case of siRNA complexes with a non-conjugated oligopeptide.
Keywords: siRNA; RNA interference; myostatin; myotubes

By combining anion-exchange chromatography with gel filtration, an effective method for purification of wild-type xyloglucanase and five other cellulolytic enzymes from strain QM9414 of Trichoderma reesei was established. Characterization by enzyme activity assay, SDS-PAGE, and mass spectrometry identified the purified proteins as cellobiohydrolases I and II, endoglucanases I and II, a xyloglucanase, and β-xylosidase, of which the xyloglucanase was purified for the first time from the mutant strain QM9414. This method holds great promise to study the mechanism of cellulolytic enzymes, to investigate the synergistic action between cellulase and other cellulolytic enzymes, and to better exploit enzyme preparations for degradation of lignocellulose.
Keywords: xyloglucanase; cellulolytic enzymes; purification; Trichoderma reesei QM9414

On the problem of identification of intracellular signaling pathways by V. Yu. Denisenko; T. I. Kuzmina (431-432).
A hypothesis is proposed about the involvement of mitochondria in transduction of intracellular signals. In addition to the endoplasmic reticulum responsible for fluctuations of intracellular concentration of Ca2+, mitochondria are supposed to determine changes in the concentration of the secondary messenger cAMP. This hypothesis extends our ideas concerning mechanisms of intracellular signal transduction.
Keywords: endoplasmic reticulum; calcium; mitochondria; cAMP