Biochemistry (Moscow) (v.76, #10)

Analysis of the literature and our own data suggest that the so-called “apoptotic” proteinases play important roles in brain function. However, mechanisms of their involvement in normal neuronal plasticity remain obscure. One of the main reasons for this is broad substrate specificity of proteinases; the number of potential substrates of each can reach several thousands. Obviously, a real approach to study functions of “apoptotic” proteinases, caspase-3 in particular, is to identify their intracellular substrates. It is the nature of a substrate that defines the direction of signal transduction or metabolic changes; therefore, identification of molecular partners of particular proteases should be the key study, not just measuring its activity or respective protein or mRNA expression. This approach will allow studying regulatory mechanisms not only for proteinases, but also for other pleiotropic enzymes usually possessing broad substrate specificity.
Keywords: proteinases; caspase; proteinase substrates; broad substrate specificity; plasticity; apoptosis; brain

Constitutive androstane receptor (CAR) is a xenosensor and target for therapy by E. M. Kachaylo; V. O. Pustylnyak; V. V. Lyakhovich; L. F. Gulyaeva (1087-1097).
Constitutive androstane receptor (CAR, NR1I3), which is under consideration in this review, is a member of the superfamily of nuclear receptors. However, certain features distinguish CAR from the variety of nuclear receptors. First, this receptor has structural features that allow it to display constitutive activity in the absence of a ligand and to interact in a species-specific manner with a huge number of ligands diverse in chemical structure and origin. Second, recently many researchers are focused on CAR because the significance is increasingly shown of its influence on a variety of physiological functions, such as gluconeogenesis, metabolism of xenobiotics, fatty acids, bilirubin, and bile acids, hormonal regulation, etc. In addition to the fundamental scientific interest, the study of CAR is of practical importance because changes in CAR activity can lead to disorders in physiological processes, which finally can result in changes in pathological states. However, despite intensive studies, many mechanisms are still unclear, which makes it difficult to understand the role of CAR in the overall picture of molecular regulation of physiological processes. This review analyzes the features and diversity of the functions of CAR.
Keywords: CAR; gene expression activation; ligands; xenobiotics; metabolism

Characteristics of σ-dependent pausing by RNA polymerases from Escherichia coli and Thermus aquaticus by E. V. Zhilina; N. A. Miropolskaya; I. A. Bass; K. L. Brodolin; A. V. Kulbachinskiy (1098-1106).
The σ70 subunit of RNA polymerase (RNAP) is the major transcription initiation factor in Escherichia coli. During transcription initiation, conserved region 2 of the σ70 subunit interacts with the −10 promoter element and plays a key role in DNA melting around the starting point of transcription. During transcription elongation, the σ70 subunit can induce pauses in RNA synthesis owing to interactions of region 2 with DNA regions similar to the −10 promoter element. We demonstrated that the major σ subunit from Thermus aquaticusA) is also able to induce transcription pausing by T. aquaticus RNAP. However, hybrid RNAP containing the σA subunit and E. coli core RNAP is unable to form pauses during elongation, while being able to recognize promoters and initiate transcription. Inability of the σA subunit to induce pausing by E. coli RNAP is explained by the substitutions of non-conserved amino acids in region 2, in the subregions interacting with the RNAP core enzyme. Thus, changes in the structure of region 2 of the σ70 subunit have stronger effects on transcription pausing than on promoter recognition, likely by weakening the interactions of the σ subunit with the core RNAP during transcription elongation.
Keywords: RNA polymerase; σ-dependent pausing; transcription regulation

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0–4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935–1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20–500 cm−1. The most intense band has the frequency of −30 cm−1 in RCs of mutant LL131H and in native RCs and the frequency of ∼100 cm−1 in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B A which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the BA molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.
Keywords: photosynthesis; charge separation; reaction center; wave packet; electron transfer

Programmed cell death in plants: Protective effect of mitochondrial-targeted quinones by L. A. Vasil’ev; E. V. Dzyubinskaya; D. B. Kiselevsky; A. A. Shestak; V. D. Samuilov (1120-1130).
Ubiquinone or plastoquinone covalently linked to synthetic decyltriphenylphosphonium (DTPP+) or rhodamine cations prevent programmed cell death (PCD) in pea leaf epidermis induced by chitosan or CN. PCD was monitored by recording the destruction of cell nuclei. CN induced the destruction of nuclei in both epidermal cells (EC) and guard cells (GC), whereas chitosan destroyed nuclei in EC not in GC. The half-maximum concentrations for the protective effects of the quinone derivatives were within the pico- and nanomolar range. The protective effect of the quinones was removed by a protonophoric uncoupler and reduced by tetraphenylphosphonium cations. CN-Induced PCD was accelerated by the tested quinone derivatives at concentrations above 10−8–10−7 M. Unlike plastoquinone linked to the rhodamine cation (SkQR1), DTPP+ derivatives of quinones suppressed menadione-induced H2O2 generation in the cells. The CN-induced destruction of GC nuclei was prevented by DTPP+ derivatives in the dark not in the light. SkQR1 inhibited this process both in the dark and in the light, and its effect in the light was similar to that of rhodamine 6G. The data on the protective effect of cationic quinone derivatives indicate that mitochondria are involved in PCD in plants.
Keywords: programmed cell death; mitochondrial-targeted quinones; mitochondria; chitosan; reactive oxygen species; pea

New actinoporins from sea anemone Heteractis crispa: Cloning and functional expression by E. S. Tkacheva; E. V. Leychenko; M. M. Monastyrnaya; M. P. Issaeva; E. A. Zelepuga; S. D. Anastuk; P. S. Dmitrenok; E. P. Kozlovskaya (1131-1139).
A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10–27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.
Keywords: sea anemone; actinoporin; multigene Hct-S family; recombinant actinoporin; hemolytic activity, structure-function analysis

The individual signaling pathways underlying cardiac hypertrophy, which is induced by either α or β adrenergic receptor (AR), are different. Activation of different AR subtypes couples to different G proteins and induction of specific signaling pathways, which ultimately results in subtype-specific regulation of cardiac function. We present the first proteomics study identifying proteins that are related to AR subtype-specific hypertrophy in cardiomyocytes by comparing the two-dimensional electrophoresis patterns between neonatal rat cardiomyocytes treated by phenylepinephrin (PE) and by isoproterenol (ISO). An improved 2-DE strategy was used in these comparative experiments. Twenty-five differentially expressed proteins in cardiomyocytes treated by PE or treated by ISO were successfully analyzed and identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, especially those that might be responsible to intracellular oxidative stress such as dismutase, peroxiredoxin, and thioredoxin-like protein p46. In addition, induced reactive oxygen species were also found to be AR subtype-specifically relevant to endoplasmic reticulum proteinase ERK1/2 phosphorylation during the development of hypertrophy induced by different AR subtypes. The results will help to better understand the underlying mechanisms of different adrenergic receptor subtype-induced hypertrophy.
Keywords: adrenergic receptors; cardiac hypertrophy; proteomics; reactive oxygen species

Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase by Yu. A. Modestova; G. Yu. Lomakina; N. N. Ugarova (1147-1154).
Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K m ATP and $K_m^{LH_2 } $ values were increased by a factor of ∼1.5–1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K m ATP and $K_m^{LH_2 } $ values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ∼2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.
Keywords: firefly luciferase; Luciola mingrelica ; site-directed mutagenesis; cysteine residues; polyhistidine tag; kinetic parameters; thermal stability

Self-assembly of soluble unlinked and cross-linked fibrin oligomers by M. A. Rosenfeld; V. B. Leonova; M. I. Biryukova; M. V. Vasileva (1155-1163).
Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350–450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.
Keywords: soluble fibrin; double-stranded protofibrils; factor XIIIa; γ-dimers; α-polymers

Methylglyoxal induces mitochondria-dependent apoptosis in sarcoma by A. Ghosh; S. Bera; S. Ray; T. Banerjee; M. Ray (1164-1171).
In the preceding paper (A. Ghosh et al. (2011) Biochemistry (Moscow), 76, 1051–1060), using several comparable tissue materials, it has been convincingly demonstrated that methylglyoxal, a normal metabolite, inhibits mitochondrial complex I of specifically malignant cells. This suggests a distinct alteration of complex I, a highly important enzyme for energy (ATP) production, in malignancy. The present paper shows that as a consequence of this inhibition mitochondrial membrane potential is drastically reduced in sarcoma tissue but not in normal skeletal muscle. This was estimated spectrofluorimetrically using the dye rhodamine 123. As a consequence, cytochrome c was released from the sarcoma mitochondria as evidenced by Western blot analysis. Moreover, on treatment with methylglyoxal membrane potential collapse of sarcoma 180 cells was also indicated by fluorescence-activated cell sorter analysis. Atomic force microscopic study demonstrated gross structural alteration specifically of tumor mitochondria on methylglyoxal treatment. All these studies suggest that methylglyoxal might initiate an apoptotic event in malignant cells.
Keywords: sarcoma; methylglyoxal; membrane potential; atomic force microscopy; cytochrome

Effect of cholinergic drugs on the activity of basic carboxypeptidases in rat nervous tissue by V. B. Solov’ev; M. T. Gengin; O. V. Solov’eva; O. V. Pavlova; E. S. Lobzina (1172-1177).
Effects of a single administration of cholinergic drugs (arecoline, atropine, nicotine, mecamylamine) on the activity of carboxypeptidase H and of phenylmethylsulfonyl fluoride-inhibited carboxypeptidase, which are involved in metabolism of neuropeptides, were studied in brain parts and the adrenal glands of rats. The enzyme activities were determined fluorimetrically using specific inhibitors and substrates. In the majority of cases the enzyme activities decreased, and this decrease was retained for at least 72 h. Changes in the activities of the studied enzymes depended on the type of cholinergic action, the nervous system part, and the time after the injection. The changes in activities of the studied carboxypeptidases are supposed to be a possible mechanism responsible for changes in the levels of neuropeptides under the influence of high doses of the drugs.
Keywords: nervous system; carboxypeptidases; cholinergic drugs

Contents of mRNAs encoding endosome/lysosome components in normal human aorta and in stage ii of atherogenesis: a hidden regulation by T. A. Shchelkunova; E. A. Albert; I. A. Morozov; P. M. Rubtsov; L. M. Samokhodskaya; I. A. Sobenin; A. N. Orekhov; A. N. Smirnov (1178-1184).
Contents of mRNAs encoding endosome/lysosome components EEA1, Rab5a, Lamp1, Lamp2, p62 (SQSTM1), and CD63 were measured by quantitative PCR and compared in intact fragments of human aorta and in aorta fragments with atherosclerotic lesions of stage II (fatty streaks) of the same donors. During atherogenesis an increase was detected only in the level of p62 mRNA but not in other mRNAs. Nevertheless, correlation analysis revealed a profound rearrangement of inter-gene correlations: only 30% of correlations found in the fatty streaks coincided with the correlations in normal fragments. Thus, new constellations were formed in fatty streaks concurrently with disappearance of correlations between mRNAs under study and mRNAs encoding factors of lipid accumulation, reverse cholesterol transfer, and some lipid sensors/transcription regulators of lipid metabolism.
Keywords: endosomes; lysosomes; autophagy; atherogenesis; PCR, gene expression

Carbohydrate specificity of chicken and human tandem-repeat-type galectins-8 in composition of cells by O. A. Vokhmyanina; E. M. Rapoport; I. M. Ryzhov; E. Yu. Korchagina; G. V. Pazynina; V. V. Severov; H. Kaltner; S. André; H. -J. Gabius; N. V. Bovin (1185-1192).
The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandemrepeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3′-sialylated and 3′-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galβ1-3GlcNAc (Lec) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.
Keywords: ABH blood group antigens; galectin; cell glycoconjugates

Methods in Enzymology, Vol. 480, Glycobiology by G. Ya. Wiederschain (1193-1193).

Methods in Enzymology, Vol. 479, Functional Glycomics by G. Ya. Wiederschain (1194-1194).