Biochemistry (Moscow) (v.73, #8)

Structure and function of MYST1 histone acetyltransferase in the interactome of animal cells by R. I. Dmitriev; M. I. Shakhparonov; N. B. Pestov (839-852).
The major function of protein MYST1 is acetylation of histone H4 at the K16 residue. This modification is essential for chromatin remodeling and is used for regulation of gene expression in eukaryotes. MYST1 is a part of multiprotein complexes that accomplish functions of male X-chromosome activation and thereby functions of dosage compensation in drosophila and, in mammals, global acetylation of histone H4 K16. Recently, novel functional links between MYST1 and proteins ATM and p53 have been observed, and it is recognized that MYST1 plays a role in tumor suppression mechanisms. In the present review, we examine novel data about functional composition and mechanisms of MYST1-containing complexes. Interplay between MYST1 and other components of the animal cell interactome is also discussed.
Keywords: hampin; MYST1; MSL; compensasome; chromatin structure rearrangement

G-Quadruplex structure: a target for anticancer therapy and a probe for detection of potassium by Bo Chen; Jiangli Liang; Xiaoping Tian; Xiaochuan Liu (853-861).
G-Quadruplexes are four-stranded DNA structures that play important regulatory roles in the maintenance of telomere length by inhibiting telomerase activity. Telomeres are specialized functional DNA-protein structures consisting of a variable number of tandem G-rich repeats together with a group of specific proteins. Telomere losses during cell replication are compensated by telomerase, which adds telomeric repeats onto the chromosome ends in the presence of its substrate—the 3′-overhang. Recently, quadruplexes have been considered as a potential therapeutic target for human cancer because they can inhibit telomerase activity, and some quadruplex-interacting drugs can induce senescence and apoptosis of cancer cells. In addition, due to the potassium preference to the other cations, especially sodium ions, quadruplexes have been suggested for developing potassium detection probes with higher sensitivity and selectivity. This review will illustrate these two aspects to provide further understanding of G-quadruplex structures.
Keywords: G-quadruplex; G-quadruplex-interacting ligand; telomere; telomerase; FRET; potassium detection

Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0–7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.
Keywords: firefly luciferase; random mutagenesis; mutation of amino acid residues; bioluminescence spectra; pH dependence of bioluminescence spectra

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc 1 complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c 1 of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k cat values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.
Keywords: cytochrome c ; cytochrome bc 1 ; electrostatic attraction; QCR kinetics; ubiquinol-cytochrome c oxidoreductase; Paracoccus denitrificans

The mammalian gene ZNF268 is regulated by hUpf1 by Chengang Zhu; Zhouzhou Zhao; Mingxiong Guo; Huanjie Shao; Hongling Qiu; Di Wang; Junhua Xu; Lu Xue; Wenxin Li (881-885).
Nonsense-mediated mRNA decay (NMD), also called RNA surveillance, is a process that degrades mRNAs with premature translation termination codons. In Saccharomyces cerevisiae, it has also been shown that NMD can regulate gene expression at the transcriptional level. To date, there has been no example where promoters are regulated by the NMD path-way in higher eukaryotes. Taking advantage of our previous research on ZNF268 transcription control, we studied the relationship between the ZNF268 promoter and the NMD pathway. We showed by transient transfection that the ZNF268 promoter activity was influenced by hUpf1, not hSmg6, in HeLa cells. This result was confirmed by the analysis of the steady state mRNA of ZNF268 after depletion of endogenous hUpf1 or hSmg6 in HeLa cells. Direct mutational analysis revealed that the C/EBP site in the promoter region is important for hUpf1 function on ZNF268 promoter. Together our results demonstrated that the mammalian gene ZNF268 is regulated by hUpf1 via its promoter.
Keywords: NMD; hUpf1; ZNF268 ; promoter regulation

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA by Yu. S. Krasikova; N. I. Rechkunova; E. A. Maltseva; I. O. Petruseva; V. N. Silnikov; T. S. Zatsepin; T. S. Oretskaya; O. D. Scharer; O. I. Lavrik (886-896).
The interaction of nucleotide excision repair factors-xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)—with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.
Keywords: nucleotide excision repair factors; photoaffinity labeling; photoreactive oligonucleotides; complex formation

Metal cofactors play a dual role in Mycobacterium tuberculosis inorganic pyrophosphatase by E. V. Rodina; L. P. Vainonen; N. N. Vorobyeva; S. A. Kurilova; T. S. Sitnik; T. I. Nazarova (897-905).
Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators—Mg2+ and Mn2+. Dissociation constants of Mt-PPase complexed with Mg2+ or Mn2+ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg2+-or Mn2+-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg2+ or Mn2+ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8–10 toward the hexameric form by stabilizing intertrimeric contacts. The pK a values have been determined for groups that control the observed hexamer-monomer (pK a 5.4), hexamer-trimer (pK a 7.5), and trimer-monomer (pK a 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.
Keywords: pyrophosphatase; M. tuberculosis ; Mg2+ ; Mn2+ ; metal cofactor; cofactor specificity

Comparative analysis of anti-restriction activities of ArdA (ColIb-P9) and Ocr (T7) proteins by G. B. Zavilgelsky; V. Yu. Kotova; S. M. Rastorguev (906-911).
Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3(ocr) genes were cloned in pUC18 vector. Both ArdA (ColIb-P9) and Ocr (T7) proteins inhibit both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. ColIb-P9 ardA, T7 0.3(ocr), and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the PltetO-1 promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the PltetO-1 promoter can be regulated over an up to 5000-fold range by supplying anhydrotetracycline to the E. coli MG1655Z1 tetR+ cells. Effectiveness of the anti-restriction activity of the ArdA and Ocr proteins depended on the intracellular concentration. It is shown that the dissociation constants K d for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: K d(Ocr) = 10−10 M, K d(ArdA) = 1.7·10−7 M.
Keywords: anti-restriction proteins; type I restriction-modification enzymes; ArdA; Ocr; luxCDABE; bioluminescence; luciferase

Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature β-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZαB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant β-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0–8.5. The purified recombinant β-glucosidase is effectively active on para-/ortho-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) with K m values of 1.9, 2.1, 0.78 mM and k cat values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-β-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-β-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of β-glucosidases and mutated recombinant enzyme is discussed.
Keywords: Arabidopsis thaliana ; β-glucosidase; site-directed mutagenesis; heterologous expression; Pichia pastoris

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men by T. A. Shchelkunova; I. A. Morozov; P. M. Rubtsov; L. M. Samokhodskaya; R. A. Kireev; I. V. Andrianova; A. N. Orekhov; A. N. Smirnov (920-928).
The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18–54-year-old men’s large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERα) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERα and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.
Keywords: sex hormone receptors; steroid sulfatase; aromatase; estrogen sulfotransferase; arteries; atherogenesis; polymerase chain reaction

DNA binding studies of PdCl2(LL)(LL = chelating diamine ligand: N,N-dimethyltrimethylenediamine) complex by S. Kashanian; N. Shahabadi; H. Roshanfekr; K. Shalmashi; K. Omidfar (929-936).
The interaction of native calf thymus DNA with the Pd(II) complex, PdCl2(LL) (LL = chelating diamine ligand: N,N-dimethyltrimethylenediamine), in 10 mM Hepes aqueous solutions at neutral pH has been monitored as a function of metal complex/DNA molar ratio by UV absorption spectrophotometry, circular dichroism (CD), viscosimetry, and fluorescence spectroscopy. The results support two modes of interaction. In particular, this complex showed absorption hypochromism and then hyperchromism, increase in melting temperature, and some structural changes in specific viscosity when bound to calf thymus DNA. The binding constant determined using absorption measurement is 2.69·103 M−1. As evidenced by the increasing fluorescence of methylene blue-DNA solutions in the presence of increasing amounts of metal complex, PdCl2(LL) is able to displace the methylene blue intercalated into DNA, but not so completely, as indicated by partial intercalation. CD spectral changes in two steps and viscosity decrease confirm our conclusions.
Keywords: Pd(II) complex; CT-DNA; partially intercalating; outside binding

Deoxyribonucleases (DNases) differing in subcellular localization and specificity towards native and denatured substrates and in products of cleavage of endogenous DNA were isolated for the first time from the hepatopancreas of freshwater snail by differential sedimentation and preparative isoelectrofocusing. It was found that treatment with phenol activates most of the investigated DNases, especially in lysosomes, and induces two new DNases of lysosomal origin. The possible different participation of certain DNases of the snail hepatopancreas in the regulation of DNA degradation under intoxication is discussed.
Keywords: deoxyribonucleases; physicochemical properties; specificity; mollusks; lysosomes; intoxication; induction of activity

Complex of dipeptidyl peptidase II with adenosine deaminase by S. G. Sharoyan; A. A. Antonyan; S. S. Mardanyan; G. Lupidi; M. Cuccioloni; M. Angeletti; G. Cristalli (943-949).
Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).
Keywords: dipeptidyl peptidase; adenosine deaminase; protein-protein interactions; fluorescence polarization; resonant mirror biosensor; differential spectrophotometry

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1 by Yu. Ya. Kit; M. A. Starykovych; V. A. Richter; R. S. Stoika (950-956).
Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.
Keywords: antibodies; protabzymes; multiple sclerosis; blood serum; colostrum; proteolytic activity