Biochemistry (Moscow) (v.77, #8)

Since the definition of archaea as a separate domain of life along with bacteria and eukaryotes, they have become one of the most interesting objects of modern microbiology, molecular biology, and biochemistry. Sequencing and analysis of archaeal genomes were especially important for studies on archaea because of a limited availability of genetic tools for the majority of these microorganisms and problems associated with their cultivation. Fifteen years since the publication of the first genome of an archaeon, more than one hundred complete genome sequences of representatives of different phylogenetic groups have been determined. Analysis of these genomes has expanded our knowledge of biology of archaea, their diversity and evolution, and allowed identification and characterization of new deep phylogenetic lineages of archaea. The development of genome technologies has allowed sequencing the genomes of uncultivated archaea directly from enrichment cultures, metagenomic samples, and even from single cells. Insights have been gained into the evolution of key biochemical processes in archaea, such as cell division and DNA replication, the role of horizontal gene transfer in the evolution of archaea, and new relationships between archaea and eukaryotes have been revealed.
Keywords: archaea; genome; evolution; sequencing; phylogeny; gene systematics; DNA replication; horizontal gene transfer

MicroRNAs: Possible role in pathogenesis of Parkinson’s disease by E. V. Filatova; A. Kh. Alieva; M. I. Shadrina; P. A. Slominsky (813-819).
Parkinson’s disease is one of the most common human neurodegenerative disorders caused by the loss of dopaminergic neurons from the substantia nigra pars compacta of human brain. However, causes and mechanisms of the progression of the disease are not yet fully clarified. To date, investigation of the role of miRNAs in norm and pathology is one of the most intriguing and actively developing areas in molecular biology. MiRNAs regulate expression of a variety of genes and can be implicated in pathogenesis of various diseases. Possible role of miRNAs in pathogenesis of Parkinson’s disease is discussed in this review.
Keywords: microRNA; Parkinson’s disease; miRNA-dependent regulation

In this review we present data about small intestine serine proteases, which are a considerable part of the proteolytic apparatus in this major part of the gastrointestinal tract. Serine proteases of intestinal epitheliocytes, their structural-functional features, cellular localization, physiological substrates, and mechanisms of activity regulation are examined. Information about biochemical and functional properties of serine proteases is presented in a common context with morphological and physiological data, this being the basis for understanding the functional processes taking place in upper part of the intestine. Serine proteases play a key role in the physiology of the small intestine and provide the normal functioning of this organ as part of the digestive system in which hydrolysis and suction of food substances occur. They participate in renewal and remodeling of tissues, retractive activity of smooth musculature, hormonal regulation, and defense mechanisms of the intestine.
Keywords: serine proteases; small intestine; digestive conveyor; activation of zymogens; tissue remodeling; epithelium renewal

This review considers principles of the use of mass spectrometry for the study of biological macromolecules. Some examples of protein identification, virion proteomics, testing vaccine preparations, and strain surveillance are represented. Possibilities of structural characterization of viral proteins and their posttranslational modifications are shown. The authors’ studies by MALDI-MS on S-acylation of glycoproteins from various families of enveloped viruses and on oligomerization of the influenza virus hemagglutinin transmembrane domains are summarized.
Keywords: mass spectrometry; MALDI; enveloped viruses; influenza virus; hemagglutinin; S-acylation; transmembrane domain; proteomics; vaccine; strain surveillance

Quinone-dependent alcohol dehydrogenases and fad-dependent alcohol oxidases by A. R. Gvozdev; I. A. Tukhvatullin; R. I. Gvozdev (843-856).
This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.
Keywords: methanol dehydrogenase; ethanol dehydrogenase; quinohemeprotein alcohol dehydrogenase; FAD-methanol oxidase; three-dimensional structure; mechanism of action

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica by I. I. Mustakhimov; A. S. Reshetnikov; D. N. Fedorov; V. N. Khmelenina; Y. A. Trotsenko (857-863).
In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ70-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.
Keywords: methylotrophic bacterium, osmoprotectant, ectoine genes, transcriptional regulation

Identification of intracellular Spiroplasma melliferum metabolites by the HPLC-MS method by A. A. Vanyushkina; D. E. Kamashev; I. A. Altukhov; V. M. Govorun (864-877).
In contrast to the abundance of systems-oriented approaches describing changes on the transcriptome or proteome level, relatively few studies have employed the metabolome. The goal of the presented research was to identify as many intracellular metabolites as possible in a Spiroplasma melliferum extract by flow injection time-of-flight mass spectrometry. The Mollicutes class bacterium S. melliferum is a member of a unique category of bacteria that have in common the absence of a cell wall, a reduced genome, and simplified metabolic pathways. Metabolite identification was confirmed by fragmentation of previously detected ions by target mass spectrometry. The selected liquid chromatography approach, hydrophilic interaction chromatography with amino and silica columns, effectively separates highly polar cellular metabolites prior to their detection on a high accuracy mass spectrometer in positive and negative acquisition mode for each column. Here we present reliable measurement of 76 metabolites, including components of sugar, amino acid, and nucleotide metabolism. We have identified about a third of the possible intracellular S. melliferum metabolites predicted by genome annotation.
Keywords: Spiroplasma melliferum ; metabolomics; mass spectrometry; LC-ESI-MS/MS; metabolite; hydrophilic interaction chromatography

Catalytic properties and amino acid sequence of endo-1→3-β-D-glucanase from the marine mollusk Tapes literata by A. M. Zakharenko; M. I. Kusaykin; S. N. Kovalchuk; V. V. Sova; A. S. Silchenko; A. A. Belik; S. D. Anastyuk; Bui Minh Ly; V. A. Rasskazov; T. N. Zvyagintseva (878-888).
A specific 1→3-β-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45dgC. The 1→3-β-D-glucanase catalyzes hydrolysis of β-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K m for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-β-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-β-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-β-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.
Keywords: 1→3-β-D-glucanase; crystalline style; laminaran; marine mollusk; Tapes literata ; transglycosylation

Mobility and spatial orientation of a novel 40-kDa actin-binding protein from the smooth muscle of the mussel Crenomytilus grayanus was studied by polarized fluorometry. The influence of this protein on orientation and mobility of the myosin heads was investigated during modeling the different stages of the ATPase cycle. The 40-kDa actin-binding protein affected the strong actin-myosin binding. We suggest that the 40-kDa actin-binding protein is involved in regulation of the actin-myosin interaction in the smooth muscle of the mussel.
Keywords: actin-binding protein of the mussel Crenomytilus grayanus ; ghost fibers; ATP analog; actomyosin intermediate states; conformational change of myosin subfragment-1; fluorescence polarization

The binding of both factors (eRF1 and eRF3) is essential for fast kinetics of the termination of protein translation. The C-terminal domain of eRF1 is known to interact with the C domain of eRF3. Eo-eRF1b contains two highly conserved tryptophan residues (W-11 and W-373), W-11 located in the Eo-eRF1b N domain and W-373 located in the EoeRF1b C domain. Fluorimetry was used to study the interactions of the proteins. When binding with Eo-eRF3Cm6, the emission peak of Eo-eRF1b is blue shifted, while the emission peak of Eo-eRF1bC has no notable change. Our results suggest that the eRF1-eRF3 interaction induces the N and C domain of eRF1b to become closer to each other.
Keywords: steady-state fluorescence quenching; eRF1-eRF3 interaction; Euplotes octocarinatus

Effect of calcium ions on electron transfer between hemes a and a 3 in cytochrome c oxidase by T. V. Vygodina; A. V. Dyuba; A. A. Konstantinov (901-909).
Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH3) 6 3+ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a 3 reduction. The difference spectrum of heme a 3 reduction in the visible region is characterized by a maximum at ∼612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a 3 reduction is slowed down by low concentrations of Ca2+. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca2+ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca2+ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.
Keywords: cytochrome c oxidase; Ca2+ ions; fast kinetics; heme a 3

Role of prodigiosin and chitinases in antagonistic activity of the bacterium Serratia marcescens against the fungus Didymella applanata by A. B. Duzhak; Z. I. Panfilova; T. G. Duzhak; E. A. Vasyunina; M. V. Shternshis (910-916).
The molecular features of antagonism of the bacterium Serratia marcescens against the plant pathogenic fungus Didymella applanata have been studied. The chitinases and the red pigment prodigiosin (PG) of S. marcescens were isolated and characterized. Specific antifungal activity of the purified PG and chitinases against D. applanata was tested in vitro. The antagonistic properties of several S. marcescens strains exhibiting different levels of PG and chitinase production were analyzed in vitro with regard to D. applanata. It was found that the ability of S. marcescens to suppress the vital functions of D. applanata depends mainly on the level of PG production, whereas chitinase production does not provide the bacterium with any competitive advantage over the fungus.
Keywords: Serratia marcescens ; Didymella applanata ; antagonism; chitinase; prodigiosin

Evaluation of the electrical potential on the membrane of the extremely alkaliphilic bacterium Thioalkalivibrio by M. S. Muntyan; D. A. Morozov; S. S. Klishin; N. V. Khitrin; G. Ya. Kolomijtseva (917-924).
The electrical potential on the membrane was measured in cells of strains AL2 and ALJ15 of the extremely alkaliphilic bacterium Thioalkalivibrio versutus using the penetrating cation tetraphenylphosphonium (TPP+) and a TPP+-selective electrode. The potentials were -228 ± 5 and -224 ± 5 mV, respectively, i.e. higher than in most alkaliphilic bacteria. Membrane potential in the cells was estimated by measuring the inner cell volume by two independent methods: (1) estimation of total cell volume by light microscopy and (2) estimation of the inner aqueous volume of the cells with allowance for the distribution difference of tritium labeled water penetrating through the membranes and a nonpenetrating colored protein. The inner cell volume was 2.4 ± 0.2 and 2.2 ± 0.1 μl/mg of cell protein by the two methods, respectively. Computer computation was used as an alternative to manual calculation to count the number of cells for estimation of total cell volume.
Keywords: electrochemical potential of H+ ions; transmembrane electric potential difference; alkaliphilic bacteria

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties by N. I. Olovnikova; M. A. Ershler; O. V. Grigorieva; A. V. Petrov; G. Yu. Miterev (925-933).
Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.
Keywords: monoclonal antibodies; anti-D; glycosylation; FcγR; ADCC; immunosuppression

HDAC1/DNMT3A-containing complex is associated with suppression of Oct4 in cervical cancer cells by Dongbo Liu; Peng Zhou; Li Zhang; Wei Gong; Gang Huang; Yingru Zheng; Fengtian He (934-940).
Octamer-binding transcription factor 4 (Oct4), an important embryonic transcriptional factor, is highly expressed in several tumors and is considered as a hallmark of cancer stem cells. Knowledge about the expression and regulatory mechanisms of Oct4 can contribute to the treatment of cancers. As for cervical cancer, however, details remain obscure about Oct4 expression and its regulatory mechanism. In this study, we found that the level of Oct4 in human papillomavirus 16 (HPV16)-positive cervical cancer cells (CaSki cells) was higher than that in HPV-negative cervical cancer cells (C-33A cells), whereas both the level of histone deacetylase 1 (HDAC1) and DNA methyltransferase 3A (DNMT3A) were lower in CaSki cells than those in C-33A cells. Treatment with valproic acid, an HDAC inhibitor, could significantly increase the expression of Oct4 in C-33A cells, but only slightly increased Oct4 in CaSki cells. Co-immunoprecipitation assays showed that HDAC1 and DNMT3A existed in a common complex. The co-immunoprecipitated DNMT3A or HDAC1 was dose-dependently decreased with valproic acid treatment. These results indicated that HDAC1/DNMT3A-containing complex is associated with the suppression of Oct4 in cervical cancer cells, and the activity of HDAC1 is required in the repression of Oct4.
Keywords: Oct4; cervical cancer; HDAC1; DNMT3A; HPV16

Expression, purification, and secondary structure characterization of recombinant KCTD1 by Fanghua Mei; Jin Xiang; Song Han; Yuan He; Yajing Lu; Jian Xu; Deyin Guo; Gengfu Xiao; Po Tien; Guihong Sun (941-945).
Potassium channel tetramerization domain containing 1 (KCTD1) contains a BTB domain, which can facilitate protein-protein interactions that may be involved in the regulation of signaling pathways. Here we describe an expression and purification system that can provide a significant amount of recombinant KCTD1 from Escherichia coli. The cDNA encoding human KCTD1 was amplified and cloned into the expression vector pET-30a(+). The recombinant protein was expressed in E. coli BL21(DE3) cells and subsequently purified using affinity chromatography. To confirm that KCTD1 was correctly expressed and folded, the molecular weight and conformation were analyzed using mass spectroscopy, Western blot, and circular dichroism. Optimizing KCTD1 expression and investigating its secondary structure will provide valuable information for future structural and functional studies of KCTD1 and KCTD family proteins.
Keywords: KCTD1; circular dichroism spectroscopy; secondary structure