Biochemistry (Moscow) (v.79, #1)

The second life of antibodies by E. V. Navolotskaya (1-7).
Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (∼75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289–292 of the CH2-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341–344), immunorphin (364–373), immunocortin (11–20), and peptide p24 (335–358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed.
Keywords: antibodies; peptides; receptors; immune system

Basolateral expression of GRP94 in parietal cells of gastric mucosa by R. M. Arin; Y. Rueda; O. Casis; M. Gallego; A. I. Vallejo; B. Ochoa (8-15).
GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-lig- and binding experiments. Both the K D value obtained in saturation experiments with N-ethylcarboxamido-[3H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.
Keywords: gastric mucosa parietal cell; basolateral polarization; molecular chaperone; adenosine receptor

Mechanism of Saccharomyces cerevisiae yeast cell death induced by heat shock. Effect of cycloheximide on thermotolerance by E. G. Rikhvanov; I. V. Fedoseeva; N. N. Varakina; T. M. Rusaleva; A. V. Fedyaeva (16-24).
The mechanism of yeast cell death induced by heat shock was found to be dependent on the intensity of heat exposure. Moderate (45°C) heat shock strongly increased the generation of reactive oxygen species (ROS) and cell death. Pretreatment with cycloheximide (at 30°C) suppressed cell death, but produced no effect on ROS production. The protective effect was absent if cycloheximide was added immediately before heat exposure and the cells were incubated with the drug during the heat treatment and recovery period. The rate of ROS production and protective effect of cycloheximide on viability were significantly decreased in the case of severe (50°C) heat shock. Treatment with cycloheximide at 39°C inhibited the induction of Hsp104 synthesis and suppressed the development of induced thermotolerance to severe shock (50°C), but it had no effect on induced thermotolerance to moderate (45°C) heat shock. At the same time, Hsp104 effectively protected cells from death independently of the intensity of heat exposure. These data indicate that moderate heat shock induced programmed cell death in the yeast cells, and cycloheximide suppressed this process by inhibiting general synthesis of proteins.
Keywords: cycloheximide; Saccharomyces cerevisiae ; thermotolerance; Hsp104; reactive oxygen species; programmed cell death

A novel approach for fluorescent visualization of glycyrrhetic acid on a cell with a quantum dot by Jie Hou; Qian Shi; Meirong Cao; Pengwei Pan; Guangbo Ge; Xuran Fan; Gang Bai; Yi Xin (25-30).
Glycyrrhetic acid (GA), a pentacyclic triterpenoid derivative obtained from hydrolysis of glycyrrhizic acid, was found to have synergistic anti-asthmatic effects with the β2-adrenergic receptor (β2AR) agonist via the β2AR-mediated pathway. This study visualized the location of GA on a human cell expressing β2AR via chemical biological approaches. A CdTe/ZnS quantum dot modified with an alkynyl group (QD-AL) was first synthesized, and an azide-terminal GA (ATGA) was also prepared. The QD-AL was used for fluorescence visualization of the distribution of GA on human embryonic kidney 293 cells expressing fusion β2AR (HEK293-β2AR) through the “click reaction” between QD-AL and ATGA. The average size of the QD-AL nanoparticle was about 10 nm, and its fluorescent emission wavelength was 620 nm. The location of GA on the HEK293-β2AR cell membrane can be visualized by the click reaction (between QD-AL and ATGA). The ability of QD-AL targeting to ATGA on the cell membrane of a HEK293-β2AR cell was further investigated using both confocal laser-scanning microscopy and a cellular uptake-inhibition assay. The results reveal that QD-AL can recognize ATGA on the cell membrane through the click reaction, which provides a new approach for visualizing the location of GA on the cell in an indirect way, and it can be applied to explore the synergistic anti-asthmatic mechanism of GA with β2AR agonist through the β2AR mediated pathway.
Keywords: fluorescent visualization; glycyrrhetic acid; quantum dot; click reaction

Base excision repair (BER) is the major pathway involved in removal of endogenous and mutagen-induced DNA damage. The X-ray cross-complementing group 1 protein (XRCC1), which participates in BER, is a scaffolding protein. The oxidized XRCC1 N-terminal domain (NTD) forms additional interactions with DNA polymerase β (Pol β). Any change in the residues of a protein (XRCC1, XRCC4, etc.) may alter its stability and function. Many coding regions of genes have single nucleotide polymorphisms (SNPs) that change the conformation of their products, and they are probably involved in some diseases. The R7L and R107H mutations are located in the XRCC1-NTD. In the present study, biophysical chemical properties of oxidized XRCC1-NTD (wild type or mutants) were investigated at different temperatures (290, 295, 298, 301, 304, 309, 310, 311, and 312 K) in water using in silico molecular mechanic computational methods. Comparison of the average calculated potential energies of oxidized XRCC1-NTD reveals that the R7L mutation increases stability, but the R107H and R7L&R107H mutations are destabilizing. Therefore, mutant types of this protein (R107H or R7L&R107H) may not function correctly. Furthermore, quantitative structure-activity relationship (QSAR) of oxidized XRCC1-NTD and docking assay showed that the R7L mutation is advantageous but the R107H and R7L&R107H mutations are disadvantageous for XRCC1-NTD, and in the latter cases it cannot interact with Pol β as well as the wild type does. Hence, DNA repair may be defective. Also, using the equation dE = ∂E/(∂T)V·dT + ∂E/(∂V)T·dV, it was determined that the best temperature for normal activity of oxidized XRCC1-NTD is exactly the natural body temperature (310 K).
Keywords: XRCC1; NTD; polymorphism; in silico ; QSAR; docking; dE

Straight core structure of DNA replication origins in the mammalian AMPD2 locus by Q. A. Lima Neto; F. S. Rando; D. V. B. Freitas; L. F. Rodrigues; F. R. Rosado; A. Fiorini; F. Gimenes; J. Tavares; M. A. Fernandez (37-43).
Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (∼300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.
Keywords: intrinsically bent DNA sites; DNA replication origins; AMPD2 mammalian amplified domain

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria by O. V. Akopova; L. I. Kolchinskaya; V. I. Nosar; V. A. Bouryi; I. N. Mankovska; V. F. Sagach (44-53).
The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K+ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨm). At K+ concentrations within the range of 0–120 mM, an increase in the initial rate of K+-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K+-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K ATP + -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K+ uptake. The ATP-dependent transport of K+ was shown to be ∼40% of the potential-dependent K+ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K+ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K ATP + -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.
Keywords: potassium; brain mitochondria; reactive oxygen species; K ATP + -channel

Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: Structural and functional characteristics by T. N. Stekhanova; E. Y. Bezsudnova; A. V. Mardanov; E. M. Osipov; N. V. Ravin; K. G. Skryabin; V. O. Popov (54-61).
Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe2+-containing nicotinamidase (k cat/K m = 427 mM−1·sec−1)/pyrazinamidase (k cat/K m = 331 mM−1·sec−1). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T1/2 (60°C) = 180 min, T1/2 (80°C) = 35 min) and thermophilicity (Topt = 90°C, Ea = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As084 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.
Keywords: nicotinamidase; pyrazinamidase; archaeon; C-D-K catalytic triad; metal binding motif

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases by O. E. Melkina; I. I. Goryanin; I. V. Manukhov; A. V. Baranova; V. A. Kolb; M. S. Svetlov; G. B. Zavilgelsky (62-68).
The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.
Keywords: trigger factor; DnaKJE chaperone; luciferase; refolding

Investigation of the regulatory function of archaeal ribosomal protein L4 by A. O. Mikhaylina; O. S. Kostareva; A. V. Sarskikh; R. V. Fedorov; W. Piendl; M. B. Garber; S. V. Tishchenko (69-76).
Ribosomal protein L4 is a regulator of protein synthesis in the Escherichia coli S10 operon, which contains genes of 11 ribosomal proteins. In this work, we have investigated regulatory functions of ribosomal protein L4 of the thermophilic archaea Methanococcus jannaschii. The S10-like operon from M. jannaschii encodes not 11, but only five ribosomal proteins (L3, L4, L23, L2, S19), and the first protein is L3 instead of S10. We have shown that MjaL4 and its mutant form lacking an elongated loop specifically inhibit expression of the first gene of the S10-like operon from the same organism in a coupled transcription?translation system in vitro. By deletion analysis, an L4-binding regulatory site has been found on MjaL3 mRNA, and a fragment of mRNA with length of 40 nucleotides has been prepared that is necessary and sufficient for the specific interaction with the MjaL4 protein.
Keywords: ribosomal protein L4; S10-like operon of mRNA; archaea; regulation of ribosomal protein synthesis; coupled transcription-translation system in vitro