Biochemistry (Moscow) (v.81, #3)

Overview of fusion tags for recombinant proteins by E. N. Kosobokova; K. A. Skrypnik; V. S. Kosorukov (187-200).
Virtually all recombinant proteins are now prepared using fusion domains also known as “tags”. The use of tags helps to solve some serious problems: to simplify procedures of protein isolation, to increase expression and solubility of the desired protein, to simplify protein refolding and increase its efficiency, and to prevent proteolysis. In this review, advantages and disadvantages of such fusion tags are analyzed and data on both well-known and new tags are generalized. The authors own data are also presented.
Keywords: fusion tags; chromatography; enhancement of protein solubility

Chlorophylls d and f and their role in primary photosynthetic processes of cyanobacteria by S. I. Allakhverdiev; V. D. Kreslavski; S. K. Zharmukhamedov; R. A. Voloshin; D. V. Korol’kova; T. Tomo; J.-R. Shen (201-212).
The finding of unique Chl d- and Chl f-containing cyanobacteria in the last decade was a discovery in the area of biology of oxygenic photosynthetic organisms. Chl b, Chl c, and Chl f are considered to be accessory pigments found in antennae systems of photosynthetic organisms. They absorb energy and transfer it to the photosynthetic reaction center (RC), but do not participate in electron transport by the photosynthetic electron transport chain. However, Chl d as well as Chl a can operate not only in the light-harvesting complex, but also in the photosynthetic RC. The long-wavelength (Qy) Chl d and Chl f absorption band is shifted to longer wavelength (to 750 nm) compared to Chl a, which suggests the possibility for oxygenic photosynthesis in this spectral range. Such expansion of the photosynthetically active light range is important for the survival of cyanobacteria when the intensity of light not exceeding 700 nm is attenuated due to absorption by Chl a and other pigments. At the same time, energy storage efficiency in photosystem 2 for cyanobacteria containing Chl d and Chl f is not lower than that of cyanobacteria containing Chl a. Despite great interest in these unique chlorophylls, many questions related to functioning of such pigments in primary photosynthetic processes are still not elucidated. This review describes the latest advances in the field of Chl d and Chl f research and their role in primary photosynthetic processes of cyanobacteria.
Keywords: chlorophyll d ; chlorophyll f ; photosystem 1; photosystem 2; photosynthesis; energetics of photosystems; spectral properties; cyanobacteria

Structure and functions of linker histones by A. V. Lyubitelev; D. V. Nikitin; A. K. Shaytan; V. M. Studitsky; M. P. Kirpichnikov (213-223).
Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions — from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.
Keywords: linker histones; chromatin structure; eukaryotic transcription

Aging is accompanied by increased production of free oxygen radicals and impairment of normal cellular functions. The aim of this work was to provide preliminary data on age-related differences in the activities of antioxidant enzymes and phase II biotransformation enzyme glutathione S-transferase (GST) in a wild population of the Asian clam Corbicula fluminea. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and GST were assessed in visceral mass of four age classes (0+-, 1+-, 2+-, and 3+-year-old) of C. fluminea clams. Age-related changes were seen in antioxidant enzyme status: levels of total SOD (totSOD) (P < 0.05), MnSOD, and CuZnSOD (P < 0.05) activities increased progressively during aging from younger to older clams. Changes in CAT and GR activities with advancing age were found, the levels being the highest in age class II, then being lower in age classes III and IV (P < 0.05). Activities of GPX and GST were lower in the senescent individuals (2+- and 3+-year-old clams) compared with young individuals (0+- and 1+-year-old clams). Overall, the decline of glutathione-dependent enzyme activities, coupled with higher and lower activities of totSOD and CAT, respectively, as the individual grows older, may render the older animals more susceptible to oxidative stress. Data reported here are not intended to be exhaustive since they concern only age/size structure of the population at one locality, so more detailed studies on both the developmental stages and levels of antioxidant enzymes of this new alien species in Serbian rivers are required.
Keywords: Asian clam; antioxidant enzyme; GST; aging

Interaction of nucleotide excision repair protein XPC—RAD23B with DNA containing benzo[a]pyrene-derived adduct and apurinic/apyrimidinic site within a cluster by L. V. Starostenko; E. A. Maltseva; N. A. Lebedeva; P. E. Pestryakov; O. I. Lavrik; N. I. Rechkunova (233-241).
The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms — NER and BER, respectively. Interaction of NER protein XPC—RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N2-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC—RAD23B crosslinks to DNA containing (+)-trans-BPDE-N2-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N2-dG isomers depends on the AP site position in the opposite strand. The influence of XPC—RAD23B on hydrolysis of an AP site clustered with BPDE-N2-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC—RAD23B was shown to stimulate the endonuclease and inhibit the 3′–5′ exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.
Keywords: protein factors of nucleotide excision repair; benzo[a]pyrene derivatives; apurinic/apyrimidinic site; cluster-type DNA damage; affinity labeling; DNA–protein complexes

Effect of thrombin inhibitors on positive feedback in the coagulation cascade by N. B. Ustinov; E. G. Zav’yalova; A. M. Kopylov (242-248).
The coagulation cascade is a series of sequential reactions of limited proteolysis of protein factors resulting in generation of thrombin. Thrombin mediates both positive and negative feedback in regulating this cascade by taking part in activation of several factors. Some thrombin inhibitors, by affecting positive feedback, inhibit generation of thrombin itself. In the current study, we used two thrombin inhibitors: argatroban, a low molecular weight reversible competitive inhibitor that binds to the active site, and bivalirudin, a bivalent oligopeptide that blocks the active site and binding center of protein substrates (exosite I). Appearance rate and total amount of thrombin were measured in a thrombin generation assay (TGA) using a fluorescent substrate. We found that argatroban slows the appearance of thrombin and lowers its amount. Bivalirudin also slows appearance of thrombin, but it does not decrease its amount, perhaps because the region being bound to the active site undergoes hydrolysis so that the inhibitor stops binding to thrombin. Many reactions of the coagulation cascade proceed on the surface of phospholipid micelles (PLMs). In the case of argatroban, PLMs do not affect the results of the TGA, whereas for bivalirudin they lower its inhibitory activity. It seems that PLMs stabilize protein complexes (wherein thrombin exosite I is hindered) mediating positive feedback in the coagulation cascade, e.g. complexes of thrombin with factor V and VIII.
Keywords: thrombin; thrombin generation assay; argatroban; bivalirudin

Identification of a region of the polypeptide chain of Na,K-ATPase α-subunit interacting with 67-kDa melittin-like protein by Yu. V. Kamanina; E. A. Klimanova; E. A. Dergousova; I. Yu. Petrushanko; O. D. Lopina (249-254).
It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.
Keywords: Na,K-ATPase; melittin-like proteins; protein—protein interactions

Tankyrase activity in organs and tissues of mice by A. N. Kuimov; A. S. Zhozhikashvili; V. N. Manskikh; L. V. Platonova; T. G. Dyuzheva (255-262).
Tankyrase, one of the NAD+ ADP-ribosyltransferases, is a target for drugs developed for their anticancer and other pharmacological activities. We designed an assay for estimation of the inhibition or activation of the enzyme in pre-clinical studies. In mice, the highest specific activity of tankyrase was observed in thymus, spleen, pancreas, and bone marrow. In murine liver, tankyrase is active in ontogenesis and during reparative regeneration; however, the basal activity is hardly detectable in normal liver and most of other organs of adult animals. We suggest that tankyrase is a part of the tissue growth and repair machinery, while its age-dependent inhibition, when an organism stops growing, turns on phenoptosis.
Keywords: ADP-ribosyltransferase; Essentiale N; hepatic stimulator substance; poly(ADP-ribosyl)ation; regeneration

DNA with damage in both strands as affinity probes and nucleotide excision repair substrates by N. V. Lukyanchikova; I. O. Petruseva; A. N. Evdokimov; V. N. Silnikov; O. I. Lavrik (263-274).
Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dCFAB and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dCFAB/dG (probe I), dCFAB/nFlu+4 (probe II), and dCFAB/nFlu−3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC—HR23B (Kd um > Kd I > Kd II Kd III ) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dCFAB results in (i) decreased melting temperature (ΔTm = −3°C) and (ii) 12° DNA bending. The extended dCFAB/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC—HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dCFAB/nFlu+4, dCFAB/nFlu−3) or irreparable (nFlu/nFlu+4, nFlu/nFlu−3, nAnt/nFlu+4, nAnt/nFlu−3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.
Keywords: DNA; bulky DNA lesions; protein factors of NER; photoaffinity labeling

Dynamics of antagonistic potency of Rhodobacter capsulatus PG lipopolysaccharide against endotoxin-induced effects by D. S. Kabanov; D. A. Serov; S. V. Zubova; S. V. Grachev; I. R. Prokhorenko (275-283).
The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.
Keywords: lipopolysaccharide (endotoxin); antagonist; cytokine; TNF-α; IL-1β; IL-6; IL-8; IL-10; IL-1Ra; IFN-γ

A non-autocatalytic mathematical model of self-oscillations in vitro in solutions of cyanobacterial Kai proteins (KaiA, KaiB, KaiC) and ATP is suggested. This model describes the process of phosphorylation/dephosphorylation of KaiC protein, which is accelerated by KaiA and inhibited by KaiB. The method of metabolic control analysis is used to show that frequency (period) as well as amplitude of self-oscillations of components of Kai proteins are temperature-compensated.
Keywords: biochemical self-oscillations; Kai proteins; control analysis of frequency; control analysis of amplitude

Glycation-induced high-density lipoprotein (HDL) modification by aldehydes can result in loss of its antiinflammatory/antioxidative properties, contributing to diabetes-associated cardiovascular diseases. Isoferulic acid, a major active ingredient of Cimicifuga heracleifolia, shows antiinflammatory, antiviral, antioxidant, and antidiabetic properties. Thus, this study investigated the antiglycation effect of isoferulic acid against compositional modifications of HDL and loss of biological activity of HDL-paraoxonase induced on incubation with different aldehydes. Protective effect of isoferulic acid was assessed by subjecting purified HDL from human plasma to glycation with methylglyoxal, glyoxal, or glycolaldehyde and varying concentrations of isoferulic acid. The effect of isoferulic acid was analyzed by determining amino group number, tryptophan and advanced glycation end-product fluorescence, thermal denaturation studies, carboxymethyl lysine content, and activity of HDL-paraoxonase. Concentration-dependent inhibitory action of isoferulic acid was observed against extensive structural perturbations, decrease in amino group number, increase in carboxymethyl lysine content, and decrease in the activity of HDL-paraoxonase caused by aldehyde-associated glycation in the HDL molecule. Isoferulic acid, when taken in concentration equal to that of aldehydes, was most protective, as 82-88% of paraoxonase activity was retained for all studied aldehydes. Isoferulic acid shows antiglycation action against aldehyde-associated glycation in HDL, which indicates its therapeutic potential for diabetic patients, especially those with micro-/macrovascular complications.
Keywords: aldehydes; glycation; high-density lipoprotein-AGEs; isoferulic acid; paraoxonase