Biochemistry (Moscow) (v.77, #6)

Structure of human DNA polymerase iota and the mechanism of DNA synthesis by A. V. Makarova; A. V. Kulbachinskiy (547-561).
Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX–XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.
Keywords: DNA polymerase iota; Y-family of DNA polymerases; replication; DNA damage; Hoogsteen base-pairing

Ribosomal proteins: Structure, function, and evolution by A. V. Korobeinikova; M. B. Garber; G. M. Gongadze (562-574).
The question concerning reasons for the variety of ribosomal proteins that arose for more than 40 years ago is still open. Ribosomes of modern organisms contain 50–80 individual proteins. Some are characteristic for all domains of life (universal ribosomal proteins), whereas others are specific for bacteria, archaea, or eucaryotes. Extensive information about ribosomal proteins has been obtained since that time. However, the role of the majority of ribosomal proteins in the formation and functioning of the ribosome is still not so clear. Based on recent data of experiments and bioinformatics, this review presents a comprehensive evaluation of structural conservatism of ribosomal proteins from evolutionarily distant organisms. Considering the current knowledge about features of the structural organization of the universal proteins and their intermolecular contacts, a possible role of individual proteins and their structural elements in the formation and functioning of ribosomes is discussed. The structural and functional conservatism of the majority of proteins of this group suggests that they should be present in the ribosome already in the early stages of its evolution.
Keywords: ribosomal proteins; RNA-protein interactions; ribosome; evolution

p21Waf1 was identified as a protein suppressing cyclin E/A-CDK2 activity and was originally considered as a negative regulator of the cell cycle and a tumor suppressor. It is now considered that p21Waf1 has alternative functions, and the view of its role in cellular processes has begun to change. At present, p21Waf1 is known to be involved in regulation of fundamental cellular programs: cell proliferation, differentiation, migration, senescence, and apoptosis. In fact, it not only exhibits antioncogenic, but also oncogenic properties. This review provides a contemporary understanding of the functions of p21Waf1 depending on its intracellular localization. On one hand, when in the nucleus, it serves as a negative cell cycle regulator and tumor suppressor, in particular by participating in the launch of a senescence program. On the other hand, when p21Waf1 is localized in the cytoplasm, it acts as an oncogene by regulating migration, apoptosis, and proliferation.
Keywords: p21Waf1 ; oncogene; cell cycle; senescence; cytoplasmic localization; apoptosis

Protein-protein interactions play a significant role in regulation of functional activity of cytochrome P450s. The aim of the present study was to elucidate the molecular interactions between steroidogenic enzymes CYP17 and CYP21 localized in endoplasmic reticulum membranes of adrenal cortex and involved in biosynthesis of corticosteroid hormones. In the present work, we demonstrate for the first time the direct interaction at molecular level between highly purified human recombinant cytochrome P450s in a mixed reconstituted system. The dependence of the interaction between CYP17 and CYP21 on concentration of the redox-partner — NADPH-cytochrome P450 reductase — is demonstrated, and it is shown that electrostatic interactions play a crucial role in the interaction between CYP17 and CYP21.
Keywords: cytochrome P450; CYP17; CYP21; protein-protein interaction; steroid hormone biosynthesis

During incubation of a constant volume of rat liver cytosol with an increasing quantity of mitochondrial protein in the presence of 3.3 mM MgCl2, the binding of nucleoside diphosphate kinase (NDPK) from the cytosol to mitochondrial membranes is described by a saturation curve. The highest bound NDPK activity accounts for less than 9% of the added activity. Analysis of the results suggests that only one NDPK isozyme is bound to the membranes. Western blotting showed it to be NDPK α, a homolog of human NDPK-B. Substrates of NDPK, hexokinase, and glycerol kinase, as well as N,N’-dicyclohexylcarbodiimide and palmitate, did not influence the association of NDPK with mitochondrial membranes. We conclude that the sites of NDPK binding to the outer mitochondrial membrane are not identical to those of hexokinase and glycerol kinase.
Keywords: nucleoside diphosphate kinase; mitochondria; outer compartment; liver; isozymes; binding

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells by T. V. Komarova; E. V. Sheval; D. V. Pozdyshev; V. S. Kolesnikova; Yu. L. Dorokhov (603-608).
Although high level of recombinant protein production can be achieved via transient expression in plant cells, the mechanism by which tolerance to the presence of recombinant protein is acquired remains unclear. Here we show that green fluorescent protein (GFP) encoded by an intron-optimized tobacco mosaic viral vector formed large membraneless GFP bodies called Y-bodies that demonstrated mainly perinuclear localization. The Y-bodies were heterogeneous in size, approaching the size of the cell nucleus. Experiments with extracted GFP and live cell imaging showed that Y-bodies included actively fluorescent, non-aggregated, tightly packed GFP molecules. The plant cells probably formed Y-bodies to exclude the recombinant protein from normal physiological turnover.
Keywords: Agrobacterium tumefaciens ; green fluorescent protein (GFP); viral vector; Y-bodies; tobacco mosaic virus; transient expression

Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45 by O. G. Ovchinnikova; B. Liu; N. A. Kocharova; A. S. Shashkov; A. N. Kondakova; M. Siwinska; L. Feng; A. Rozalski; L. Wang; Y. A. Knirel (609-615).
A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional ROESY and H-detected 1H,13C HSQC experiments. The polysaccharide contains N-acetylglu-cosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: $$ o 4) - eta - D - GlcpNAc - (1 o 4) - eta - D - GlcpNAc3(Rlac - L - Ala) - (1 o .$$ The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.
Keywords: Providencia alcalifaciens ; lipopolysaccharide; N-acetylmuramic acid; structure; bacterial polysaccharide; peptidoglycan

Comparison of experimental and theoretical data on hydrogen-deuterium exchange for ten globular proteins by M. Yu. Suvorina; A. K. Surin; N. V. Dovidchenko; M. Yu. Lobanov; O. V. Galzitskaya (616-623).
The number of protons available for hydrogen-deuterium exchange was predicted for ten globular proteins using a method described elsewhere by the authors. The average number of protons replaced by deuterium was also determined by mass spectrometry of the intact proteins in their native conformations. Based on these data, we find that two models proposed earlier agree with each other in estimation of the number of protons replaced by deuterium. Using a model with a probability scale for hydrogen bond formation, we estimated a number of protons replaced by deuterium that is close to the experimental data for long-term incubation in D2O (24 h). Using a model based on estimations with a scale of the expected number of contacts in globular proteins there is better agreement with the experimental data obtained for a short period of incubation in D2O (15 min). Therefore, the former model determines weakly fluctuating parts of a protein that are in contact with solvent only for a small fraction of the time. The latter model (based on the scale of expected number of contacts) predicts either flexible parts of a protein chain exposed to interactions with solvent or disordered parts of the protein.
Keywords: hydrogen-deuterium exchange; secondary structure; hydrogen bond; mass spectrometry

Hybrid system based on quantum dots and photosystem 2 core complex by E. G. Maksimov; V. N. Kurashov; M. D. Mamedov; V. Z. Paschenko (624-630).
We show that semiconductor nanocrystals (quantum dots, QD) can be used to increase the absorption capacity of pigment-protein complexes. In a mixture of photosystem 2 core complex (PS2) and QD, the fluorescence of the latter decreases several-fold due to the transfer of the absorbed energy to the PS2 core complex. We discuss Forster’s inductive-resonance mechanism as a possible way of energy transfer in donor-acceptor pairs QD-PS2 core complex. Calculations based on the experimental data show that the enhancement of PS2 fluorescence and the rate of QA reduction increase up to 60% due to efficient energy migration from QD to PS2.
Keywords: quantum dots; photosystem 2; hybrid systems; energy transfer

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin by A. V. Sokolov; V. A. Kostevich; D. N. Romanico; E. T. Zakharova; V. B. Vasilyev (631-638).
A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K i for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A 610/A 280 ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.
Keywords: ceruloplasmin; neomycin; affinity chromatography

Unlike Naja naja, Bungarus caeruleus, Echis carinatus, and Daboia/Vipera russellii venoms, Ophiophagus hannah venom is medically ignored in the Indian subcontinent. Being the biggest poisonous snake, O. hannah has been presumed to inject several lethal doses of venom in a single bite. Lack of therapeutic antivenom to O. hannah bite in India makes any attempt to save the victim a difficult exercise. This study was initiated to compare O. hannah venom with the above said venoms for possible interference in hemostasis. Ophiophagus hannah venom was found to actively interfere in hemostatic stages such as fibrin clot formation, platelet activation/aggregation, and fibrin clot dissolution. It decreased partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin clotting time (TCT). These activities are similar to that shown by E. carinatus and D. russellii venoms, and thus O. hannah venom was found to exert procoagulant activity through the common pathway of blood coagulation, while N. naja venom increased aPTT and TCT but not PT, and hence it was found to exert anticoagulant activity through the intrinsic pathway. Venoms of O. hannah, E. carinatus, and D. russellii lack plasminogen activation property as they do not hydrolyze azocasein, while they all show plasmin-like activity by degrading the fibrin clot. Although N. naja venom did not degrade azocasein, unlike other venoms, it showed feeble plasmin-like activity on fibrin clot. Venom of E. carinatus induced clotting of human platelet rich plasma (PRP), while the other three venoms interfered in agonist-induced platelet aggregation in PRP. Venom of O. hannah least inhibited the ADP induced platelet aggregation as compared to D. russellii and N. naja venoms. All these three venoms showed complete inhibition of epinephrine-induced aggregation at varied doses. However, O. hannah venom was unique in inhibiting thrombin induced aggregation.
Keywords: O. hannah ; D. russellii ; E. carinatus ; N. naja ; fibri(noge)nolytic; defibrinogenation; platelet aggregation

We show the development of clearly pronounced age-related pathological changes in eye tissues of Wistar and OXYS rats. Photoreceptor cells were virtually absent in all OXYS rats in the age of 24 months. Massive accumulations of lipofuscin granules were detected in the pigmented epithelium cells. Flattening, overgrowing, and degradation of endothelial cells of choriocapillaries were also observed. Along with these changes, vessels without signs of degradation were detected in the pigmented epithelium. In 24-month-old Wistar rats these changes were local and were seen in only some of the animals. The mitochondria-targeted antioxidant SkQ1 (the rats were given SkQ1 daily with food at the dose of 250 nmol/kg for 5 months, starting from the age of 19 months) prevented the development of these pathological changes in both Wistar and OXYS rats. The data were subjected to mathematical processing and statistical analysis.
Keywords: ultrastructure; oxidative stress; retina; choriocapillary layer; antioxidant

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor a165 by S. V. Tillib; T. I. Ivanova; E. Yu. Lyssuk; S. S. Larin; A. V. Kibardin; E. V. Korobko; P. N. Vikhreva; N. V. Gnuchev; G. P. Georgiev; I. V. Korobko (659-665).
Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A165 has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A165. We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A165 as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A165, which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A165-dependent pathological neoangiogenesis.
Keywords: nanoantibodies; human vascular endothelial growth factor VEGF-A165 ; diagnostics; ELISA; VEGF-A165 inhibitor

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss by S. S. Jankauskas; E. Y. Plotnikov; M. A. Morosanova; I. B. Pevzner; L. D. Zorova; V. P. Skulachev; D. B. Zorov (666-670).
The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals’ mortality. Treatment with SkQR1 also decreases gentamycininduced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.
Keywords: kidney; organ of Corti; mitochondria; antioxidant; nephrotoxicity; ototoxicity; antibiotics

Cerebral ischemia-reperfusion induces GAPDH S-nitrosylation and nuclear translocation by Chong Li; Jun-Jun Feng; Yong-Ping Wu; Guang-Yi Zhang (671-678).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, plays an important role in glycolysis. It was reported that GAPDH undergoes S-nitrosylation, which facilitated its binding to Siah1 and resulted in nuclear translocation and cell apoptosis. The results of this study show that GAPDH S-nitrosylation, Siah1 binding, translocation to nucleus, and concomitant neuron death occur during the early stages of reperfusion in the rat four-vessel occlusion ischemic model. N-Methyl-D-aspartate receptor antagonist MK801, neuronal nitric oxide synthase inhibitor 7-nitroindazole, or monoamine oxidase-B inhibitor (R)-(-)-deprenyl hydrochloride could inhibit GAPDH S-nitrosylation and translocation and exert neuroprotective effects.
Keywords: GAPDH; S-nitrosylation; Siah1; deprenyl hydrochloride; cerebral ischemia

Phosphatase activity in barley proteins tightly bound to DNA and its development-dependent changes by K. Bielskienė; D. Labeikytė; N. Sjakste; L. Bagdonienė; B. Juodka (679-688).
The tightly bound proteins (TBPs), a protein group that remains attached to DNA either covalently or noncovalently after deproteinization, have been found in numerous eukaryotic species. Some TBPs isolated from mammalian and yeast cells possess phosphatase or kinase activity. The aim of this study was to characterize further TBPs in barley (Hordeum vulgare) cells. The spectra of TBPs varied in different organs of barley shoots (first leaves, coleoptile, and roots) and at different developmental stages of the plant. Some barley TBPs manifested phosphatase, probably Ser/Thr or dual Ser/Thr/Tyr activity. MALDI-TOF mass spectrometry of barley TBPs identified several proteins involved in chromatin rearrangement and regulation processes, including transcription factors, serpins, protein phosphatases and protein kinases, RNA helicases, and DNA topoisomerase II.
Keywords: tightly bound proteins; nuclear matrix; phosphatase