Biochemistry (Moscow) (v.82, #4)

Role of proton pumps in tumorigenesis by V. A. Kobliakov (401-412).
One of the differences between normal and cancer cells is lower pH of the extracellular space in tumors. Low pH in the extracellular space activates proteases and stimulates tumor invasion and metastasis. Tumor cells display higher level of the HIF1α transcription factor that promotes cell switch from mitochondrial respiration to glycolysis. The terminal product of glycolysis is lactate. Lactate formation from pyruvate is catalyzed by the specific HIF1α-dependent isoform of lactate dehydrogenase A. Because lactate accumulation is deleterious for the cell, it is actively exported by monocarboxylate transporters. Lactate is cotransported with proton, which acidifies the extracellular space. Another protein that contributes to proton concentration increase in the extracellular space is tumor-specific HIF1α-dependent carbonic anhydrase IX, which generates a proton in the reaction between carbon dioxide and water. The activity of Na+/H+ exchanger (another protein pump) is stimulated by stress factors (e.g. osmotic shock) and proliferation stimuli. This review describes the mechanisms of proton pump activation and reviews results of studies on effects of various proton pump inhibitors on tumor functioning and growth in cell culture and in vivo. The prospects of combined application of proton pump inhibitors and cytostatics in cancer therapy are discussed.
Keywords: invasion; metastasis; lactate dehydrogenase; monocarboxylate transporters; carbonic anhydrase; Na+/H+ exchanger; hypoxia; HIF1α

Natural and chemotherapy-induced clonal evolution of tumors by M. K. Ibragimova; M. M. Tsyganov; N. V. Litviakov (413-425).
Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.
Keywords: clonal evolution; tumor; chemotherapy; mutation; metastasis

Formyl peptide receptors (FPRs) expressed by mammalian myeloid cells are the important part of innate immunity. They belong to the seven-transmembrane domain class of receptors coupled to heterotrimeric GTP-binding proteins. Binding of the receptor with a wide spectrum of exogenous and endogenous ligands triggers such defensive phagocyte reactions as chemotaxis, secretory degranulation, and respiratory burst, keeping a balance of inflammatory and antiinflammatory processes in the organism. The association between single nucleotide polymorphisms in the gene of FPR1 receptor resulting in disruption of the receptor structure and the development of certain pathologies accompanied with inflammation, such as aggressive periodontitis, macular degeneration, and even gastric cancer (Maney, P., and Walters, J. D. (2009) J. Periodontol., 80, 1498-1505; Liang, X. Y., et al. (2014) Eye, 28, 1502-1510; Otani, T., et al. (2011) Biochem. Biophys. Res. Commun., 405, 356-361) has been shown. In this review, we matched the missense mutation of formyl-peptide receptors with their known functional domains and classified them according to their potential significance in pathology.
Keywords: formyl peptide receptors; FPR1; FPR2; single nucleotide polymorphisms

HuR stabilizes lnc-Sox5 mRNA to promote tongue carcinogenesis by Lifang Wang; Shucheng Ye; Junye Wang; Zhenfang Gu; Yanhui Zhang; Chunmei Zhang; Xuezhen Ma (438-445).
Long noncoding RNAs (lncRNAs) have been recently regarded as systemic regulators in multiple biological processes including tumorigenesis. In this study, we report an ultra-highly expressed lncRNA, lnc-Sox5, in tongue tumor tissues. The results imply that lnc-Sox5 may play vital role in tongue carcinoma progression. We observed that the growth of Tca8113 cells was suppressed by lnc-Sox5 downregulation. Additionally, lnc-Sox5 knockdown simultaneously increased Tca8113 cell apoptosis, but the cell cycle was arrested. RNA immunoprecipitation suggested that HuR directly bound to and stabilized lnc-Sox5 RNA. Consistently, HuR knockdown reduced the level of lnc-Sox5 in Tca8113 cells. However, overexpression of HuR induced more lnc-Sox5 in Tca8113 cells. Both lnc-Sox5 knockdown and HuR knockdown suppressed Tca8113 cell tumorigenesis in xenograft models. These results suggest that lnc-Sox5, which was stabilized by HuR, could regulate carcinogenesis of tongue cancer and may serve as a predicted target for tongue carcinoma therapies.
Keywords: long non-coding RNA; lnc-Sox5; tongue carcinoma; cell cycle; apoptosis; HuR; RNA immunoprecipitation

Overall analysis and understanding of mechanisms are of great importance for treatment of infantile pneumonia due to its high morbidity and mortality worldwide. In this study, we preliminarily explored the function and mechanism of focal adhesion kinase (FAK) in regulation of inflammatory response induced by lipopolysaccharides in A549 cells. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, quantitative reverse transcription polymerase chain reaction, and Western blot analysis were used to explore the correlation of FAK expression with cell apoptosis, viability, and the inflammatory cytokine activity in A549 cells. The results showed that knockdown of FAK enhanced cell viability, suppressed apoptosis, and decreased inflammatory cytokine activity. In addition, downregulation of FAK could activate the Wnt and nuclear factor κB signaling pathways. These findings suggest that FAK might be involved in progression of infantile pneumonia and could be a new therapeutic target for this disease.
Keywords: infantile pneumonia; lipopolysaccharides; focal adhesion kinase; Wnt and NF-κB pathways; new therapeutic target

Toward gene therapy of hypertension: Experimental study on hypertensive ISIAH rats by M. N. Repkova; A. S. Levina; A. A. Seryapina; N. V. Shikina; E. V. Bessudnova; V. F. Zarytova; A. L. Markel (454-457).
TiO2-based nanocomposites were prepared to deliver oligonucleotides into cells. The nanocomposites were designed by the immobilization of polylysine-containing oligonucleotides on TiO2-nanoparticles (TiO2·PL-DNA). We showed for the first time the possibility of using the proposed nanocomposites for treatment of hypertensive disease by introducing them into hypertensive ISIAH rats developed as a model of stress-sensitive arterial hypertension. The mRNA of the gene encoding angiotensin I-converting enzyme (ACE1) involved in the synthesis of angiotensin II was chosen as a target. Administration (intraperitoneal injection and inhalation) of the nanocomposite showed a significant (by 20-30 mm Hg) decrease in systolic blood pressure when the nanocomposite contained the ACE1 gene-targeted oligonucleotide. When using the oligonucleotide with a random sequence, no effect was observed. Further development and improvement of the inhalation nanocomposite drug delivery to systemic hypertensive disease treatment promises new possibilities for clinical practice.
Keywords: antisense oligonucleotides; hypertension; ISIAH rats; nanocomposites

Triosephosphates as intermediates of the Crabtree effect by S. S. Sokolov; O. V. Markova; K. D. Nikolaeva; I. A. Fedorov; F. F. Severin (458-464).
An increase in glucose concentration in the medium rapidly decreases respiration rate in many cell types, including tumor cells. The molecular mechanism of this phenomenon, the Crabtree effect, is still unclear. It was shown earlier that adding the intermediate product of glycolysis fructose-1,6-bisphosphate to isolated mitochondria suppresses their respiration. To study possible roles of glycolytic intermediates in the Crabtree effect, we used a model organism, the yeast Saccharomyces cerevisiae. To have the option to rapidly increase intracellular concentrations of certain glycolytic intermediates, we used mutant cells with glycolysis blocked at different stages. We studied fast effects of glucose addition on the respiration rate in such cells. We found that addition of glucose affected cells with deleted phosphoglycerate mutase (strain gpm1-delta) more strongly than ones with inactivated aldolase or phosphofructokinase. In the case of preincubation of gpm1-delta cells with 2-deoxyglucose, which blocks glycolysis at the stage of 2-deoxyglucosephosphate formation, the effect of glucose addition was absent. This suggests that triosephosphates are intermediates of the Crabtree effect. Apart from this, the incubation of gpm1-delta cells in galactose-containing medium appeared to cause a large increase in their size. It was previously shown that galactose addition did not have any short-term effect on respiration rate of gpm1-delta cells and, at the same time, strongly suppressed their growth rate. Apparently, the influence of increasing triosephosphate concentration on yeast physiology is not limited to the activation of the Crabtree effect.
Keywords: gpm1 ; glycolysis; yeast; inhibition of respiration; Crabtree effect

Effects of solvent environments on protein refolding have gained significant attention due to their biotechnological and pharmaceutical applications. Recent advances have shown that a number of organic osmolytes have the unique ability to induce proper folding of several misfolded proteins and simultaneously inhibit aggregation during the process. In the present study, we investigated the effects of polyol osmolytes on the refolding of guanidinium chloride-denatured ribonuclease-A (RNase-A) and compared it with that of other osmolyte types. Measurements of enzymatic activity parameters (K m and k cat) clearly indicate that polyol-induced RNase-A folding enhanced its catalytic efficiency as compared to folding in the absence of osmolytes or in the presence of osmolytes of other types. Furthermore, structural characterization revealed that the increase in catalytic efficiency stems from conformational alterations of the polyol-induced folded protein molecules as compared to other types of osmolytes.
Keywords: osmolytes; polyol; protein folding; refolding; catalytic efficiency; microenvironment

MicroRNA-294 promotes cellular proliferation and motility through the PI3K/AKT and JAK/STAT pathways by upregulation of NRAS in bladder cancer by Yongwei Li; Zhengfei Shan; Chu Liu; Diandong Yang; Jitao Wu; Changping Men; Yankai Xu (474-482).
In our study we examined the role of microRNA-294 (miR-294) in bladder cancer and related mechanisms. Realtime polymerase chain reaction (RT-PCR) was performed to determine the expression level of miR-294. Western blot was used to determine the expression of NRAS, mainly factors in the PI3K/AKT and JAK/STAT pathways. Cell counting kit8 assay, clonogenic assay, wound-healing assay, transwell and flow cytometry were used to explore, respectively, cell proliferation, survival, migration, invasion, and apoptosis of bladder cancer cell line T24. The expressions of miR-294 in bladder cancer cells including J82, HT1376, T24, and SW780 were significantly increased compared to those in human bladder epithelium cells (both HCV29 and SV-HUC-1). The proliferation rate, surviving fraction, migration, and invasion of T24 cells in miR-294 mimetic transfected group were significantly increased, while they were significantly decreased by miR294 inhibitor transfection. Moreover, miR-294 suppression could increase the apoptotic rate of T24 cells. In addition, drug resistance of T24 cells to cisplatin was increased in miR-294 mimetic-treated group, while it was decreased by miR-294 inhibitor compared to empty control. Overexpression of miR-294 could upregulate NRAS expression in T24 cells and activate PI3K/AKT and JAK/STAT pathways. We found that miR-294 expression was positively related with proliferation and motility of T24 cells. Moreover, miR-294 suppression could promote the sensitivity of T24 cells to cisplatin. We also found miR-294 could upregulate NRAS and activate the PI3K/AKT and JAK/STAT pathways in T24 cells.
Keywords: microRNA-294; NRAS; PI3K/AKT and JAK/STAT pathways; bladder cancer

Structure and gene cluster of the K93 capsular polysaccharide of Acinetobacter baumannii B11911 containing 5-N-Acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid by A. A. Kasimova; M. M. Shneider; N. P. Arbatsky; A. V. Popova; A. S. Shashkov; K. A. Miroshnikov; Veeraraghavan Balaji; Indranil Biswas; Yu. A. Knirel (483-489).
Capsular polysaccharide (CPS) assigned to the K93 type was isolated from the bacterium Acinetobacter baumannii B11911 and studied by sugar analysis along with oneand two-dimensional 1H and 13C NMR spectroscopy. The CPS was found to contain a derivative of pseudaminic acid, and the structure of the branched tetrasaccharide repeating unit was established. Genes in the KL93 capsule biosynthesis locus were annotated and found to be consistent with the CPS structure established. The K93 CPS has the α-D-Galp-(1→6)-β-D-Galp-(1→3)-D-GalpNAc trisaccharide fragment in common with the K14 CPS of Acinetobacter nosocomialis LUH 5541 and A. baumannii D46. It also shares the β-D-Galp-(1→3)-DGalpNAc disaccharide fragment and the corresponding predicted Gal transferase Gtr5, as well as the initiating GalNAc-1P transferase ItrA2, with a number of A. baumannii strains.
Keywords: Acinetobacter baumannii ; capsular polysaccharide structure; capsule biosynthesis locus; pseudaminic acid; glycosyltransferase

Femtosecond and picosecond dynamics of recombinant bacteriorhodopsin primary reactions compared to the native protein in trimeric and monomeric forms by O. A. Smitienko; O. V. Nekrasova; A. V. Kudriavtsev; M. A. Yakovleva; I. V. Shelaev; F. E. Gostev; D. A. Dolgikh; I. B. Kolchugina; V. A. Nadtochenko; M. P. Kirpichnikov; T. B. Feldman; M. A. Ostrovsky (490-500).
Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bRrec) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bRrec was produced in an Escherichia coli expression system. Since bRrec was prepared in a DMPC–CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bRtrim and bRmon, respectively) was carried out. We found that bRrec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bRtrim and bRmon. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bRrec, occurred more slowly compared to bRtrim, but similarly to bRmon. The lifetime of intermediate I, judging from the signal of ΔA ESA(470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bRrec, 0.52 ps (73%) and 1.7 ps (27%) for bRmon, and 0.45 ps (90%) and 1.75 ps (10%) for bRtrim. The formation time of intermediate K, judging from the signal of ΔA GSA(625-635 nm), was 13.5 ps for bRrec, 9.8 ps for bRmon, and 4.3 ps for bRtrim. In addition, there was a decrease in the photoreaction efficiency of bRrec and bRmon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bRrec are similar to those of the monomeric form of the native protein, bRrec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.
Keywords: bacteriorhodopsin; recombinant protein; primary reactions; femtosecond absorption laser spectroscopy

Studying factors involved in biogenesis of Lysobacter sp. XL1 outer membrane vesicles by I. V. Kudryakova; N. E. Suzina; N. G. Vinokurova; N. A. Shishkova; N. V. Vasilyeva (501-509).
The Gram-negative bacterium Lysobacter sp. XL1 produces outer membrane vesicles that are heterogeneous in size, density, and protein composition. One of the subpopulations is secretory vesicles for lytic protease L5 of Lysobacter sp. XL1 (Kudryakova et al. (2015) FEMS Microbiol. Lett., 362, fnv137). Protein L5 was assumed to influence biogenesis of these secretory vesicles that contain it. Using a Pseudomonas fluorescens Q2-87/B expression system, it was shown that the recombinant L5 protein may act as a factor of vesicle biogenesis. This points to a possible involvement of L5 protein in Lysobacter sp. XL1 vesicle biogenesis. Furthermore, it was established that the main phospholipid of Lysobacter sp. XL1 vesicles is cardiolipin, and vesicles are formed predominantly of outer membrane regions enriched with this phospholipid. This indicates that cardiolipin participates in biogenesis of all vesicle subpopulations in Lysobacter sp. XL1.
Keywords: factors of outer membrane vesicle biogenesis; bacteriolytic protease L5; cardiolipin; Lysobacter sp. XL1; lytic effect

Trastuzumab and pertuzumab plant biosimilars: Modification of Asn297-linked glycan of the mAbs produced in a plant with fucosyltransferase and xylosyltransferase gene knockouts by T. V. Komarova; E. V. Sheshukova; E. N. Kosobokova; M. V. Serebryakova; V. S. Kosorukov; V. N. Tashlitsky; Y. L. Dorokhov (510-520).
Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPBWT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPBΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297linked GnGn-type glycan contains α1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.
Keywords: plant; monoclonal antibody; immunoglobulin G; trastuzumab; pertuzumab; glycosylation; ΔXTFT N. benthamiana with knockout of XT and FT genes; immunotherapy; biosimilars

Short exogenous peptides regulate expression of CLE, KNOX1, and GRF family genes in Nicotiana tabacum by L. I. Fedoreyeva; T. A. Dilovarova; V. V. Ashapkin; Yu. Ts. Martirosyan; V. Kh. Khavinson; P. N. Kharchenko; B. F. Vanyushin (521-528).
Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10–7-10–9 M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.
Keywords: cell differentiation; Nicotiana tabacum ; gene expression; short peptides; plant growth regulation