Biochemistry (Moscow) (v.80, #3)

Oligonucleotide inhibitors of telomerase: Prospects for anticancer therapy and diagnostics by M. I. Zvereva; T. S. Zatsepin; D. M. Azhibek; O. S. Shubernetskaya; O. V. Shpanchenko; O. A. Dontsova (251-259).
The activity of telomerase allows eukaryotic cells to have unlimited division potential. On its functioning, telomerase synthesizes short DNA repeats at the 3′-end of DNA within chromosomes that ensures genome stability during cell division. Telomerase is active in the majority of cancer cell types and is virtually absent in somatic cells with rare exceptions. This difference allows us to consider inhibition of telomerase activity as a possible approach to antitumor therapy. Telomerase is a nucleoprotein composed of two main components: the reverse transcriptase (hTERT), which is a catalytic subunit, and telomerase RNA (hTR), which encodes a template for synthesis of repeats. The biogenesis and features of telomerase seem very promising for its inhibition due to complementary interactions. In this review, we analyze putative pathways of oligonucleotide influence on telomerase and consider the known native and modified oligonucleotide inhibitors of telomerase, as well as possible mechanisms of their action. We also discuss the application of telomerase-targeted oligonucleotide conjugates for in vivo imaging of tumor cells.
Keywords: human telomerase; telomerase inhibitors; anticancer therapy target; oligonucleotide; tumor cell imaging

Rat liver sinusoidal surface N-linked glycoproteomic analysis by affinity enrichment and mass spectrometric identification by Jianglin Li; Jun Gao; Miao Jiang; Jia Chen; Zhonghua Liu; Ping Chen; Songping Liang (260-275).
Glycosylation in liver is one of the most biologically important protein modifications. It plays critical roles in many physiological and pathological processes by virtue of its unique location at the blood-tissue interface, including angiogenesis, liver cancer, cirrhosis, and fibrosis. To analyze glycosylation of plasma membrane proteins in liver sinusoidal endothelial cells (LSEC), N-glycopeptides of the LSEC surface were enriched using a filter-assisted sample preparation-based lectin affinity capture method and subsequently identified with mass spectrometry. In total, 225 unique N-glycosylation sites on 152 glycoproteins were identified, of which 119 (53%) sites had not previously been determined experimentally. Among the glycoproteins, 53% were classified as plasma membrane proteins and 47 (31%) as signaling proteins and receptors. Moreover, 23 cluster of differentiation antigens with 49 glycopeptides were detected within the membrane glycoproteins of the liver sinusoidal surface. Furthermore, bioinformatics analysis revealed that the majority of identified glycoproteins have an impact on processes of LSEC. Therefore, N-glycoproteomic analysis of the liver sinusoidal surface may provide useful information on liver regeneration and facilitate liver disease diagnosis.
Keywords: liver sinusoidal endothelial surface; N-glycoproteomic; N-glyco-FASP; mass spectrometry

Downregulation of miR-221, -30d, and -15a contributes to pathogenesis of prostate cancer by targeting Bmi-1 by Hanqing Xuan; Wei Xue; Jiahua Pan; Jianjun Sha; Baijun Dong; Yiran Huang (276-283).
Prostate cancer is the second leading cause of cancer-related deaths of men. Bmi-1, a member of PcG family of proteins, has been implicated in the pathogenesis of prostate cancer, and disturbed profile of microRNAs (miRNAs) has been found in prostate cancer tissues. How Bmi-1 is regulated by miRNAs is unclear. In this study, we screened 18 miRNAs that potentially repress the expression of Bmi-1 using a dual luciferase system and found that 12 miRNAs could bind with the 3′-untranslated region of Bmi-1 mRNA. Using qRT-PCR, we found that expression of miR-221, -15a, and -30d was significantly reduced in prostate cancer tissues. Subsequent functional study indicated that miR-221 and miR-30d can repress prostate cancer cell proliferation, and this effect can be partially rescued by Bmi-1 overexpression. Our study constructs the relation between downregulated miR-221 and miR-30d and prostate cancer pathogenesis. These results indicate that miR-221 and miR-30d are candidate tumor suppressor miRNAs in prostate cancer and therefore serve as potential clinical classification markers and therapeutic targets for human prostate cancer.
Keywords: miRNA; prostate cancer; Bmi-1; cell proliferation

A 3D-model of bovine testicular hyaluronidase (BTH) was constructed based on established tertiary structure of human hyaluronidase Hyal1 using a molecular homological modeling method in silico. The analysis of the BTH 3D-model demonstrated lysine residue stratification during enzyme modification. The 3D-model of chondroitin sulfate (CHS)-modified hyaluronidase (BTH-CHS) was obtained by modeling covalent binding of lysine residues with benzoquinone-activated CHS. The degree of enzyme modification and the length of CHS chains were varied during 3D modeling. The importance of deep BTH modification degree for the formation of active and stable enzyme derivatives was shown, as determined earlier experimentally. The effective size of the CHS coat for productive BTH modification was confirmed. It is theoretically achieved at the increase in molecular mass of BTH-CHS derivative to approximately 140–180 kDa and can be practically obtained, according to experimental data, using CHS of different molecular mass (30–50 as well as 120–140 kDa).
Keywords: bovine testicular hyaluronidase; tertiary structure; chondroitin sulfate; lysine residue modification; hyaluronidase-chondroitin sulfate conjugate; modification degree; chondroitin sulfate coat

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: Structural, biochemical, and functional characterization by M. G. Alekseeva; T. A. Mironcheva; D. A. Mavletova; S. M. Elizarov; N. V. Zakharevich; V. N. Danilenko (296-309).
The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the β- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, β, γ, δ, ɛ, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, β-, α- and ɛ-subunits. The β-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.
Keywords: FoF1-ATP synthase; Ser/Thr protein kinase; inverted membrane vesicles; Streptomycetes

Epigenetic mechanisms of peptidergic regulation of gene expression during aging of human cells by V. V. Ashapkin; N. S. Linkova; V. Kh. Khavinson; B. F. Vanyushin (310-322).
Expression levels of genes encoding specific transcription factors and other functionally important proteins vary upon aging of pancreatic and bronchial epithelium cell cultures. The peptides KEDW and AEDL tissue-specifically affect gene expression in pancreatic and bronchial cell cultures, respectively. It is established in this work that the DNA methylation patterns of the PDX1, PAX6, NGN3, NKX2-1, and SCGB1A1 gene promoter regions change upon aging in pancreatic and bronchial cell cultures in correlation with variations in their expression levels. Thus, stable changes in gene expression upon aging of cell cultures could be caused by changes in their promoter methylation patterns. The methylation patterns of the PAX4 gene in pancreatic cells as well as those of the FOXA1, SCGB3A2, and SFTPA1 genes in bronchial cells do not change upon aging and are unaffected by peptides, whereas their expression levels change in both cases. The promoter region of the FOXA2 gene in pancreatic cells contains a small number of methylated CpG sites, their methylation levels being affected by cell culture aging and KEDW, though without any correlation with gene expression levels. The promoter region of the FOXA2 gene is completely unmethylated in bronchial cells irrespective of cell culture age and AEDL action. Changes in promoter methylation might be the cause of age- and peptide-induced variations in expression levels of the PDX1, PAX6, and NGN3 genes in pancreatic cells and NKX2-1 and SCGB1A1 genes in bronchial cells. Expression levels of the PAX4 and FOXA2 genes in pancreatic cells and FOXA1, FOXA2, SCGB3A2, and SFTPA1 genes in bronchial cells seem to be controlled by some other mechanisms.
Keywords: short peptides; DNA methylation; transcription; cell differentiation; pancreatic cell culture; bronchial cell culture; differentiation factors

Hybrid structures of polycationic aluminum phthalocyanines and quantum dots by E. G. Maksimov; D. A. Gvozdev; M. G. Strakhovskaya; V. Z. Paschenko (323-331).
Semiconductor nanocrystals (CdSe/ZnS quantum dots, QDs) were used as inorganic focusing antenna, allowing for the enhancement of fluorescence and photosensitizing activity of polycationic aluminum phthalocyanines (PCs). It was found that QDs form stable complexes with PCs in aqueous solutions due to electrostatic interactions. In such hybrid complexes, we observed highly efficient nonradiative energy transfer from QD to PC, leading to a sharp increase in the effective absorption cross section of PC in the absorption bands of the CdSe/ZnS quantum dots. When hybrid complexes are excited within these bands, the intensity of PC fluorescence and the rate of photosensitized singlet oxygen generation increases significantly (up to 500 and 350%, correspondingly) compared to free PC at the same concentration. The observed effect is of interest for modeling primary stages of photosynthesis and increasing photosensitizing activity of dyes used in photodynamic therapy.
Keywords: quantum dots; aluminum phthalocyanines; hybrid systems; energy migration; reactive oxygen species

A novel defensin-like antifungal peptide (Tf-AFP) with molecular mass of 10.3 kDa was isolated from seeds of Trigonella foenum-graecum (fenugreek) by ammonium sulfate precipitation, cation-exchange, gel-filtration, hydrophobic chromatography, and RP-HPLC. Mass spectroscopic analysis revealed the intact mass of the purified antifungal peptide as 10321.5 Da and high similarity to plant defensins and other antifungal proteins in database search. 2D-PAGE showed pI value to be 8.8 and absence of isoforms. Isolated Tf-AFP inhibited growth of fungal species such as Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. The antifungal activity was inhibited in the presence of 50 mM NaCl. Circular dichroism analysis demonstrated that the protein is rich in β-sheet structure and highly stable over a wide range of temperatures. Surprisingly, reduction of disulfide bridges and chemical denaturation did not produce large changes in secondary structure as judged by circular dichroism as well as by fluorescence spectroscopy.
Keywords: chromatographic method; defensin-like antifungal peptide; MALDI-TOF-MS; biochemical and biophysical studies; Trigonella foenum-graecum

Seasonal changes in isoform composition of giant proteins of thick and thin filaments and titin (connectin) phosphorylation level in striated muscles of bears (Ursidae, Mammalia) by N. N. Salmov; I. M. Vikhlyantsev; A. D. Ulanova; Yu. V. Gritsyna; A. G. Bobylev; A. P. Saveljev; V. V. Makariushchenko; G. Yu. Maksudov; Z. A. Podlubnaya (343-355).
Seasonal changes in the isoform composition of thick and thin filament proteins (titin, myosin heavy chains (MyHCs), nebulin), as well as in the phosphorylation level of titin in striated muscles of brown bear (Ursus arctos) and hibernating Himalayan black bear (Ursus thibetanus ussuricus) were studied. We found that the changes that lead to skeletal muscle atrophy in bears during hibernation are not accompanied by a decrease in the content of nebulin and intact titin-1 (T1) isoforms. However, a decrease (2.1–3.4-fold) in the content of T2 fragments of titin was observed in bear skeletal muscles (m. gastrocnemius, m. longissimus dorsi, m. biceps) during hibernation. The content of the stiffer N2B titin isoform was observed to increase relative to the content of its more compliant N2BA isoform in the left ventricles of hibernating bears. At the same time, in spite of the absence of decrease in the total content of T1 in the myocardium of hibernating brown bear, the content of T2 fragments decreased ∼1.6-fold. The level of titin phosphorylation only slightly increased in the cardiac muscle of hibernating brown bear. In the skeletal muscles of brown bear, the level of titin phosphorylation did not vary between seasons. However, changes in the composition of MyHCs aimed at increasing the content of slow (I) and decreasing the content of fast (IIa) isoforms of this protein during hibernation of brown bear were detected. Content of MyHCs I and IIa in the skeletal muscles of hibernating Himalayan black bear corresponded to that in the skeletal muscles of hibernating brown bear.
Keywords: hibernation; brown bear; Himalayan black bear; striated muscles; thick (myosin) filaments; thin (actin) filaments; titin; nebulin; isoforms of myosin heavy chains; Pro-Q Diamond

Chromatin in rat liver nuclei under conditions of low ionic strength (20–25 mM) and [Mg2+] from 2 to 5 mM has a condensed structure (100–200 nm globules) and gives the same CD signal (320–340 nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1 mM leads to chromatin decondensation to 30 nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA = 6 and in the presence of 5 mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3 mM Mg2+ leads to chromatin decondensation to 30 nm fibrils. Extraction of about half of histone H1 at [Mg2+] ⩽ 2 mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1–2 min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5‐7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA = 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA = 30 and DM/DNA = 0.1, about 5–10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging.
Keywords: cell nuclei; chromatin; levels of structural organization; distamycin; polyglutamic acid; histone H1; CD spectra

Taurine modulates catalytic activity of cytochrome P450 3A4 by V. V. Shumyantseva; A. A. Makhova; T. V. Bulko; R. Bernhardt; A. V. Kuzikov; E. V. Shich; V. G. Kukes; A. I. Archakov (366-373).
The influence of the biologically active compound taurine on the stability and catalytic properties of the hemoprotein cytochrome P450 3A4 has been investigated. The catalytic properties were analyzed by electrochemical methods (cyclic and square-wave voltammetry) using cytochrome P450 3A4 immobilized on the electrode. Taurine at concentrations in the range 10–70 μM stimulated the electrochemical reduction of cytochrome P450 3A4, and the reduction was the highest (115 ± 3%) in the presence of 50 μM taurine. Taurine pronouncedly attenuated the itraconazol-caused inhibition of the P450 isoenzyme P450 3A4. Taurine protected cytochrome P450 3A4 due to stabilizing it during electrolysis at controlled voltage in the presence of erythromycin as a substrate. This protection was manifested by an increase in the amount of the “residual” reduced form of the hemoprotein (52 ± 5 and 71 ± 8%, respectively).
Keywords: cytochrome P450; antioxidants; drug metabolism; bioelectrochemistry; taurine

Normal level of sepsis-associated phenylcarboxylic acids in human serum by N. V. Beloborodova; V. V. Moroz; A. A. Osipov; A. Yu. Bedova; A. Yu. Olenin; M. L. Getsina; O. V. Karpova; E. G. Olenina (374-378).
Previous studies showed that large amounts of phenylcarboxylic acids (PhCAs) are accumulated in a septic patient’s blood due to increased endogenous and microbial phenylalanine and tyrosine biotransformation. Frequently, biochemical aromatic amino acid transformation into PhCAs is considered functionally insignificant for people without monogenetic hereditary diseases. The blood of healthy people contains the same PhCAs that are typical for septic patients as shown in this paper. The overall serum PhCAs level was 6 μM on average as measured by gas chromatography with flame ionization detection. This level is a stable biochemical parameter indicating the normal metabolism of aromatic amino acids. The concentrations of PhCAs in the metabolic profile of healthy people are distributed as follows: phenylacetic ≈ p-hydroxyphenyllactic > p-hydroxyphenylacetic > phenyllactic ≈ phenylpropionic > benzoic. We conclude that maintaining of stable PhCAs level in the serum is provided as the result of integration of human endogenous metabolic pathways and microbiota.
Keywords: tyrosine metabolism; sepsis; phenylcarboxylic acids; phenyllactic acid; p-hydroxyphenyllactic acid; phenylpropionic acid; benzoic acid; microbiota

Heparan sulfate 6-O-sulfotransferase 3 is involved in bone marrow mesenchymal stromal cell osteogenic differentiation by Shancheng Zhao; Chao Deng; Zhen Wang; Liping Teng; Jinghua Chen (379-389).
The roles of sugar chains such as heparan sulfate (HS) in stem cell self-renewal and differentiation are poorly understood. HS is a sugar chain with linear sulfated polyanionic disaccharide repeating structures that interact with many proteins, including structural proteins in the extracellular matrix and growth factors and their receptors. Thus, unraveling the role of HS in stem cell self-renewal and differentiation could provide new insights and technical routes in clinical stem cell applications. Here, we purified rat bone marrow mesenchymal stromal cells (BMMSCs) by density gradient centrifugation, analyzed mesenchymal stromal cell surface stemness marker expression by flow cytometry, and identified the sulfotransferases responsible for sulfation ester modification of HS. An osteogenic differentiation model was established by chemical induction reagents and confirmed via alkaline phosphatase (ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMMSCs before and after osteogenic induction were detected by RT-PCR and Western blot. Cell spheroids were formed in both control and osteogenic culture systems when BMMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) mRNA and protein were upregulated in these calcified cell spheroids. HS6ST3 knockdown BMMSCs were established with RNA interference, and they had significantly lower ALP activity and decreased expression of the osteogenic differentiation markers Runx2 and Ocn. These findings suggest that HS6ST3 is involved in BMMSC differentiation, and new glycotherapeutic-based technologies could be developed in the future.
Keywords: BMMSCs; osteogenic differentiation; extracellular matrix; heparan sulfate; sulfotransferase