Biochemistry (Moscow) (v.78, #3)

The gastrointestinal tract is subject to a huge antigenic load, which is especially significant in the intestinal lumen. Being the connecting link between the organism and the external environment, the small intestine fulfils not only digestive and transport functions, but also protective ones and acts as a selective barrier for the flow of nutrients. This review considers proteases of the protective system of small intestine cells, their biochemical properties and activation mechanisms, and involvement in biochemical processes responsible for normal functioning and defense reactions of the intestine. Serine proteases of intestinal immunity are multifunctional enzymes making proteolytic attack aimed to immediately exterminate aggressive elements of the intestinal contents (allergens, toxins), to activate (inactivate) zymogens, receptors, and peptide hormones, and to hydrolyze protein precursors and other biologically active factors. Proteases of intestinal immunity control the inflammatory response, proliferation of B-lymphocytes, apoptosis, and secretory and contractive activity of the intestine; they release neurogenic factors, inactivate biologically active substances, and are involved in degradation of the intercellular matrix and in tissue remodeling.
Keywords: serine proteases; tryptases; chymases; granzymes; cathepsin G; small intestine; immunity

Molecular functions of small regulatory noncoding RNA by Yong Huang; Ji Liang Zhang; Xue Li Yu; Ting Sheng Xu; Zhan Bin Wang; Xiang Chao Cheng (221-230).
Recently, using large-scale genomic sequencing, a great number of small noncoding RNAs (ncRNA) has been discovered. Short ncRNAs can be classified into three major classes — small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA). These short ncRNAs ranging from 20 to 300 nt in size are now recognized as a new paradigm of gene regulation for controlling many biological processes. In this paper, we review the biogenesis and recent research on the functions of small regulatory non-coding RNAs and aim at understanding their important functions in living organisms.
Keywords: miRNAs; siRNAs; piRNAs; biogenesis; regulation; functions

Structural organization of viral RNA-dependent RNA polymerases by G. S. Shatskaya; T. M. Dmitrieva (231-235).
This review describes available data on the structure of viral RNA-dependent RNA polymerases (RdRP) obtained from X-ray analysis and discusses the functional significance of the structural elements of these enzymes. Because most of the studies done to date relate to RdRP structures of picorna-, flavi-, and caliciviruses, here we consider mostly the structures of RdRP of these groups of viruses, and also include information about polymerases of other virus families.
Keywords: RNA-dependent RNA polymerase; viral proteins; picornaviruses; polymerase structure

Approaches to the design of selective ligands for membrane progesterone receptor alpha by O. V. Lisanova; T. A. Shchelkunova; I. A. Morozov; P. M. Rubtsov; I. S. Levina; L. E. Kulikova; A. N. Smirnov (236-243).
A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6–C7-double bond. The data suggest that the modes of lig- and interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.
Keywords: membrane progesterone receptor alpha; steroid ligand binding; selectivity

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes, but the mechanism of its function in the biogenesis of iron-sulfur cluster remains elusive. In this paper, we demonstrate that Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of 4·1020 M−1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form of IscA can be converted to iron-sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99, or Cys101 in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron-sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro. Taken together, these findings suggest that A. ferrooxidans IscA is not only an iron-sulfur protein, but also an iron binding protein that can act as an iron donor for biogenesis of iron-sulfur clusters.
Keywords: Acidithiobacillus ferrooxidans ; IscA; IscU; iron-sulfur cluster

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine by V. I. Timofeev; S. A. Kuznetsov; V. Kh. Akparov; G. G. Chestukhina; I. P. Kuranova (252-259).
The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO 4 2− ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 sub-sites in these three carboxypeptidases was revealed. It was found that SO 4 2− ion occupies the same position in the S1’ subsite as the C-terminal carboxy group of the substrate.
Keywords: carboxypeptidase T; S1 subsite; crystal structure; X-ray structural analysis

The interaction between myosin and actin in striated muscle tissue is regulated by Ca2+ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, β-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, β-, and Γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.
Keywords: skeletal myosin isoforms; tropomyosin isoforms; in vitro motility assay

We were the first to show that MbO2 deoxygenation in the cell occurs only upon interaction of myoglobin with mitochondrial membrane, which must be accompanied by changes in the heme cavity conformation of the protein and its affinity for the ligand. Under aerobic conditions, some changes in the equilibrium O2 dissociation constant (K dis) can be detected by changes of the rate of MbO2 autooxidation, i.e. spontaneous turning it into metMb (k ox), as far as a direct correlation between K dis and k ox is experimentally shown. In this work, we studied the effect on MbO2 autooxidation rate of phospholipid liposomes from neutral soybean phosphatidylcholine (lecithin) and from negatively charged 1-palmitoyl-2-oleylphosphatidylglycerol (POPG) at various phospholipid/MbO2 ratios from 25 : 1 to 100 : 1, and also the effect of rat liver mitochondria at concentration of 1 and 2 mg/ml mitochondrial protein (at 22 and 37°C). In all cases, k ox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on kox is significantly greater than that of neutral lecithin liposomes. At the POPG/MbO2 molar ratio of 25 : 1, MbO2 autooxidation rate is almost 25-fold increased compared to the control, whereas in the presence of 50-fold molar excess of lecithin, k ox is only ∼10 times higher (10 mM buffer, pH 7.2, 22°C). With the same phospholipid/MbO2 ratio of 100: 1, k ox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, k ox grows proportionally to their concentration (about 10-fold per 1 mg/ml of mitochondrial protein), and practically does not change after adding superoxide dismutase in the reaction mixture. The k ox value decreases markedly at high ionic strength, thus suggesting an important role of coulombic electrostatics in the myoglobin-mitochondrial interaction. The increase in the autooxidation rate of MbO2 (and hence its K dis) due to the interaction with phospholipid membranes points to decreasing affinity of myoglobin for oxygen, which facilitates O2 detachment from MbO2 at physiological p 02 values.
Keywords: myoglobin; autooxidation; phospholipid membranes; mitochondria

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle by V. V. Sirenko; A. H. Simonyan; A. V. Dobrzhanskaya; N. S. Shelud’ko; Y. S. Borovikov (273-281).
Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.
Keywords: calponin-like muscle protein of mussel; ghost fibers; ATP analogs; intermediate states of actomyosin; conformational changes of myosin subfragment-1

Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis by T. A. Sushko; A. A. Gilep; A. V. Yantsevich; S. A. Usanov (282-289).
CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ4-steroids and Δ5-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ5–Δ7 steroids as a substrate.
Keywords: cytochrome P450; CYP17; CYP21; 7-dehydrosteroids; steroid hormone biosynthesis

Activities of polygalacturonase and 1,3-β-glucanase increased in campion (Silene vulgaris) callus cells during co-cultivation with the fungus Trichoderma harzianum. This was associated with a decrease in galacturonic acid residues in the pectic polysaccharide of campion silenan and also in the production of pectin by the callus. Co-cultivation of the callus and the fungus resulted in an increase in contents of arabinose residues in the intracellular arabinogalactan and in contents of galactose residues in the extracellular arabinogalactan.
Keywords: callus; Trichoderma harzianum ; Silene vulgaris ; enzymes; pectic polysaccharides; silenan

Brain aging and mitochondria-targeted plastoquinone antioxidants of SkQ-type by N. K. Isaev; E. V. Stelmashook; N. N. Stelmashook; I. N. Sharonova; V. G. Skrebitsky (295-300).
Normal brain aging leads to decrease in cognitive functions, shrink in brain volume, loss of nerve fibers and degenerating myelin, reduction in length and branching of dendrites, partial loss of synapses, and reduction in expression of genes that play central roles in synaptic plasticity, vesicular transport, and mitochondrial functioning. Impaired mitochondrial functions and mitochondrial reactive oxygen species can contribute to the damage of these genes in aging cerebral cortex. This review discusses the possibility of using mitochondria-targeted antioxidants to slow the processes of brain aging.
Keywords: brain aging; neurons; mitochondria-targeted antioxidants

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria by G. A. Naberezhnykh; V. I. Gorbach; G. N. Likhatskaya; S. Yu. Bratskaya; T. F. Solov’eva (301-308).
The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.
Keywords: lipopolysaccharide; chitosan; liposomes; binding; computer modeling

Interaction of synthetic peptide octarphin with human blood lymphocytes by Yu. N. Nekrasova; Yu. A. Zolotarev; E. V. Navolotskaya (309-313).
The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of nonopioid β-endorphin receptor, was prepared with specific activity 28 Ci/mmol. The binding of [3H]octarphin to T and B lymphocytes isolated from the blood of donors was studied. It was found that [3H]octarphin binds both to T and B cells with high affinity: K d = 3.0 ± 0.2 and 3.2 ± 0.3 nM, respectively. The specific binding of [3H]octarphin to T and B lymphocytes was competitively inhibited by unlabeled β-endorphin (K i = 1.9 ± 0.2 and 2.2 ± 0.3 nM, respectively) and was not inhibited by unlabeled naloxone, [Met5]enkephalin, [Leu5]enkephalin, α-endorphin, and γ-endorphin. Thus, T and B lymphocytes of human blood possess a nonopioid β-endorphin receptor whose binding is provided by the fragment 12–19 (the octarphin sequence).
Keywords: peptide; receptors; naloxone; β-endorphin; lymphocytes

The formation of lymphogenic metastases remains enigmatic. In particular, the much more pronounced predilection of carcinomas than of sarcomas to metastasizing into regional lymph nodes is an unsolved problem. We suggest that this difference could be due to the ability of epitheliocytes for a hypothetical process termed by us “collateral presentation of antigens”. Under conditions of infection of epithelium with intracellular pathogens or during inflammation, epithelial cells acquire a special receptor phenotype, undergo the epithelial-mesenchymal transition, and migrate along lymphatic vessels into lymph nodes where they present antigen to immunocytes. The collateral presentation of antigens can be of significant biological importance in the case of insufficient classical pathway of antigen presentation (by dendritic cells) or on disturbance in the death mechanisms of the infected cells. Depending on conditions of induction of the epithelial-mesenchymal transition and on possible ability of epitheliocytes to express MHC II with co-stimulating molecules, two pathways, “container-mediated” and “MHC II-dependent”, of antigen presentation in lymph nodes resulting in development of immunogenesis or anergy of immunocytes are supposed to exist. All pathways of delivery of the epithelial cells into lymph nodes and of antigen presentation by epitheliocytes terminate by death of these cells. The lymphogenic metastasizing realizes the same mechanism under conditions of tumor disease; however, this is not associated with cell death, but they actively colonize the lymph node. The proposed hypothesis allows us to explain the metastasizing of sarcomas into lymph nodes. The main prerequisite for lymphogenic metastasizing seems to be related with the mesenchymal-epithelial transition of sarcoma cells promoting their involvement in the presentation of antigens.
Keywords: collateral presentation of antigens; lymphogenic metastasizing; benign epithelial inclusions; MHC II; carcinomas; sarcomas