Biochemistry (Moscow) (v.77, #11)

Biogenesis of β-barrel integral proteins of bacterial outer membrane by T. F. Solov’eva; O. D. Novikova; O. Yu. Portnyagina (1221-1236).
Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.
Keywords: Gram-negative bacteria; outer membrane; integral protein; biogenesis; chaperones; SecYEG-translocon; ATPase SecA; Bam-complex

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.
Keywords: amyloid fibril; protofibril; oligomer; nucleus; insulin; injection amyloidosis

Redox control of cardiac rhythm by V. I. Kapelko (1248-1257).
The rhythm of cardiac beats is generated by pacemaker cells differing from other cardiomyocytes by the presence of slow diastolic depolarization. Consistently activated transmembrane ionic currents provide cyclic excitation of pacemakers, forming the original “membrane clocks”. A new concept has been forwarded in the last decade according to which periodic fluctuations in myoplasmic Ca2+ level (“calcium clocks”) not only influence a course of “membrane clocks”, but they also can serve as independent generators of the rhythm. Transport of Ca2+ in cells is under constant influence of active forms of oxygen and nitrogen. Both superoxide and NO in moderate doses facilitate Ca2+ output from the sarcoplasmic reticulum, accelerating the course of “calcium clocks”, but in higher doses they have opposite effect that may be neutralized mainly by reduced glutathione. The control of cardiac rhythm by active forms of oxygen and nitrogen represents a feedback mechanism by which mitochondria and NO-synthases support Ca2+ homeostasis in cells that can be temporarily disturbed under mechanical loads or hypoxia.
Keywords: diastolic depolarization; Ca2+ transport; ryanodine receptor; sarcoplasmic reticulum; mitochondria; NO-synthases

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus by S. M. Elizarov; M. G. Alekseeva; F. N. Novikov; G. G. Chilov; D. A. Maslov; A. A. Shtil; V. N. Danilenko (1258-1265).
We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca2+-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.
Keywords: aminoglycoside phosphotransferase; serine/threonine protein kinases; antibiotic resistance; phosphorylation sites; site-directed mutagenesis

Actin isoforms and reorganization of adhesion junctions in epithelial-to-mesenchymal transition of cervical carcinoma cells by G. S. Shagieva; L. V. Domnina; T. A. Chipysheva; V. D. Ermilova; C. Chaponnier; V. B. Dugina (1266-1276).
Malignant cell transformation requires changes in the ability of cells to migrate. The disruption of actin cytoskeleton and intercellular adhesions is an important component of the acquisition of invasive properties in epithelial malignancies. The invasive ability of carcinoma cells is associated with reduced expression of adhesion junction molecules and increased expression of mesenchymal markers, frequently referred to as epithelial-to-mesenchymal transition (EMT). Standard features of the EMT program in cancer cells include fibroblastic phenotype, downregulation of the epithelial marker E-cadherin, induction of Snail-family transcription factors, as well as expression of mesenchymal proteins. We compared the epithelial and mesenchymal marker profiles of nonmalignant HaCaT keratinocytes to the corresponding profiles of cervical carcinoma cell lines C-33A, SiHa, and CaSki. The characteristics of the EMT appeared to be more developed in SiHa and CaSki cervical cancer cells. Further activation of the EMT program in cancer cells was induced by epidermal growth factor. Decreased epithelial marker E-cadherin in CaSki cells was accompanied by increased mesenchymal markers N-cadherin and vimentin. Downregulated expression of E-cadherin in SiHa and CaSki cells was associated with increased expression of Snail transcription factor. Our goal was to study actin reorganization in the EMT process in cell cultures and in tissue. We found that β-cytoplasmic actin structures are disorganized in the cervical cancer cells. The expression of β-cytoplasmic actin was downregulated.
Keywords: epithelial-to-mesenchymal transition; E-cadherin; cervical carcinoma; cytoplasmic actin isoforms

Cellular acidosis inhibits assembly, disassembly, and motility of stress granules by E. M. Chudinova; E. S. Nadezhdina; P. A. Ivanov (1277-1284).
Stress granules (SGs) are large ribonucleoprotein (RNP)-containing particles that form in cytoplasm in response to a variety of acute changes in the cellular environment. One of the general parameters of the cell environment is pH. In some diseases, as well as in muscle fatigue, tissue acidosis occurs, leading to decrease in intracellular pH. Here we studied whether decrease in pH causes the formation of SGs in cultured animal cells, whether it affects the formation of the SGs under the action of arsenite and, if such effects occur, what are the mechanisms of the influence of acidosis. Acidosis was simulated by decreasing the pH of the culture medium, which acidified the cytoplasm. We found that medium acidification to pH 6.0 in itself did not cause formation of SGs in cells. Moreover, acidification prevented the formation of SGs under treatment with sodium arsenite or sodium arsenite together with the proteasome inhibitor MG132, and it inhibited the dissociation of preformed SGs under the influence of cycloheximide. We established that pH decrease did not affect the phosphorylation of eIF2α that occurs under the action of sodium arsenite, and even caused such phosphorylation by itself. We also found that the velocity of SG motion in cytoplasm at acidic pH was very low, and the mobile fraction of SG-incorporated PABP protein revealed by FRAP was decreased. We suppose that acidic pH impairs biochemical processes favoring assembly of RNPs in stress conditions and RNP dissociation on the termination of stress. Thus, in acidosis the reaction of the cellular translation apparatus to stress is modified.
Keywords: RNP; cytoplasm pH; sodium arsenite; FRAP; microtubule

Mapping of cis-regulatory sites in the promoter of testis-specific Stellate genes of Drosophila melanogaster by O. M. Olenkina; K. S. Egorova; A. A. Aravin; N. M. Naumova; V. A. Gvozdev; L. V. Olenina (1285-1293).
Tandem Stellate genes organized into two clusters in heterochromatin and euchromatin of the X-chromosome are part of the Ste-Su(Ste) genetic system required for maintenance of male fertility and reproduction of Drosophila melanogaster. Stellate genes encode a regulatory subunit of protein kinase CK2 and are the main targets of germline-specific piRNA-silencing; their derepression leads to appearance of protein crystals in spermatocytes, meiotic disturbances, and male sterility. A short promoter region of 134 bp appears to be sufficient for testis-specific transcription of Stellate, and it contains three closely located cis-regulatory elements called E-boxes. By using reporter analysis, we confirmed a strong functionality of the E-boxes in the Stellate promoter for in vivo transcription. Using selective mutagenesis, we have shown that the presence of the central E-box 2 is preferable to maintain a high-level testis-specific transcription of the reporter gene under the Stellate promoter. The Stellate promoter provides transcription even in heterochromatin, and corresponding mRNAs are translated with the generation of full-size protein products in case of disturbances in the piRNA-silencing process. We have also shown for the first time that the activity of the Stellate promoter is determined by chromatin context of the X-chromosome in male germinal cells, and it increases at about twofold when relocating in autosomes.
Keywords: promoter; spermatogenesis; Drosophila ; Stellate genes; heterochromatin

Structure of hexasaccharide 1-phosphate polymer from Arthrobacter uratoxydans VKM Ac-1979T cell wall by N. V. Potekhina; A. S. Shashkov; S. N. Senchenkova; L. V. Dorofeeva; L. I. Evtushenko (1294-1302).
A hexasaccharide 1-phosphate polymer of original structure and two teichoic acids (TA) belonging to different structural types were found in Arthrobacter uratoxydans VKM Ac-1979T cell wall. The poly(hexasaccharide 1-phosphate) combines features of teichuronic acids and glycosyl 1-phosphate polymers, and its structure has never been reported earlier. Its composition includes residues of α- and β-D-glucuronic acid as well as α-D-galacto-, β-D-gluco-, α-D-mannopyranose, and 6-O-acetylated 2-acetamido-2-deoxy-α-D-glucopyranose. The phosphodiester bond in the polymer joins the glycoside hydroxyl of α-D-glucuronic acid and O6 of α-D-galactopyranose. TA 1 is β-D-glucosylated 1,3-poly(glycerol phosphate), and TA 2 is 3,6-linked poly[α-D-glucosyl-(1→2)-glycerol phosphate]. The phosphate-containing polymers were studied by chemical methods and on the basis of one-dimensional 1H-, 13C-, and 31P-NMR spectra, homonuclear two-dimensional 1H/1H COSY, TOCSY, ROESY, and heteronuclear 1H/13C HSQC, HSQC-TOCSY, HMBC, and 1H/31P HMBC experiments. The set and structure of the polymers revealed as well as the cell wall sugars (galactose, glucose, mannose, glucosamine) and glycerol can be used in microbiological practice for taxonomic purposes.
Keywords: Arthrobacter uratoxydans ; cell wall; cell wall sugars; glycosyl 1-phosphate polymer; teichoic acid; NMR spec-troscopy

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila by G. S. Dotsenko; M. V. Semenova; O. A. Sinitsyna; S. W. A. Hinz; J. Wery; I. N. Zorov; E. G. Kondratieva; A. P. Sinitsyn (1303-1311).
Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K m and k cat values are 0.4 mM and 15 sec−1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K m and k cat values are 1.3 mg/ml and 67 sec−1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K m and k cat values are 0.08 mM and 35 sec−1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.
Keywords: Myceliophthora thermophila ; β-mannosidase; β-mannanase; α-galactosidase; enzymatic hydrolysis

Bacteriolytic activity of human interleukin-2 by P. A. Levashov; S. A. Sedov; N. G. Belogurova; S. V. Shipovskov; A. V. Levashov (1312-1314).
In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.
Keywords: interleukin-2; bacteriolytic activity; Escherichia coli

Molecular cloning, isolation, and properties of chaperone Skp from Yersinia pseudotuberculosis by E. V. Sidorin; N. M. Tishchenko; V. A. Khomenko; M. P. Isaeva; P. S. Dmitrenok; N. Yu. Kim; G. N. Likhatskaya; T. F. Solov’eva (1315-1325).
The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.
Keywords: chaperone Skp; Yersinia pseudotuberculosis ; immunoglobulin G; cross-linking stabilization; MALDI-TOF mass spectrometry; computer modeling; protein-protein interactions

Effect of affinity Sorbent on proteomic profiling of isatin-binding proteins of mouse brain by O. A. Buneeva; A. T. Kopylov; O. V. Tikhonova; V. G. Zgoda; A. E. Medvedev; A. I. Archakov (1326-1338).
Use of small molecules for isolation of particular sub-proteomes is often complicated by the need for chemical modification of a parent compound for affinity sorbent preparation. Isatin (indoledione-2,3) is an endogenous indole that exhibits a wide spectrum of biological activities. Using 5-aminocaproylisatin for proteomic profiling of fractionated rodent brain homogenates, we previously identified more than sixty individual proteins. However, proteins tested in an optical biosensor study for validation of their isatin-binding capacity demonstrated different affinity for immobilized 5-aminocaproylisatin and 5-aminoisatin. In this study, we comparatively evaluated proteomic profiles of isatin-binding proteins separated using both isatin analogs as the affinity ligands. The total number of identified proteins was higher with the shorter isatin analog (88 versus 66), and only 22 proteins were identical in the two proteomic profiles. Thus, proteomic profiling of brain isatin-binding proteins is significantly influenced by the length of the spacer between the amino group used for affinity ligand coupling to Sepharose and the isatin moiety. This suggests that the actual number of brain proteins interacting with endogenous (unmodified) isatin still remains underestimated due to different affinity of proteins for the isatin analogs used for the affinity-based proteomic profiling.
Keywords: small molecule affinity chromatography; proteomic profiling; sub-proteome isolation; brain; isatin; isatin-binding proteins