Biochemistry (Moscow) (v.76, #11)

This review discusses the functional role of nitric oxide in ischemia-reperfusion injury and mechanisms of signal transduction of apoptosis, which accompanies ischemic damage to organs and tissues. On induction of apoptosis an interaction is observed of the nitric oxide signaling system with the sphingomyelin cycle, which is a source of a proapoptotic agent ceramide. Evidence is presented of an interaction of the sphingomyelin cycle enzymes and ceramide with nitric oxide and enzymes synthesizing nitric oxide. The role of a proinflammatory cytokine TNF-α in apoptosis and ischemia-reperfusion and mechanisms of its cytotoxic action, which involve nitric oxide, the sphingomyelin cycle, and lipid peroxidation are discussed. A comprehensive study of these signaling systems provides insight into the molecular mechanism of apoptosis during ischemia and allows us to consider new approaches for treatment of diseases associated with the activation of apoptosis.
Keywords: nitric oxide; sphingomyelin cycle; ceramide; lipid peroxidation; tumor necrosis factor-α; apoptosis; ischemia-reperfusion

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.
Keywords: short biologically active peptides; peptide-oligonucleotide (DNA) binding; DNA methylation; regulation of genetic functions

NMDA receptors as a possible component of store-operated Ca2+ entry in human T-lymphocytes by L. F. Zainullina; R. S. Yamidanov; V. A. Vakhitov; Yu. V. Vakhitova (1220-1226).
Elevation of intracellular Ca2+ in T-lymphocytes as a consequence of T cell antigen receptor activation triggers transcriptional programs resulting in effector cytokine secretion and immune response coordination. Increase of Ca2+ concentration in T-lymphocytes follows both the Ins(1,4,5)P3-dependent release from an intracellular store and subsequent influx from extracellular milieu. Flow cytometry and the fluorescent dye Fluo-4AM have been used to demonstrate that noncompetitive NMDA receptor antagonist (+)-MK801 inhibits Ca2+ influx in T cells induced by thapsigargin. Combination of thapsigargin and (+)-MK801 with following incubation does not affect Ca2+ mobilization from intracellular stores, while decreased Ca2+ entry was observed. Overall data indicate that the ion channel blocker (+)-MK801 is able to inhibit the Ca2+ influx and confirm our suggestion about involvement of NMDA receptor in the store-operated Ca2+ entry mechanisms in human T-lymphocytes. To identify the signal transduction pathways associated with NMDA receptors in mitogen-stimulated T-lymphocytes, the cells were incubated with (+)-MK801, then activity of key phosphorylated protein kinases of MAP-activated (pERK1/2, pSAPK/JNK, p-p38), Ca2+-dependent (pCaMKII), PI3/Akt-dependent (pGSK-3β), and PKC-activated (pPKCθ) pathways were detected. The data we obtained demonstrate that (+)-MK801 treatment leads to more prominent decrease in Ras-activated protein kinases pERK1/2 and Rac-activated proteins p-p38 and pSAPK/JNK, as compared to DAG-dependent pPKCθ and Ca2+-dependent pCaMKII. These results show that NMDA receptors are mainly involved in regulation of Ras/Rac-dependent signaling in T-lymphocytes.
Keywords: human T-lymphocytes; NMDA receptors; T cell antigen receptor; store-operated Ca2+ entry; signaling cascades

A novel keratin-like fibrous protein K58 with molecular weight of about 58 kDa was discovered in bivalve Siliqua radiata ligament and identified by amino acid composition and MALDI-TOF-TOF analysis. We found that the protein is composed of cylindrical fibers (∼160 nm in diameter) and contains high glycine (27.4%) and phenylalanine (10.5%) contents. Furthermore, it is homologous to keratin type II cytoskeletal 1, with repeat motifs of SGGG and SYGSGG. FTIR and secondary structure analysis indicate that K58 is composed of 46.2% β-sheet, 33.4% β-turn, 13.1% α-helix, and 4.7% disordered structure. This structure feature is closely related to the superior tensile strength, elasticity, and solvent resistance property of K58. These discoveries provide some evidence for evolution of keratin and fibrous proteins and prompt further studies of ligament fibrous proteins.
Keywords: bivalve ligament; fibrous protein; keratin; Siliqua radiata ; K58; secondary structure

Contribution of conserved Glu255 and Cys289 residues to catalytic activity of recombinant aldehyde dehydrogenase from Bacillus licheniformis by Yen-Chung Lee; Den-Tai Lin; Ping-Ling Ong; Hsiang-Ling Chen; Huei-Fen Lo; Long-Liu Lin (1233-1241).
Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis aldehyde dehydrogenase (BlALDH) and identified two different residues, Glu255 and Cys289, that might be responsible for the catalytic function of the enzyme. The role of these residues was further investigated by site-directed mutagenesis and biophysical analysis. The expressed parental and mutant proteins were purified by nickel-chelate chromatography, and their molecular masses were determined to be approximately 53 kDa by SDS-PAGE. As compared with the parental BlALDH, a dramatic decrease or even complete loss of the dehydrogenase activity was observed for the mutant enzymes. Structural analysis showed that the intrinsic fluorescence and circular dichroism spectra of the mutant proteins were similar to the parental enzyme, but most of the variants exhibited a different sensitivity towards thermal- and guanidine hydrochloride-induced denaturation. These observations indicate that residues Glu255 and Cys289 play an important role in the dehydrogenase activity of BlALDH, and the rigidity of the enzyme has been changed as a consequence of the mutations.
Keywords: aldehyde dehydrogenase; Bacillus licheniformis ; site-directed mutagenesis; dehydrogenase activity; fluorescence; circular dichroism

Antitumor effect of non-steroid glucocorticoid receptor ligand CpdA on leukemia cell lines CEM and K562 by E. A. Lesovaya; A. Yu. Yemelyanov; K. I. Kirsanov; M. G. Yakubovskaya; I. V. Budunova (1242-1252).
Glucocorticoids (GCs) are widely used in chemotherapy of hematological malignancies, particularly leukemia. Their effect is mediated by glucocorticoid receptor (GR), a well-known transcription factor. Besides their therapeutic impact, GCs may cause a number of side effects leading to various metabolic complications. The goal of immediate interest is testing glucocorticoid analogs capable of induction/enhancement of GR transrepression, but preventing GR dimerization and transactivation leading to side effects. In this work we have investigated effects of a promising new selective GR agonist, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride (CpdA), on CEM and K562 leukemia cells. Both cell lines express functional GR. CpdA compared with the glucocorticoid fluocinolone acetonide (FA) exerted more prominent cytostatic and apoptotic effects on the cells. Both cell lines exhibited sensitivity to CpdA, demonstrating a good correlation with the effects of FA on cell growth and viability. In contrast to FA, CpdA did not induce GR transactivation evaluated by no obvious increase in expression of GR target (and dependent) gene FKBP51. At the same time, luciferase assay showed that CpdA efficiently activated transrepression of NF-ϰB and AP-1 factors. We also evaluated the effect of combined action of CpdA and the proteasome inhibitor Bortezomib. The latter induced a caspase-dependent apoptosis in both T-cell leukemia cell lines. By treatment of CEM cells with different CpdA/GC and Bortezomib doses, we have designed a protocol where CpdA shows potentiating effect on Bortezomib cytotoxic activity. Generally, the present work characterizes a novel non-steroid GR ligand, CpdA, as a promising compound for possible application in leukemia chemotherapy.
Keywords: glucocorticoid receptor; leukemia; selective GR agonist

MgADP and MgATP binding to catalytic sites of βY341W-α3β3Γ subcomplex of F1-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α3β3Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α3β3Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α3β3Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α3β3Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F1-ATPases.
Keywords: ATP synthase; catalytic cooperativity; bi-site catalysis; multi-site catalysis; nucleotide binding

Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling by H. Nazari; A. Khaleghian; A. Takahashi; N. Harada; N. J. G. Webster; M. Nakano; K. Kishi; Y. Ebina; Y. Nakaya (1262-1269).
Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.
Keywords: insulin; GLUT4 translocation; cortactin; cytoskeleton; glucose uptake

Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2 by I. V. Volokhina; Yu. S. Gusev; S. I. Mazilov; M. I. Chumakov (1270-1275).
Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2–4 protein molecules of 12–18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA.
Keywords: Agrobacterium tumefaciens ; virulence protein VirE2; supramolecular complexes; T-DNA; pores; bioinformatics