Asunto(s)

RESUMEN

Cholesterol is one of the major lipid components of plasma membrane and it plays an important role in various signaling processes in mammalian cells. Our study focused on the role of membrane cholesterol in organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by weakly developed actin network and reduced stress fibers--human embryonic kidney HEK293 cells, epidermoid larynx carcinoma HEp2 cells and mouse fibroblasts 3T3-SV40. Using F-actin labeling with rhodamine-phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin, and polyene macrolide antibiotic filipin. In cells treated with methyl-beta-cyclodextrin or filipin, similar processes of actin cytoskeleton reorganization involving filament assembly were revealed. In carcinoma HEp2 cells and fibroblasts 3T3-SV40, cholesterol-sequestering reagents induced intensive stress fiber formation and enhanced cell spreading which corresponded to reversion of transformed phenotype. The rearrangements of cytoskeleton are likely initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.

Asunto(s)

RESUMEN

The article describes a story of the discovery and properties of bacterial metalloproteinase ECP32 that cleaves actin at the only site between Gly42 and Val43. This site is intensively involved in the monomer-monomer contacts within actin filament. Cleavage with ECP32 results in a reversible loss of actin polymerizability. Therefore ECP cleaved actin has been a unique model to study mechanisms of actin polymerization and the filament dynamics. and the effects of actin-binding proteins on these processes, as well as to reveal allosteric effects in actin molecule and to determine three-dimensional actin structure that was previously determined only for the actin-ligand complexes. Furthermore, the non-pathogenic bacteria synthesizing ECP32 were shown to penetrate in eukaryotic cells rearranging their cytoskeleton. Biochemical analysis using the Vitek-2 system and sequencing of the 16S rRNA gene reidentified the ECP32-producing strain, previously identified as E. coli, as Serratia grimesii. Bacteria of a reference strain S. grimesii were found to express the gene of metalloprotease that cleaves actin similarly to ECP32. The gene was cloned, sequenced and expressed in E. coli. The protein encoded by this gene was named grimelysin. Grimelysin shared essential characteristics of ECP32: molecular weight, limited actin proteolysis, inhibition by chelating agents, cleavage site, and the N-terminal amino acids of the active enzyme published for ECP32. These data show that grimelysin and ECP32 seem to be the same protein. As it was demonstrated by confocal microscopy the bacteria capable of synthesizing natural or recombinant grimelysin acquire invasive phenotype. The data described here allow us to suggest that actin may be a target protein which proteolysis promotes bacterial invasion.

Asunto(s)

RESUMEN

The protease ECP32 is significant in investigations of actin, the basic protein of muscles and the cytoskeleton. The enzyme originates from the natural enterobacteria strain, which accumulates minor amounts of the protease intracellularly at the post-exponential growth phase. The limiting factor for biosynthesis is the amount of oxygen that has entered the medium. The highly effective method of two-phase cultivation with vigorous aeration at the exponential growth phase was recommended. Based upon the enzyme properties studied, there is a decreased potential for success when using either the affinity or one-stage purification methods. In order to overcome obstacles in the aforementioned methods, a simple method for ECP32 preparation and storage was developed, with purity and activity levels satisfying the requirements of actin structure and function investigations.

Asunto(s)

RESUMEN

Actin sequences are conserved to a much greater degree than those in almost any other proteins, so that two cytoplasmic isoforms differ by only four of 374 amino acid residues. Nevertheless, the results of biochemical, immunocytochemical and molecular biology experiments demonstrate that appearance, amount and localization of actin isoforms are strongly controlled by cell machinery. Although at the early stages of cell differentiation expression of any actin gene is potentially possible, under normal physiological conditions, while differentiation proceeds, synthesis of specific actin isoforms is temporally regulated and the produced proteins are segregated spatially. Pathological situations of tissue injury or mammalian disease correlate either with up- and down-regulation of distinct actin genes returning to a fetal gene program or with a failure to sort actin isoforms. Different actin isoforms cannot substitute for each other, and changes in expression of specific actin genes are accompanied by alterations in cell structure and function suggesting that specific actin isoforms perform unique cellular functions. This article summarizes the data on segregation of actin isoforms in cell compartments and analyses the mechanisms suggested to explain spatial segregation of cytoplasmic actin isoforms within a cell.

Asunto(s)

RESUMEN

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.

Asunto(s)

RESUMEN

New analytical possibilities of biological macromolecule partition in a two-phase polymer system (dextran-500/polyethyleneglycol-6000) were investigated. The technical principles were based on the fact that optical density of the system changes during phase formation. Various actin forms were investigated. Unlike G-actin, F-actin increases the speed of phase formation. Different actin isoforms exert different influence on this process. The kinetics of the partition system into phases makes it possible to figure out the picture of native G-actin damage. The two-phase polymer system has been shown to be capable of supporting the native properties of actin for not less than 6 days at 20 degrees C. An assumption is made that two-phase systems can be used for imitating the intracellular medium in studies on actin functional characteristics.

Asunto(s)

RESUMEN

Bacteria of spontaneously isolated non-pathogenic strain E. coli A2 have been previously shown to produce a new proteinase, referred to as protease ECP 32, which specifically cleaves actin (Khaitlina et al., 1988; Matveyev et al., 1996). Similar proteinase activity was found in revertants of Shigella flexneri L-forms. In this work immunofluorescence and electron microscopy were used to address a question of whether E. coli A2 can invade epithelial cells similarly as it has been demonstrated for Sh. flexneri. Infection of Hep-2 cells with E. coli A2 resulted in bacterial invasion of the cells followed by cytoskeleton reorganization. On one end of intracellular bacteria bundles of actin filaments resembling a comet-like tail were observed. Bacteria of referent strain CCM 5172, not producing protease ECP 32, were not taken up by the cells. These data suggest that protease ECP 32 may be involved in the process of bacterial invasion and cytoskeleton reorganization.

Asunto(s)

RESUMEN

The structure of inactivated actin was studied by the methods of intrinsic fluorescence upon stationary and pulse excitation, selective fluorescence quenching with acrylamide, and testing the protein surface with a hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid (ANS). The results are discussed along with earlier data on actin sedimentation, near- and far-UV CD spectra, and fluorescence anisotropy. The thermodynamic stability of inactivated actin, the presence of a secondary structure characteristic of the native protein, and the reversibility of the inactivated actin-completely unfolded actin transition allow inactivated actin to be considered an intermediate form in the process of protein folding into the native globular structure. In vitro actin inactivation is accompanied by specific association of actin macromolecules resulting in the formation of homogeneous stable complexes. The tendency toward aggregation (or specific association, in the case of actin), which is determined by the presence of extended hydrophobic clusters on the molecule surface, appears to be one of the intrinsic properties of any protein in the intermediate state. The mobility of the amino acid side chains in the inactivated actin differs considerably from that in the completely unfolded actin. The relaxation properties of the microenvironment of tryptophan residues determine relatively long-wave fluorescence spectra of the inactivated actin. However, the mobility observed is insufficient to compensate the asymmetry of the microenvironment of aromatic residues, which is confirmed by a characteristic and intense CD spectrum in the near-UV region. The mobility of the indole rings of tryptophans located in the internal regions of the inactivated actin that are solvent-inaccessible although polar is even considerably lower than that in the native actin.

Asunto(s)

RESUMEN

Immunocytochemical analysis of preparation of dispersed nuclei content in oocytes of III-IV stages of oogenesis, in terms of Dumont (1972), from hibernating grass frogs using monoclonal antibodies against actin, revealed two types of intranuclear structures containing this protein: coiled bodies (CB) and satellite microbodies (SM). Staining of these preparations with Rhodamin-phalloidin, known specifically to interact with fibrillar actin, did not reveal it in these structures. Results of our biochemical studies, using protease ESP32 specifically cutting only globular actin, are suggesting that both CB and SM contain globular actin. Gall et al. (1975) proposed that CB may be involved in assembling and sorting of small nuclear RNA for the three main RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. Our finding of actin in CB allows a suggestion on actin involvement in the transport of RNA processing complexes from CB to some actual places where processing of RNA takes place. According to our previous data (Tsvetkov, Parfenov., 1994), SM participate in the karyosphere capsule formation. This process is preceded by SM fusion triggered presumably by actin.

Asunto(s)

RESUMEN

Hydrolysis of the C-peptide from recombinant human proinsulin, porcine insulin, and melittin by the E. coli actin-degrading proteinase ECP 32 was studied by reverse phase high performance liquid chromatography and mass spectrometry with electrospray ion source. Proteinase ECP 32 hydrolyzed only melittin at the Ala15-Leu16 or Leu16-Ile17 bonds (KM = 2.4 x 10(-6) M). The effects of pH and buffer composition on the rate of enzymatic hydrolysis were studied. The pH optimum of melittin hydrolysis was 7. Phosphates inhibited, whereas ATP stimulated the hydrolysis of melittin. Melittin was suggested as a substrate for determining the activity of proteinase ECP 32.

Asunto(s)

RESUMEN

Proteolytic activity of cell extracts from revertants of Shigella flexneri L-forms as well as biochemical properties of these strains and their sensitivity to antibiotics were studied. The protease found earlier in cells of strain E. coli A2 was shown to be synthesized by one of 8 revertants under study. This protease split actin and did not split some other proteins, its activity was inhibited by inhibitors of metalloproteases. Strain 5a2c which produced the protease was similar to the strain E. coli A2 and differed from other revertants in some biochemical properties, resistance to ampicillin and sensitivity to furazolidone. Thus the protease activity can be a marker of structural and functional transformation of Sh. flexneri under the influence of furazolidone.

Asunto(s)

RESUMEN

A novel bacterial protease specifically hydrolyzing actin with the formation of a stable fragment with Mr of 36 kDa was obtained. This protease was shown to be synthesized at the stationary phase of bacterial culture growth. The actin hydrolysis by bacterial protease was inhibited by o-phenanthroline, EDTA and p-chloromercuribenzoate but not by N-ethyl-maleimide, phenylmethylsulfonylfluoride, Leu-peptin, pepstatin and other serine proteinase inhibitors. The protease was stable within the pH range of 4.5-8.5 and had an activity optimum at pH 7.0-8.0. The protease activity was maintained for 40 min at 45 degrees C and for 30 min at 50 degrees C; at 65 degrees C the enzyme was fully inactivated by 5 min heating. The protease preparations causing quantitative conversion of actin into a 36 kDa fragment did not hydrolyze casein, albumin, ovalbumin, lysozyme, DNAase I, RNAase, myosin, alpha-actinin, tropomyosin and troponin. It was assumed that the protease under consideration is a neutral metalloprotease specifically hydrolyzing actin.

Asunto(s)

RESUMEN

To release Z-discs of skeletal muscles myofibrils from actin microfilaments, I--Z--I-brushes (complexes of Z-discs and thin filaments) were treated with DNAse I-both in suspension and on electron microscopical grids. It was shown that such a treatment resulted in depolymerization of actin filaments. The preparations obtained were heterogeneous and contained I--Z--I-brushes with shorter actin filaments and single Z-discs. The structure of Z-discs released from actin filament remained intact. Therefore these preparations may be used in studies on regulation of actin microfilaments assembly.

Asunto(s)

RESUMEN

The proteolysis-stable actin fragment (m. w. 36300 as determined by SDS polyacrylamide gel electrophoresis) was obtained by actin treatment with a non-identified bacterial protease. This fragment is larger than the trypsin-stable actin fragment and is similar to the unstable trypsin intermediate fragment. The obtained actin fragment is not polymerized, does not activate the ATPase activity of myosin and does not form a superprecipitating complex with it. Thus, the functional properties of actin are lost during the split-off of a relatively small, apparently, the N-terminal part of the polypeptide chain.

Asunto(s)

RESUMEN

The interaction of alpha-actinin, F-actin and tropomyosin has been investigated by using electron microscopy, SDS-gel electrophoresis and viscosimetry. It was shown that the temperature dependence of the interaction with F-actin was similar for alpha-actinins, isolated from the muscle of warmblooded animal (rabbit skeletal muscle) and from muscle of cold--blooded ones (cross striated part of the adductor of scallop Patinopecten yessoensis). The temperature rise from 0 degrees C to 37 degrees C results in a decrease of the affinity of alpha-actinin to F-actin. Tropomyosin decreases the amount of alpha-actinin bound to F-actin at 37 degrees C, but does not dislodge it completely from F-actin filaments. In this case the alpha-actinin binding also occurs along the entire length of the F-actin. It has also been shown that at 0 degrees C alpha-actinin and tropomyosin interact with F-actin independently of each other. The data obtained attest that tropomyosin and alpha-actinin have different binding sites on F-actin. The temperature and tropomyosin regulate only the amount of the bound alpha-actinin, but not its localization on the actin filament, as it was earlier supposed. These data allow one to conclude that the localization of alpha-actinin in Z-disk of cross-striated muscle can be determined neither by physiological temperature nor the presence of tropomyosin.

Asunto(s)

Asunto(s)

RESUMEN

A protein with chain weight of about 90,000 Dalton (MP-90) was isolated from the cross-striated adductor muscle of the scallop Patinopecten yessoensis. The Physicochemical properties of MP-90 and its interaction with actin were studied in comparison with those of alpha-actinin of rabbit skeletal muscle. The data obtained allow us to conclude that this isolated protein is alpha-actinin of cross-striated adductor muscle of scallop Patinopecten yessoensis.

Asunto(s)

RESUMEN

The mode of tryptophan residue orientation in myosin and action myofilaments of the muscle fiber was studied using polarized ultraviolet (UV) fluorescent microscopy of the muscle fiber was studied using polarized ultraviolet (UV) fluorescent microscopy technique. During an elective extraction of proteine from thick and thin myofillaments changes in UV fluorescence anisotropy of muscle fibers were detected, thus suggesting that tryptophanil residues in myosin may be oriented by their own short axes mostly parallel, but in actin--perpendicular to the muscle fiber axis. The use of acrylamide, an UV fluorescence quencher, is proposed for the control of extraction electivity of proteins from muscle fibers.

Asunto(s)

Asunto(s)