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PubMed1993

Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.

Immunofluorescence microscopy revealed that two actin-binding proteins of low molecular weight with different functional activity, ADF and cofilin, are transported into nuclei of cultured myogenic cells to form rod structures there together with actin, when the cells were incubated in medium containing dimethylsulfoxide. In most cases, ADF and cofilin colocalized in the same nuclear actin rods, but ADF appeared to predominate in mononucleated cells, while cofilin was present in multinucleated myotubes. In some mononucleated cells, the nuclear actin rods were composed of ADF and actin but devoid of cofilin. An ADF homologue in mammals, destrin, was also translocated into nuclear actin rods under similar conditions. As a nuclear transport signal sequence exists in cofilin and ADF but not in actin, ADF and/or cofilin may be responsible for the nuclear import of actin in myogenic cells under certain conditions.

Actin Depolymerizing Factors↗
PubMed1993

Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments.

ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

Actin Depolymerizing Factors↗
PubMed2004

Elevated fluid shear stress enhances postocclusive collateral artery growth and gene expression in the pig hind limb.

OBJECTIVE: The role of fluid shear stress (FSS) in collateral vessel growth remains disputed and prospective in vivo experiments to test its morphogenic power are rare. Therefore, we studied the influence of FSS on arteriogenesis in a new model with extremely high levels of collateral flow and FSS in pig and rabbit hind limbs. METHODS AND RESULTS: A side-to-side anastomosis was created between the distal stump of one of the bilaterally occluded femoral arteries with the accompanying vein. This clamps the collateral reentry pressure at venous levels and increases collateral flow, which is directed to a large part into the venous system. This decreases circumferential wall stress and markedly increases FSS. One week after anastomosis, angiographic number and size of collaterals were significantly increased. Maximal collateral flow exceeded by 2.3-fold that obtained in the ligature-only hind limb. Capillary density increased in lower leg muscles. Immunohistochemistry revealed augmented proliferative activity of endothelial and smooth muscle cells. Intercellular adhesion molecule-1 and vascular cell adhesion molecule (VCAM)-1 were upregulated, and monocyte invasion was markedly increased. In 2-dimensional gels, actin-regulating cofilin1 and cofilin2, destrin, and transgelin2 showed the highest degree of differential regulation. CONCLUSIONS: High levels of FSS cause a strong arteriogenic response, reinstate cellular proliferation, stimulate cytoskeletal rearrangement, and normalize maximal conductance. FSS is the initiating molding force in arteriogenesis. The role of fluid shear stress on the development of a collateral circulation was studied by abruptly increasing collateral blood flow by a distal femoral artery-to-vein anastomosis. This increased number and size of collateral vessels to a hitherto unknown degree. Fluid shear stress is the primary and strongest arteriogenic stimulus.

Actin Depolymerizing Factors↗
PubMed1996

A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli.

1. Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes. The structure and function of cofilin are conserved during evolution. 2. Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3. Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo. 3. The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells. 4. In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue. The actin-binding activity is essential for yeast cells to grow. 5. Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells. 6. Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses. Functional roles of cofilin/actin in the nucleus remain to be elucidated. 7. Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity.

Actin Depolymerizing Factors↗
PubMed2004

[Nano-ESI-MS/MS identification on differentiation-associated proteins in M1 mouse myeloid leukemia cells induced by IL-6].

OBJECTIVE: To identify two differentiation-associated proteins induced by rhIL-6 in M1 mouse myeloid leukemia cells. METHODS: Protein spots were excised from 2-D gels and digested in-gel with trypsin. The trypsin lysis products were first analyzed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) through peptide mass fingerprinting and then performed peptide sequencing by nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS/MS). The database search was finished with the Mascot search engine (http://www.matrixscience.co.uk) using the data processed through MaxEnt3 and MasSeq. RESULTS: The two proteins were not revealed by peptide mass fingerprint using MALDI-TOF-MS, while they were respectively identified as Destrin and Putative protein after the sequence of their trypic peptides were obtained by the nano-ESI-MS/MS techniques. CONCLUSION: Nano-ESI-MS/MS technique can successfully identify the two differentiation-associated proteins induced by rhIL-6 and has great advantage in protein analysis.

Actin Depolymerizing Factors↗
PubMed2006

[Proteomic analysis of human ovarian cancer cell lines and their platinum-resistant clones].

OBJECTIVE: To perform comparative proteomic analysis of human ovarian cancer cell lines for detecting platinum-resistance associated proteins. METHODS: The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP) human ovarian cancer cell lines were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software, stained with Coomassie Brilliant Blue, then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. The mRNA and protein levels of the differentially expressed protein which was most significant in all of the four resistant cell lines were validated by RT-PCR and western blotting, respectively. RESULTS: Five proteins were found to be significant in four cell lines. Annexin A3 and destrin were up-regulated and nicotinamide-adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was not changed. However, cofilin 1 represented a different trend. In the two resistant sublines of SKOV3, cofilin 1 had a down-regulation, but it had an up-regulation in the cell lines induced from SKOV3. The expression of annexin A3 was up-regulated by 3 - 20 fold and the results of RT-PCR and western blotting showed complete consistency with that by 2-DE. CONCLUSIONS: Proteomic techniques are useful to the identification of the resistance-associated proteins in ovarian cancer platinum-resistant cell lines and five candidates have been found. The five differential proteins might become hopeful candidate biomarkers for resistance.

Annexin A3↗
PubMed2002

Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase.

This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.

Animals↗
PubMed2005

Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases.

Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.

Adolescent↗
PubMed2001

Augmented expression of peroxiredoxin I in lung cancer.

Comparative proteome analysis was performed between human normal (BEAS 2B) and malignant (A549) lung epithelial cells in an attempt to identify novel biomarkers of lung cancer. Approximately 500 protein spots could be separated by mini two-dimensional electrophoresis and visualized with Coomassie blue R-250. Among those relatively abundant proteins, eight spots were changed more than twofold reproducibly and identified by peptide mass fingerprints using mass spectrometry and database search. The increased proteins in A549 were aldehyde dehydrogenase, peroxiredoxin I, fatty acid binding protein, aldoketoreductase, and destrin, whereas the decreased proteins were galectin-1, transgelin, and stathmin. Since human lung is exposed to continuous oxidative stress, antioxidant enzyme peroxiredoxin I was selected for further investigation and its augmented expression was confirmed in cancer tissues compared to normal tissues from lung cancer patients, suggesting peroxiredoxin I as a potential biomarker of lung cancer.

Amino Acid Sequence↗
PubMed1998

Cofilin and gelsolin segment-1: molecular dynamics simulation and biochemical analysis predict a similar actin binding mode.

An understanding of the actin-depolymerizing function attributed to members of the ADF/cofilin/destrin superfamily requires a structural model of these proteins in complex with actin. As a step toward defining actin-cofilin interactions, the complex of yeast cofilin with monomeric actin was predicted, starting with the actin-gelsolin segment-1 binding mode recently suggested for the actin-destrin complex. After refinement by molecular dynamics simulation, the structure of cofilin converged in a new binding mode that required only minimal changes induced in the actin-cofilin interface. The predicted complex exhibits strong interactions between the N termini of actin and cofilin, mediated by a salt bridge of cofilin Arg3 with actin Asp1. The forming of this salt bridge could be prevented by the phosphorylation of cofilin Ser4, which is believed to inhibit cofilin depolymerization activity. Recent mutagenesis studies, crosslinking experiments and peptide binding studies are consistent with the predicted model of the actin-cofilin complex. The structural homology between cofilin and gelsolin segment-1 binding to actin was confirmed experimentally by two types of competitive binding assays.

Actin Depolymerizing Factors↗
PubMed1993

Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein.

We have cloned a Saccharomyces cerevisiae gene (COF1) encoding a low-M(r) actin-binding protein of 143 amino acid (aa) residues (yeast cofilin; Cof); its aa sequence is 35% identical to porcine Cof. The yeast recombinant Cof produced in Escherichia coli exhibited in vitro activities on actin filaments similar to those of mammalian and avian Cof. Gene disruption and tetrad analysis showed that gene COF1 is essential for yeast cell growth. Expression of the cDNA of porcine Cof or destrin (Des), the latter a Cof-related protein, complemented the cof1 null allele in yeast cells.

Actin Depolymerizing Factors↗
PubMed2007

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry.

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.

Amino Acid Sequence↗
PubMed1997

Analogous F-actin binding by cofilin and gelsolin segment 2 substantiates their structural relationship.

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047-1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366-369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369-373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin alpha-helix 112-128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2-3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

Actin Depolymerizing Factors↗
PubMed2003

F-actin capping (CapZ) and other contractile saphenous vein smooth muscle proteins are altered by hemodynamic stress: a proteonomic approach.

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

Actin Cytoskeleton↗
PubMed2000

[Characterization of a Cl. Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture].

A field strain of cl. perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case. The isolate characterized as Cl. perfringens, type D was based on its cultural and biochemical characters and its factors of virulence. The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat ... were optimized. Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times. The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type. The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin. It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart. The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.

Animals↗
PubMed1991

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin.

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.

Actin Depolymerizing Factors↗
PubMed1997

Vertebrates have conserved capping protein alpha isoforms with specific expression patterns.

Capping protein (CP), a ubiquitous actin binding protein composed of an alpha and a beta subunit, is important for actin assembly and cell motility. Lower organisms have one gene and one isoform of each subunit. Chickens have two very similar alpha-subunit isoforms. To determine if vertebrates in general contain multiple alpha isoforms and if those alpha isoforms have conserved sequences, we isolated and analyzed alpha subunit cDNA's in mice and humans. Both mice and humans also have two alpha isoforms. Phylogenetic analysis of the alpha isoform sequences reveals that vertebrates have two highly conserved subfamilies, alpha1 and alpha2. The alpha1 and alpha2 subfamilies are very similar to each other but can be defined and distinguished from each other by a small number of key amino acid residues. In addition, 3' untranslated cDNA sequences are conserved within the isoform subfamilies. To investigate the function of the alpha isoforms, we examined their expression in mouse cells and tissues. Endothelial cells contain only the alpha2 isoform, and erythrocytes contain almost exclusively the alpha1 isoform. Most tissues have both alpha1 and alpha2 isoforms but the ratio of alpha1:alpha2 varies widely. Together, these findings support the hypothesis that the CP alpha isoforms have conserved, unique and essential roles in vertebrates.

Actin Depolymerizing Factors↗
PubMed1999

Erythrocyte membrane fractions contain free barbed filament ends despite sufficient concentrations of retained capper(s) to prevent barbed end growth.

Many cellular functions depend on rapid cytoskeletal rearrangements localized to specific cytoplasmic domains. Tight regulation of the submembranous microfilament network is accomplished in large part in erythrocytes and granulocytes by actin binding proteins that cap the fast-growing barbed filament ends. Study of this dynamic system is necessarily hampered by the confounding perturbations of cell lysis and dilution. In this paper, we characterize the functional properties of the membrane-associated spectrin-actin complex from human erythrocytes as it exists after hypotonic lysis. Purified spectrin-actin "seeds" extracted from erythrocyte membranes effectively nucleated actin elongation from their barbed ends. However, polymerization from spectrin-actin complexes associated with the membrane fraction prematurely slowed despite the presence of G-actin in great excess of the critical monomer concentration. The addition of cytochalasin B decreased (rather than augmented) the slowing of elongation attributable to the membrane fraction, indicating that capping of barbed filament ends (not monomer sequestration) was the major mechanism underlying this effect. The paradoxical implication of our findings is that, despite the presence of excess capper(s) in the membrane fraction, the membrane-associated spectrin-actin seeds were not capped until after dilution into physiological ionic strength buffer containing monomeric actin. Furthermore, by comparing the degrees of contamination of the extracted and membrane-associated spectrin-actin preparations, it appeared that recognized capping proteins (including gelsolin and capping protein beta2) were not the predominant cappers found in the membrane pellet after hypotonic lysis. We hypothesize that the barbed ends of membrane-associated spectrin-actin complexes, while not excluding actin monomers, may be selectively inaccessible to certain cappers (perhaps simply as the result of steric hindrance). Growth from such complexes in vivo could be limited by the availability of polymerization-competent G-actin.

Actin Depolymerizing Factors↗