The disassembly and withdrawal of vimentin intermediate filaments (VIF) from the plasma membrane induces membrane ruffling and the formation of a lamellipodium. Conversely, lamellipodium formation is inhibited when VIF are present.

Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN

Although the mechanism of neuronal death

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine -glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.Plasma fibronectin is thought to play an important homeostatic role by acting as a scavenger for cytosolic transglutaminases (TGs; protein-glutamine:amine -glutamyltransferase, EC 2.3.2.13). Such enzymes occur in many different cell types and could pose the danger of polymerizing proteins if discharged freely into the circulation (1-3). We have focused on the interaction of human plasma fibronectin with the human erythrocyte (RBC) enzyme (4). Binding between the two proteins is instantaneous and very tight and occurs even in the absence of Ca2 , which indicates that it does not depend on the unmasking of the active center of TG. Studies with chymotryptic fragments of fibronectin showed that the gelatin (collagen)-binding domain of the molecule was involved also in the binding of TG (5). However, the two sites seem to act independently because attachment to TG and gelatin can take place simultaneously in a ternary complex. Electron microscopic examination demonstrates that, along the contour lengths of the constituent chains of fibronectin, human RBC TG was bound at a distance of 5-10 nm from the N termini, frequently forming ring-like structures (6).By using well-characterized thermolytic fragments of human fibronectin (7), it is now possible to further define the TG-associating domains of the plasma protein. It will be shown in the present paper that an -42-kDa gelatin-binding structure, which is sequentially composed of a type I, followed by two type II and three type I motifs , displays a full strength of binding for the human RBC enzyme. The high specificity for binding was exploited for the affinity chromatographic purification of TG directly from the hemoglobin-depleted RBC lysate. MATERIALS AND METHODSProtein Preparations. The 42-kDa and 30-kDa gelatinbinding fragments of human plasma fibronectin were obtained by digestion with thermolysin as described by Borsi et al. (8). Further treatment of the 42-kDa fragment with pepsin was employed for producing a yet smaller 21-kDa gelatinbinding fragment (6). For use in i...

The mechanism for stem cell mediated improvement following acute myocardial infarction has been actively debated. We support hypotheses that the stem cell effect is primarily paracrine factor-linked. We used a heparin-presenting injectable nanofiber network to bind and deliver paracrine factors derived from hypoxic conditioned stem cell media to mimic this stem cell paracrine effect. Our self-assembling peptide nanofibers presenting heparin were capable of binding paracrine factors from a media phase. When these factor-loaded materials were injected into the heart following coronary artery ligation in a mouse ischemia-reperfusion model of acute myocardial infarction, we found significant preservation of hemodynamic function. Through media manipulation, we were able to determine that crucial factors are primarily less than 30 kDa and primarily heparin-binding. Using recombinant VEGF and bFGF loaded nanofiber networks the effect observed with conditioned media was recapitulated. When evaluated in another disease model, a chronic rat ischemic hind limb, our factor-loaded materials contributed to extensive limb revascularization. These experiments demonstrate the potency of the paracrine effect associated with stem cell therapies and the potential of a biomaterial to bind and deliver these factors, pointing to a potential therapy based on synthetic materials and recombinant factors as an acellular therapy.

The inositol phosphate hydrolyzing activity of human phospholipase C␦1 (PLC␦1) is markedly inhibited when the enzyme is coexpressed with the human heart Gh͞transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLC␦1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLC␦1. Nucleotides (GTP > GDP > ATP > GMP ‫؍‬ ADP, in order of decreasing efficiency) interfered with the formation of the PLC␦1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TGslow ؉ GTP u [TG:GTP]fast. Altogether, our findings indicate that GTP controls PLC␦1 activity by releasing this protein from an inhibitory association with G h͞transglutaminase.

Guinea pig liver transglutaminase (EC 2.3.2.13) displays a Ca(2+)-independent binding (Ka = 10(7) M-1) to the same gelatin-binding domain of human plasma fibronectin that is known to form a very tight complex with the human red cell enzyme. The fibronectin-combining site of the liver transglutaminase was investigated by testing fragments obtained from the parent protein by controlled digestion with endoproteinase Lys-C. Overlay assays, probed with anti-fibronectin antibody, revealed that the fibronectin binding ability of the transglutaminase was encoded in a linear sequence in its 28-kDa N-terminal domain. Removal of the first 7 residues by further digestion of the purified 28-kDa material with endoproteinase Glu-C generated a 27-kDa fragment that, however, showed no binding activity. Thus, residues 1-7 in the liver enzyme seem to be of particular importance for influencing its ability to bind to fibronectin.

Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a 111-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 1251 and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (<65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponentialphase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.