Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity.
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ABSTRACT:
SUBMITTER: Arpin M
PROVIDER: S-EPMC2115311 | biostudies-other | 1988 Nov
REPOSITORIES: biostudies-other
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Project description:Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.
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Project description:Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.
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Project description:Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin-severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.
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Project description:BACKGROUND: Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. RESULTS: A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. CONCLUSION: Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.
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Project description:The EGFR ligand amphiregulin (AREG) has been implicated as an important autocrine growth factor in several epithelial malignancies and in psoriasis, a hyperproliferative skin disorder. To characterize the mechanisms by which AREG regulates autocrine epithelial cell growth, we transduced human keratinocytes (KCs) with lentiviral constructs expressing tetracycline (TET)-inducible small hairpin RNA (shRNA). TET-induced expression of AREG shRNA markedly reduced autocrine extracellular signal-regulated kinase phosphorylation, strongly inhibited autocrine KC growth with an efficiency similar to metalloproteinase and EGFR inhibitors, and induced several markers of KC differentiation, including keratins 1 and 10. Addition of various concentrations of exogenous EGFR ligands to KC cultures reversed the growth inhibition in response to AREG-blocking antibodies but not to shRNA-mediated AREG knockdown. Lentivirus-mediated expression of the full-length AREG transmembrane (TM) precursor, but not of the AREG extracellular domain, markedly reversed the shRNA-mediated growth inhibition and morphological changes, and strongly reduced the induction of multiple markers of KC differentiation. Taken together, our data show that autocrine human KC growth is highly dependent on the AREG TM precursor protein and strongly suggest a previously unreported function of the metalloproteinase-processed carboxy (C)-terminal domain of AREG.
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