title: Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.
abstract: Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland; Department of Dermatology, Inselspital, Bern University Hospital, Bern, Switzerland. Electronic address: bertrand.favre@insel.ch.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Favre
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland; Department of Dermatology, Inselspital, Bern University Hospital, Bern, Switzerland.
authors.forename: Nadja
authors.initials: N
authors.lastname: Begré
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland; Department of Dermatology, Inselspital, Bern University Hospital, Bern, Switzerland.
authors.forename: Jamal-Eddine
authors.initials: JE
authors.lastname: Bouameur
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland; Department of Dermatology, Inselspital, Bern University Hospital, Bern, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Borradori
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry; D051180:Desmoplakins / Q000737:chemistry; D000071821:Dystonin; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007382:Intermediate Filaments / Q000737:chemistry; D007633:Keratins / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C486268:DST protein, human; D051180:Desmoplakins; D000071821:Dystonin; D055767:Immobilized Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007633:Keratins
keywords: BPAG1; Desmoplakin; EGFP; Intermediate filaments; Keratin; Plakin; Plectin; Protein–protein interaction