Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca (2016). Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins. Methods in enzymology, 569, pp. 117-137. Academic Press 10.1016/bs.mie.2015.06.017

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Dermatology, Urology, Rheumatology, Nephrology, Osteoporosis (DURN) > Clinic of Dermatology

UniBE Contributor:

Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine and Borradori, Luca

Subjects:

600 Technology > 610 Medicine & health

ISSN:

0076-6879

Publisher:

Academic Press

Language:

English

Submitter:

Sandra Nyffenegger

Date Deposited:

09 Mar 2016 11:04

Last Modified:

15 Sep 2017 13:08

Publisher DOI:

10.1016/bs.mie.2015.06.017

PubMed ID:

26778556

Uncontrolled Keywords:

BPAG1; Desmoplakin; EGFP; Intermediate filaments; Keratin; Plakin; Plectin; Protein–protein interaction

URI:

https://boris.unibe.ch/id/eprint/76551

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