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Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks....

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Published in:Molecular & cellular proteomics 2006-02, Vol.5 (2), p.234-244
Main Authors: Grelle, Gerlinde, Kostka, Susanne, Otto, Albrecht, Kersten, Birgit, Genser, Klaus F, Müller, Eva-Christina, Wälter, Stephanie, Böddrich, Annett, Stelzl, Ulrich, Hänig, Christian, Volkmer-Engert, Rudolf, Landgraf, Christiane, Alberti, Simon, Höhfeld, Jörg, Strödicke, Martin, Wanker, Erich E
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Language:English
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Summary:Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro , suggesting that it might influence caytaxin degradation in vivo . Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M500198-MCP200