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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 |
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Main Authors: | , , , , , , , , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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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. |
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ISSN: | 1535-9476 1535-9484 |
DOI: | 10.1074/mcp.M500198-MCP200 |