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.