Abstract
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.
Highlights
Proteins mediate their biological function through interactions with other proteins
We suggest that failure to detect the Tau/carboxyl terminus of Hsp70-interacting protein (CHIP) protein-protein interactions (PPIs) could be due to insufficient translocation of the microtubule-binding protein Tau into the nucleus, which is a prerequisite for reporter gene activation
We applied our technology in poof-of-principle experiments to identify novel interaction partners for the human proteins valosin-containing protein (VCP), CHIP, and amphiphysin II (Table I)
Summary
Clones and Constructs—For the production of protein arrays, clones from the hEx1 library [27] expressing His-tagged human fusion proteins were used. For the expression of GST-tagged fusions, cDNA fragments encoding CHIP (aa 2–303) and amphiphysin II (aa 497–593) were PCR-amplified from the brain cDNA pool number 588 (RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany) and subcloned into the plasmids pGEX-6P-1 and -2 (Amersham Biosciences), respectively. Membranes with high density spotted His-tagged fusion proteins were blocked for 4 h at 4 °C with PBS-T (PBS supplemented with 0.05% Tween 20) containing 0.5 mM DTT and 5% nonfat dry milk powder They were rinsed with PBS-T and incubated overnight at 4 °C in 100 ml of overlay buffer (PBS-T supplemented with 5% fetal calf serum, 100 M ATP, and 100 M GTP) to which crude bacterial cell extract containing the GST fusion protein (ϳ0.5 mg) had been added prior to use. Overlay proteins VCP, CHIP, and amphiphysin II are indicated in bold
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