Systematic analysis of protein-protein interactions involved in autophagy and neurodegenerative disease processes
In this project we will create static protein-protein interaction (PPI) networks connecting huntingtin (htt) and its known interacting partners with proteins involved in autophagy using the high-throughput automated yeast two-hybrid (Y2H) screening technology. Human cDNAs encoding selected htt proteins and known interaction partners as well as candidate autophagy proteins will be subcloned into Y2H expression vectors and PPIs will be identified systematically by automated interaction mating using spotting and pipetting robots.
The identified interactions will be validated with pull-down, co-immunoprecipitation, aggregation as well as functional autophagy assays, and will undergo bioinformatic confidence scoring. High confidence interaction networks will be created, and data will be integrated with expression profiling and proteomics data (in collaboration with D. Rubinsztein, University Cambridge). Using this approach, potential functional modules (protein complexes) that are critical for htt misfolding and degradation during HD pathogenesis will be identified. The generated data will be combined with literature information and stored in a database accessible to all collaboration partners in this project.