Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

Development of an LC-MALDI method for the analysis of protein complexes.

In this study, a two-dimensional LC-MALDI-TOF/TOF method has been developed for analyzing protein complexes. In our hands, the method has proven to be an excellent strategy for the analysis of protein complexes isolated in pull-down experiments. This is in part because the preservation of the chromatographic separation on a MALDI target yields an "unlimited" amount of time to obtain MS/MS spectra, making it possible to probe more deeply into complex samples. A brief statistical analysis was performed on the data obtained from the LC-MALDI-TOF/TOF system in order to better understand peptide fragmentation patterns under high-energy collision conditions. These statistical analyses provided some insight into how to evaluate the quality and accuracy of the database search results derived from the TOF/TOF-based analysis. The potential of the method was demonstrated by the successful identification of all the known penicillin-binding proteins in E. coli isolated using a drug-based pull-down with ampicillin as the bait. The performance of the LC-MALDI-TOF/TOF system was compared with that of an equivalent 2D LC-ESI-MS/MS approach, in the analysis of a protein bait-based pull-down. Regardless of the number of peptides identified in the ESI versus MALDI approach, the two approaches were found to be complementary. When the data is merged at the peptide level, the combined result gives higher Mascot scores and an overall higher confidence in protein identification than with either approach alone.