Development and application of a DNA microarray-based yeast two-hybrid system.

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein-protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.

A directed protein interaction network for investigating intracellular signal transduction.

Cellular signal transduction is a complex process involving protein-protein interactions (PPIs) that transmit information. For example, signals from the plasma membrane may be transduced to transcription factors to regulate gene expression. To obtain a global view of cellular signaling and to predict potential signal modulators, we searched for protein interaction partners of more than 450 signaling-related proteins by means of automated yeast two-hybrid interaction mating. The resulting PPI network connected 1126 proteins through 2626 PPIs. After expansion of this interaction map with publicly available PPI data, we generated a directed network resembling the signal transduction flow between proteins with a naïve Bayesian classifier. We exploited information on the shortest PPI paths from membrane receptors to transcription factors to predict input and output relationships between interacting proteins. Integration of directed PPI with time-resolved protein phosphorylation data revealed network structures that dynamically conveyed information from the activated epidermal growth factor and extracellular signal-regulated kinase (EGF/ERK) signaling cascade to directly associated proteins and more distant proteins in the network. From the model network, we predicted 18 previously unknown modulators of EGF/ERK signaling, which we validated in mammalian cell-based assays. This generic experimental and computational approach provides a framework for elucidating causal connections between signaling proteins and facilitates the identification of proteins that modulate the flow of information in signaling networks.

Mutant huntingtin impairs Ku70-mediated DNA repair.

DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt-expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington's disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology.

Detection of alpha-rod protein repeats using a neural network and application to huntingtin.

A growing number of solved protein structures display an elongated structural domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel alpha-helices. Alpha-rods are flexible and expose a large surface, which makes them suitable for protein interaction. Although most likely originating by tandem duplication of a two-helix unit, their detection using sequence similarity between repeats is poor. Here, we show that alpha-rod repeats can be detected using a neural network. The network detects more repeats than are identified by domain databases using multiple profiles, with a low level of false positives (<10%). We identify alpha-rod repeats in approximately 0.4% of proteins in eukaryotic genomes. We then investigate the results for all human proteins, identifying alpha-rod repeats for the first time in six protein families, including proteins STAG1-3, SERAC1, and PSMD1-2 & 5. We also characterize a short version of these repeats in eight protein families of Archaeal, Bacterial, and Fungal species. Finally, we demonstrate the utility of these predictions in directing experimental work to demarcate three alpha-rods in huntingtin, a protein mutated in Huntington's disease. Using yeast two hybrid analysis and an immunoprecipitation technique, we show that the huntingtin fragments containing alpha-rods associate with each other. This is the first definition of domains in huntingtin and the first validation of predicted interactions between fragments of huntingtin, which sets up directions toward functional characterization of this protein. An implementation of the repeat detection algorithm is available as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized using BiasViz, a graphic tool for representation of multiple sequence alignments.