Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine-encoding repeat in SCA1. In all known polyglutamine diseases, the glutamine expansion confers toxic functions onto the protein. The mechanism by which this occurs remains enigmatic, however, in light of the fact that the mutant protein apparently maintains interactions with its usual partners. Here we show that the expanded polyglutamine tract differentially affects the function of the host protein in the context of different endogenous protein complexes. Polyglutamine expansion in Ataxin1 favors the formation of a particular protein complex containing RBM17, contributing to SCA1 neuropathology via a gain-of-function mechanism. Concomitantly, polyglutamine expansion attenuates the formation and function of another protein complex containing Ataxin1/Capicua, contributing to SCA1 via a partial loss-of-function mechanism. This model provides mechanistic insight into the molecular pathogenesis of SCA1 as well as other polyglutamine diseases.Expansion of an unstable translated CAG repeat located in different disease genes so far causes nine dominantly inherited neurodegenerative disorders, the so-called polyglutamine diseases: Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and six autosomal dominant spinocerebellar ataxias (SCAs) 1 . As would be expected for dominant mutations, polyglutamine expansions confer toxic properties on the host proteins 1-3 ; animal models genetically lacking the polyglutamine-containing proteins do not develop neurodegeneration 4-7 . However, expansion of the polyglutamine tract is necessary but not sufficient to cause pathology: in the case of SCA1, for example, expanded Ataxin1 (ATXN1) does not produce cerebellar degeneration if it lacks the nuclear localization signal 8 or the AXH domain 9 , or if a serine to alanine substitution prevents phosphorylation at residue 776 10 . These and other studies in HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSBMA and HD indicate that protein domains outside of the polyglutamine tract play a significant role in the selective neurotoxicity observed in these diseases 11-18 . Moreover, they suggest that there is a relationship between the normal functions of the wild-type proteins and the toxic functions of their expanded counterparts. Given that mouse and fly models overexpressing wild-type ATXN1 develop a mild version of SCA1 19 begs the question of whether the glutamine expansion enhances some interactions to mediate the gain-of-function.To gain a foothold on this question, we sought to characterize protein partners of ATXN1 that interact with it in a manner dependent on two criteria necessary for toxicity: polyglutamine expansion and phosphorylation at serine 776 (S776). We have identified RBM17 (RNA binding motif protein 17) as a protein that meets these criteria. Here we show that ATXN1 forms at least two distinct, large native complex...