title: Huntingtin interacting proteins are genetic modifiers of neurodegeneration.
abstract: 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.
authors.affiliation: Prolexys Pharmaceuticals, Salt Lake City, Utah, United States of America.
authors.forename: Linda S
authors.initials: LS
authors.lastname: Kaltenbach
authors.forename: Eliana
authors.initials: E
authors.lastname: Romero
authors.forename: Robert R
authors.initials: RR
authors.lastname: Becklin
authors.forename: Rakesh
authors.initials: R
authors.lastname: Chettier
authors.forename: Russell
authors.initials: R
authors.lastname: Bell
authors.forename: Amit
authors.initials: A
authors.lastname: Phansalkar
authors.forename: Andrew
authors.initials: A
authors.lastname: Strand
authors.forename: Cameron
authors.initials: C
authors.lastname: Torcassi
authors.forename: Justin
authors.initials: J
authors.lastname: Savage
authors.forename: Anthony
authors.initials: A
authors.lastname: Hurlburt
authors.forename: Guang-Ho
authors.initials: GH
authors.lastname: Cha
authors.forename: Lubna
authors.initials: L
authors.lastname: Ukani
authors.forename: Cindy Lou
authors.initials: CL
authors.lastname: Chepanoske
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Zhen
authors.forename: Sudhir
authors.initials: S
authors.lastname: Sahasrabudhe
authors.forename: James
authors.initials: J
authors.lastname: Olson
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kurschner
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Ellerby
authors.forename: John M
authors.initials: JM
authors.lastname: Peltier
authors.forename: Juan
authors.initials: J
authors.lastname: Botas
authors.forename: Robert E
authors.initials: RE
authors.lastname: Hughes
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000187:drug effects; D006801:Humans; D000071058:Huntingtin Protein; D047468:Immunoprecipitation; D051379:Mice; D008959:Models, Neurological; D009410:Nerve Degeneration / Q000235:genetics* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010455:Peptides / Q000633:toxicity; D011485:Protein Binding; D025941:Protein Interaction Mapping; D015203:Reproducibility of Results
chemical_list: C086055:HTT protein, human; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; C097188:polyglutamine