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.

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a G␤␥-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-The phototransduction cascade of vertebrate photoreceptor cells is a well studied G protein-mediated signaling pathway that has been a model system for much of our understanding of G protein signaling. Sensitivity in this system is modulated to allow responsiveness over several orders of magnitude of light intensity. Signal response is maximized in the absence of light ("dark adaptation") and dampened as background illumination increases ("light adaptation"). Several independent molecular events, many involving Ca 2ϩ , are believed to underlie this regulation (see Refs. 1 and 2 for reviews). Cytosolic Ca 2ϩ levels in rod outer segments vary from their dark resting concentration of ϳ500 nM to below 50 nM when the Ca 2ϩ channels close as a result of the light signal (3). This decrease in Ca 2ϩ concentration triggers a number of feedback responses believed to be involved in light adaptation (2). These responses include an increase in phosphorylation of rhodopsin (4), which causes rhodopsin inactivation through arrestin binding, and an increase in guanylyl cyclase activity (5), which restores [cGMP] after its depletion by light-activated cGMP phosphodiesterase.Phosducin (Pd), 1 an abundant protein in retinal photoreceptors and the developmentally related pineal gland (6, 7), is believed to play a role in modulation of phototransduction and other G protein pathways (8 -10) by virtue of its ability to bind G protein ␤␥-heterodimers (G␤␥) with high affinity (11-13). When bound to Pd, G␤␥ is sterically blocked from interacting with G␣ subunits (10, 14) or other G␤␥ effectors (15-17). Thus, Pd has been shown to down-regulate G protein signals in photoreceptors (9 -10) and other cell types in vitro (8) as well as in overexpression experiments (15,18). In other G protein systems that require G␤␥ for receptor phosphorylation to initiate receptor inactivation, similar in vitro and overexpression experiments have shown that Pd enhances G protein signaling by blocking the binding of G␤␥ to G protein receptor kinase-2 or -3 (18 -20).Phosphorylation of Pd by cAMP-dependent protein kinase (PKA) significantly diminishes its ability to inhibit G protein signals (8,10,15). In photoreceptor cells, the phosphorylation state of Pd is light-dependent, with maximal phosphorylation occurring in the dark (21). It has therefore been proposed that Pd is a feedback regulator of the light signal in a phosphorylationdependent manner (22). In this hypothesis, dephosphorylation * The work was supported by National Institutes of Health Grants EY12287 (to B. M. W.), AR43768 (to K. A. R.), and EY06062 (to H. E. H.) and by the Howard Hughes Medical Institute (to N. G. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby...

Visual signal transduction is a nearly noise-free process that is exquisitely well regulated over a wide dynamic range of light intensity. A key component in dark/light adaptation is phosducin, a phosphorylatable protein that modulates the amount of transducin heterotrimer (Gt alpha beta gamma) available through sequestration of the beta gamma subunits (Gt beta gamma). The structure of the phosphophosducin/Gt beta gamma complex combined with mutational and biophysical analysis provides a stereochemical mechanism for the regulation of the phosducin-Gt beta gamma interaction. Phosphorylation of serine 73 causes an order-to-disorder transition of a 20-residue stretch, including the phosphorylation site, by disrupting a helix-capping motif. This transition disrupts phosducin's interface with Gt beta gamma, leading to the release of unencumbered Gt beta gamma, which reassociates with the membrane and Gt alpha to form a signaling-competent Gt alpha beta gamma heterotrimer.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the ␤␥ subunit complex (G␤␥) and blocking G␤␥ association with G␣ subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal G t ␤␥. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G t ␤␥ with a 2.5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k 1 ; and 3) most of the free energy of binding comes from the Nterminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of G t ␤␥ with G t ␣ and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of G t ␤␥, both the N-and Cterminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in G t ␤␥ to a conformation that did not bind the membrane. These models yielded a free energy change of ؉1.5 ؎ 0.25 kcal/mol for the transition from the G t ␣-binding to the Pd-binding conformation of G t ␤␥.G protein 1 -coupled receptors detect and transduce a wide variety of chemical and physical stimuli in eukaryotic cells. Signals from hormones, neurotransmitters, odorants, and photons use G protein-dependent pathways. These pathways are designed to amplify and integrate a multiplicity of both stimulatory and inhibitory responses. The cellular response is initiated by an agonist-dependent conformational change in the receptor. This starts a cascade of events in which GTP is exchanged for GDP on the ␣ subunit of the heterotrimer G protein (G), and the G protein dissociates into G␣⅐GTP and G␤␥ subunits. Both G␣⅐GTP and G␤␥ then activate effector enzymes or ion channels. These effectors in turn control kinase cascades and second messenger concentrations (cyclic nucleotides, inositol phosphates, lipids, and Ca 2ϩ

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.