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Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose the concept of an “assayome” for binary protein-protein interaction (PPI) mapping as an optimal combination of assays and/or assay versions that maximizes detection of true positive interactions, while avoiding detection of random protein pairs. We engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions differing by protein expression systems and tagging configurations. The resulting N2H assayome recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our assayome concept should be applicable to systematic mapping of other biological landscapes.

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy.

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.

N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.

Catching primal functional changes in early, ‘very far from disease onset’ (VFDO) stages of Huntington’s disease is likely to be the key to a successful therapy. Focusing on VFDO stages, we assessed neuronal microcircuits in premanifest Hdh150 knock-in mice. Employing in vivo two-photon Ca2+ imaging, we revealed an early pattern of circuit dysregulation in the visual cortex - one of the first regions affected in premanifest Huntington’s disease - characterized by an increase in activity, an enhanced synchronicity and hyperactive neurons. These findings are accompanied by aberrations in animal behavior. We furthermore show that the antidiabetic drug metformin diminishes aberrant Huntingtin protein load and fully restores both early network activity patterns and behavioral aberrations. This network-centered approach reveals a critical window of vulnerability far before clinical manifestation and establishes metformin as a promising candidate for a chronic therapy starting early in premanifest Huntington’s disease pathogenesis long before the onset of clinical symptoms.

Expanded CAG trinucleotide repeats in Huntington's disease (HD) are causative for neurotoxicity. The mutant CAG repeat RNA encodes neurotoxic polyglutamine proteins and can lead to a toxic gain of function by aberrantly recruiting RNA-binding proteins. One of these is the MID1 protein, which induces aberrant Huntingtin (HTT) protein translation upon binding. Here we have identified a set of CAG repeat binder candidates by in silico methods. One of those, furamidine, reduces the level of binding of HTT mRNA to MID1 and other target proteins in vitro. Metadynamics calculations, fairly consistent with experimental data measured here, provide hints about the binding mode of the ligand. Importantly, furamidine also decreases the protein level of HTT in a HD cell line model. This shows that small molecules masking RNA-MID1 interactions may be active against mutant HTT protein in living cells.

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.

Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington’s disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branchingin vitroplays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.

Impairment of synaptic function can lead to neuropsychiatric disorders collectively referred to as synaptopathies. The SNARE protein SNAP-25 is implicated in several brain pathologies and, indeed, brain areas of psychiatric patients often display reduced SNAP-25 expression. It has been recently found that acute downregulation of SNAP-25 in brain slices impairs long-term potentiation; however, the processes through which this occurs are still poorly defined. We show that in vivo acute downregulation of SNAP-25 in CA1 hippocampal region affects spine number. Consistently, hippocampal neurons from SNAP-25 heterozygous mice show reduced densities of dendritic spines and defective PSD-95 dynamics. Finally, we show that, in brain, SNAP-25 is part of a molecular complex including PSD-95 and p140Cap, with p140Cap being capable to bind to both SNAP-25 and PSD-95. These data demonstrate an unexpected role of SNAP-25 in controlling PSD-95 clustering and open the possibility that genetic reductions of the protein levels – as occurring in schizophrenia – may contribute to the pathology through an effect on postsynaptic function and plasticity.

Alzheimer’s disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-β (Aβ) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aβ peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aβ in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

Sonic Hedgehog (SHH)-GLI signalling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signalling. A protein complex containing the ubiquitin-ligase MID1 and protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signalling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyses the ubiquitination and proteasomal cleavage of the GLI3-regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1/PP2A protein complex with GLI3 activity control.

PolyQ (polyglutamine) diseases such as HD (Huntington's disease) or SCA1 (spinocerebellar ataxia type 1) are neurodegenerative disorders caused by abnormally elongated polyQ tracts in human proteins. PolyQ expansions promote misfolding and aggregation of disease-causing proteins, leading to the appearance of nuclear and cytoplasmic inclusion bodies in patient neurons. Several lines of experimental evidence indicate that this process is critical for disease pathogenesis. However, the molecular mechanisms underlying spontaneous polyQ-containing aggregate formation and the perturbation of neuronal processes are still largely unclear. The present chapter reviews the current literature regarding misfolding and aggregation of polyQ-containing disease proteins. We specifically focus on studies that have investigated the amyloidogenesis of polyQ-containing HTTex1 (huntingtin exon 1) fragments. These protein fragments are disease-relevant and play a critical role in HD pathogenesis. We outline potential mechanisms behind mutant HTTex1 aggregation and toxicity, as well as proteins and small molecules that can modify HTTex1 amyloidogenesis in vitro and in vivo. The potential implications of such studies for the development of novel therapeutic strategies are discussed.

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.

Genetic studies have linked the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in the etiology of schizophrenia and other psychiatric disorders. However, the function of the protein encoded by this locus, LG72, is currently controversially discussed. Some studies have suggested that LG72 binds to and regulates the activity of the peroxisomal enzyme D-amino-acid-oxidase, while others proposed an alternative role of this protein due to its mitochondrial location in vitro. Studies with transgenic mice expressing LG72 further suggested that high levels of LG72 lead to an impairment of mitochondrial functions with a concomitant increase in reactive oxygen species production. In the present study, we now performed extensive interaction analyses and identified the mitochondrial methionine-R-sulfoxide reductase B2 (MSRB2) as a specific interaction partner of LG72. MSRB2 belongs to the MSR protein family and functions in mitochondrial oxidative stress defense. Based on our results, we propose that LG72 is involved in the regulation of mitochondrial oxidative stress.

HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10(-5)). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming. Stem Cells 2014;32:364–376

SUMMARY A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-β and α-synuclein.

Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue-specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimer's or Huntington's disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat-shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non-invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.

Protein-protein interactions (PPIs) play a key role in many, if not all, cellular processes. Disease is often caused by perturbation of PPIs, as recently indicated by studies of missense mutations. To understand the associations of proteins and to unravel the global picture of PPIs in the cell, different experimental detection techniques for PPIs have been established. Genetic and biochemical methods such as the yeast two-hybrid system or affinity purification-based approaches are well suited to high-throughput, proteome-wide screening and are mainly used to obtain qualitative results. However, they have been criticized for not reflecting the cellular situation or the dynamic nature of PPIs. In this review, we provide an overview of various genetic methods that go beyond qualitative detection and allow quantitative measuring of PPIs in mammalian cells, such as dual luminescence-based co-immunoprecipitation, Förster resonance energy transfer or luminescence-based mammalian interactome mapping with bait control. We discuss the strengths and weaknesses of different techniques and their potential applications in biomedical research.

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.

The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease β-amyloid peptide (Aβ) neurotoxicity. Solution-state NMR allows probing initial EGCG-Aβ interactions. We show that EGCG-induced Aβ oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Aβ. The C-terminal part of the Aβ peptide (residues 22-39) adopts a β-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Aβ aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions.

The polyglutamine-binding protein 1 (PQBP1) has been linked to several X-linked intellectual disability disorders and progressive neurodegenerative diseases. While it is currently known that PQBP1 localizes in nuclear speckles and is engaged in transcription and splicing, we have now identified a cytoplasmic pool of PQBP1. Analysis of PQBP1 complexes revealed six novel interacting proteins, namely the RNA-binding proteins KSRP, SFPQ/PSF, DDX1 and Caprin-1, and two subunits of the intracellular transport-related dynactin complex, p150 Glued and p27. PQBP1 protein complex formation is dependent on the presence of RNA. Immunofluorescence studies revealed that in primary neurons, PQBP1 co-localizes with its interaction partners in specific cytoplasmic granules, which stained positive for RNA. Our results suggest that PQBP1 plays a role in cytoplasmic mRNA metabolism. This is further supported by the partial co-localization and interaction of PG1BP1 with the fragile X mental retardation protein (FMRP), which is one of the best-studied proteins found in RNA granules. In further studies, we show that arsenite-induced oxidative stress caused relocalization of PQBP1 to stress granules (SGs), where PQBP1 co-localizes with the new binding partners as well as with FMRP. Additional results indicated that the cellular distribution of PQBP1 plays a role in SG assembly. Together these data demonstrate a role for PQBP1 in the modulation of SGs and suggest its involvement in the transport of neuronal RNA granules, which are of critical importance for the development and maintenance of neuronal networks, thus illuminating a route by which PG1BP1 aberrations might influence cognitive function.

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

Yeast two-hybrid (Y2H) screening is a powerful method to detect protein–protein interactions (PPI) at the genomic-scale. A recently proposed framework for binary interactome mapping recommends the repeated screening approach to improve the quality of PPI data. Such repeated screening reveals Y2H interactions ranging from highly sampled to singleton interactions. The quality and the biological significance of interactions from distinguished sampling classes remain unknown. In order to systematically characterize such interactions, we have chosen a dataset of 1,262 interactions that were screened repeatedly four-times. The interactions were classified as highly sampled, weakly sampled, and singleton interactions. We assessed the quality of interactions in different sampling classes using features such as protein structural properties, conservation in yeast and presence of known domain–domain interactions that are previously associated with false positive rates. Our analysis reveals that the quality of singleton interactions is comparable to that of highly sampled interactions. Interestingly, singletons encompass a higher fraction of known domain–domain interactions than highly sampled ones. Furthermore, we observed that the singleton interactions are transient in nature, while the highly sampled interactions are predominantly part of stable complexes. Hence, the repeated Y2H screening method is ideal for detecting transient PPIs that are crucial in cellular signaling pathways.

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1–89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1–89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1–89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.

Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, we show that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines, as shown by the following experiments: (1) downregulating TfR expression reduced proliferation in vitro and in vivo; (2) forced TfR expression in low-grade glioma accelerated proliferation to the level of high-grade glioma; (3) iron and oxidant chelators attenuated tumor proliferation in vitro and tumor size in vivo. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused N-methyl-D-aspartate-receptor-mediated reduction of neuron cell mass. Our results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Selective vulnerability of neurons is a critical feature of neurodegenerative diseases, but the underlying molecular mechanisms remain largely unknown. We here report that Omi/HtrA2, a mitochondrial protein regulating survival and apoptosis of cells, decreases selectively in striatal neurons that are most vulnerable to the Huntington's disease (HD) pathology. In microarray analysis, Omi/HtrA2 was decreased under the expression of mutant huntingtin (htt) in striatal neurons but not in cortical or cerebellar neurons. Mutant ataxin-1 (Atx-1) did not affect Omi/HtrA2 in any type of neuron. Western blot analysis of primary neurons expressing mutant htt also confirmed the selective reduction of the Omi/HtrA2 protein. Immunohistochemistry with a mutant htt-transgenic mouse line and human HD brains confirmed reduction of Omi/HtrA2 in striatal neurons. Overexpression of Omi/HtrA2 by adenovirus vector reverted mutant htt-induced cell death in primary neurons. These results collectively suggest that the homeostatic but not proapoptotic function of Omi/HtrA2 is linked to selective vulnerability of striatal neurons in HD pathology.

Previous studies indicate that binding of alpha-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled alpha-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that alpha-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of alpha-synuclein with membranes in vitro. When compared to wild-type alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of alpha-synuclein with membranes is altered in the inherited forms of Parkinson's disease.

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1α (EF-1α) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1α. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS. Electronic supplementary material The online version of this article (doi:10.1007/s00439-007-0456-6) contains supplementary material, which is available to authorized users.

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.

Death-associated protein 6 (DAXX) is a ubiquitous protein implicated in various cellular processes such as apoptosis, tumorigenesis, development and transcription. The role of DAXX is however ambiguous and many contradictory results regarding its function in apoptosis upon various cellular stresses are described in the literature. In order to have a better understanding of the role of DAXX throughout the entire organism under physiological stress conditions, we have characterized the mRNA levels, protein expression and the proteolytic processing of DAXX in the normal aging process in peripheral organs and brain regions in C57BL/6 male mice. Overall, Daxx mRNA expression decreases with aging in the liver, kidney, heart, cortex and cerebellum. In contrast, an increase is observed in the striatum. The protein expression of DAXX and of its proteolytic fragments increases with aging in the kidney, heart and cortex. In liver and spleen, no changes are observed while in the striatum and cerebellum, certain forms increase and others decrease with age, suggesting that the functions of DAXX may be cell type dependent. This study provides important details regarding the expression and post-translational modifications of DAXX in aging in the entire organism and provides reference data for the deregulation observed in age-associated diseases.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity., The AAA+ ATPase p97 is an essential hexameric protein with multiple protein interaction partners and cellular functions. Here, the authors use interaction mapping to examine partner proteins of this large complex, and assess the effects of these proteins on the disassembly of the p97 complex.

Drosophila melanogaster is an established and versatile model organism. Here we describe and make available a collection of transgenic Drosophila strains expressing human synaptic genes. The collection can be used to study and characterise human synaptic genes and their interactions and as controls for mutant studies. It was generated in a way that allows the easy addition of new strains, as well as their combination. In order to highlight the potential value of the collection for the characterisation of human synaptic genes we also use two assays, investigating any gain-of-function motor and/or cognitive phenotypes in the strains in this collection. Using these assays we show that among the strains made there are both types of gain-of-function phenotypes investigated. As an example, we focus on the three strains expressing human tyrosine protein kinase Fyn, the small GTPase Rap1a and human Arc, respectively. Of the three, the first shows a cognitive gain-of-function phenotype while the second a motor gain-of-function phenotype. By contrast, Arc, which has no Drosophila ortholog, shows no gain-of-function phenotype., Summary: This report describes a resource collection of Drosophila melanogaster strains expressing human synaptic genes and methods that can be applied in order to investigate the genes' function.

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.

Mutations in the valosin-containing protein (VCP, p97) gene on chromosome 9p13-p12 cause a late-onset form of autosomal dominant inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes and filamentous VCP- and ubiquitin-positive cytoplasmic and nuclear protein aggregates. Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not detected in primary IBMPFD myoblasts or in transient and stable transfected cells using wild-type-VCP and R93C-, R155H-, R155C-VCP mutants. Glutathione S-transferase pull-down experiments showed that all three VCP mutations do not affect the binding to Ufd1, Npl4 and ataxin-3. Structural analysis demonstrated that R93 and R155 are both surface-accessible residues located in the centre of cavities that may enable ligand-binding. Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the identification of cyclic sugar compounds with high binding scores. The latter findings provide a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD.

Enhancing cellular defense mechanisms against different kinds of stress may be an attractive therapeutic strategy for neurodegenerative diseases. In particular, inducing the expression of molecular chaperones might reduce the formation of misfolded proteins and toxic aggregates that occur in polyglutamine (polyQ) disorders such as Huntington’s disease. Geldanamycin, a natural substance that modulates Hsp90 function, was previously shown to induce a heat-shock response and to reduce polyQ aggregation in mammalian cells. However, because of toxic and unfavorable pharmacokinetic properties, geldanamycin is not suitable for clinical use. In this study we evaluated the effects of the pharmacologically improved geldanamycin derivatives 17-DMAG and 17-AAG on polyQ aggregation in mammalian cells. Quantitative RT-PCR and SDS-PAGE experiments revealed that 17-DMAG induces expression of the molecular chaperones Hsp40, Hsp70, and Hsp105 in mammalian cells and inhibits the formation of mutant huntingtin aggregates with higher efficiency than 17-AAG or geldanamycin itself. Induction of a heat-shock response and inhibition of polyQ aggregation occurred at nanomolar concentrations. We suggest that geldanamycin derivatives such as 17-DMAG should be considered for the development of a drug treatment for polyQ disorders and other neurodegenerative diseases involving protein aggregation.

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.

Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening as well as for the validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.

The accumulation of amyloid beta peptide(1-42) (Abeta(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Abeta(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Abeta may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Abeta endocytosis. We visualized aggregate formation of fluorescently labeled Abeta(1-42) and tracked its internalization by human neuroblastoma cells and neurons. beta-Sheet-rich Abeta(1-42) aggregates entered the cells at low nanomolar concentration of Abeta(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Abeta(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Abeta(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of beta-sheet-rich aggregates is a prerequisite for Abeta(1-42) uptake and cytotoxicity.

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins. DFG [SFB740, 740/2-11, SFB618, 618/3-09, SFB/TRR43 A7]; BMBF(NGFN-Plus) [01GS08169-73, 01GS08150, 01GS08108]; HDSA Coalition for the Cure; EU (EuroSpin) [Health-F2-2009-241498, HEALTH-F2-2009-242167]; Helmholtz Association (MSBN, HelMA) [HA-215]; FCT [IF/00881/2013] info:eu-repo/semantics/publishedVersion