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Numerous genetic methods facilitate the detection of binary protein-protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions.

The self-assembly of the 42-residue amyloid-β peptide, Aβ42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aβ42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aβ42 oligomers. These structures exhibit low β-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, β-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aβ42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aβ42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aβ42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, β-sheet-rich Aβ42 fibrils in AD, while structures with low β-sheet content may be less relevant.

Mutations are at the root of many human diseases. Still, we largely do not exactly understand how they trigger pathogenesis. One, more recent, hypothesis has been that they comprehensively perturb protein-protein interaction (PPI) networks and significantly alter key biological processes. Under this premise, many rare genetic disorders with Mendelian inheritance, like Huntington's disease and several spinocerebellar ataxias, are likely to be caused by complex genotype-phenotype relationships involving abnormal PPIs. These altered PPI networks and their effects on cellular pathways are poorly understood at the molecular level. In this review, we focus on PPIs that are perturbed by the expanded pathogenic polyglutamine tract in huntingtin (HTT), the protein which, in its mutated form, leads to the autosomal dominant, neurodegenerative Huntington's disease. One aspect of perturbed mutant HTT interactions is the formation of abnormal protein species such as fibrils or large neuronal inclusions as a result of homotypic and heterotypic aberrant molecular interactions. This review focuses on abnormal PPIs that are associated with the assembly of mutant HTT aggregates in cells and their potential relevance in disease. Furthermore, the mechanisms and pathobiological processes that may contribute to phenotype development, neuronal dysfunction and toxicity in Huntington's disease brains are also discussed. This article is part of the Special Issue "Proteomics".

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.

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.

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.

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.

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.

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

The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.

Human protein interaction maps have become important tools of biomedical research for the elucidation of molecular mechanisms and the identification of new modulators of disease processes. The Unified Human Interactome database (UniHI, http://www.unihi.org) provides researchers with a comprehensive platform to query and access human protein-protein interaction (PPI) data. Since its first release, UniHI has considerably increased in size. The latest update of UniHI includes over 250 000 interactions between similar to 22 300 unique proteins collected from 14 major PPI sources. However, this wealth of data also poses new challenges for researchers due to the complexity of interaction networks retrieved from the database. We therefore developed several new tools to query, analyze and visualize human PPI networks. Most importantly, UniHI allows now the construction of tissue-specific interaction networks and focused querying of canonical pathways. This will enable researchers to target their analysis and to prioritize candidate proteins for follow-up studies. Deutsche Forschungsgemeinschaft [SFB 618] info:eu-repo/semantics/publishedVersion