Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time. Molecular & Cellular Proteomics 7: 2279 -2287, 2008.After sequencing the human genome the next challenge is now to analyze the complex protein networks underlying cellular functions. In the last decade a wide variety of methods to study protein-protein interactions ranging from biochemical to genetic or cell-based approaches have been developed. Biochemical methods like affinity purification or co-immunoprecipitation (Co-IP) 1 allow the detection of protein complexes in vitro. Genetic methods, such as the yeast two-hybrid system (1), enable efficient high throughput screening of interactions within the cellular environment (2). However, the analysis of mammalian protein interactions in yeast may suffer from the absence or insufficient conservation of cellular factors modulating protein-protein interactions, e.g. through posttranslational modifications (3).In the last years new fluorescence-based methods for incell visualization of protein-protein interactions have been introduced. Two established techniques, fluorescence resonance energy transfer (4, 5) and bimolecular fluorescence complementation (6), are based on the expression of fluorescently labeled proteins or fragments thereof. However, fluorescence resonance energy transfer requires costly instrumentation and advanced technical expertise, whereas bimolecular fluorescence complementation is based on the irreversible complementation and slow maturation of fluorophores, which does not allow real time detection of proteinprotein interactions (6). Another strategy is based on the relocation of proteins to either cell membranes (7) or cytoplasmic aggregates of viral proteins (8).All these methods have inherent shortcomings and are typically combined to obtain more reliable results. We have now developed a novel fluorescent two-hybrid (F2H) assay for the direct visualization of protein-protein interactions in living mammalian cells. The simple optical readout of this F2H assay allows observation of protein-protein interactions in real time and should also be suitable for high throughput screens. EXPERIMENTAL PROCEDURESExpression Construct...

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A variety of autoantibodies were identified in sera of prostate cancer patients and provide a first step towards autoantibody diagnostics. Serum autoantibodies reflect the disease and represent valuable tools not only for prostate cancer, but also for other diseases affecting the immune response.

Systemic lupus erythematosus (SLE) is a heterogeneous disease with respect to disease manifestations, disease progression and treatment response. Therefore, strategies to identify biomarkers that help distinguishing SLE subgroups are a major focus of biomarker research. We reasoned that a multiparametric autoantibody profiling approach combined with data mining tools could be applied to identify SLE patient clusters. We used a bead-based array containing 86 antigens including diverse nuclear and immune defense pathway proteins. Sixty-four autoantibodies were significantly (p < 0.05) increased in SLE (n ¼ 69) compared to healthy controls (HC, n ¼ 59). Using binary cut-off thresholds (95% quantile of HC), hierarchical clustering of SLE patients yields five clusters, which differ qualitatively and in their total number of autoantibodies. In two patient clusters the overall accumulated autoantibody reactivity of all antigens tested was 31% and 48%, respectively. We observed a positive association between the autoantibody signature present in these two patient clusters and the clinical manifestation of glomerulonephritis (GLMN). In addition, groups of autoantibodies directed against distinct intracellular compartments and/or biological motifs characterize the different SLE subgroups. Our findings highlight the relevant potential of multiparametric autoantibody detection and may contribute to a deeper understanding of the clinical and serological diversity of SLE. Lupus (2016) 25, 812-822.

BackgroundThe aim was to identify novel diagnostic autoantibody candidates for rheumatoid arthritis (RA) by comprehensive screening for autoreactivity.MethodWe incubated 5892 recombinant proteins coupled to fluorescent beads, with patients’ sera for the detection of IgG-autoantibodies in three independent patient cohorts: A (n = 72 patients with established RA); B/B- (n = 116 patients with early RA (B) and n = 51 CCP-negative patients with early RA from B (B-)); and C (n = 184 patients with early seronegative RA), in comparison to matched healthy controls. Intersects of significantly increased autoantibodies as determined by the Mann-Whitney test were sought.ResultScreening of 5892 antigens in RA cohorts A and B, or the seronegative cohorts B- and C revealed intersects of 23 and 13 significantly increased autoantibodies, respectively. Reactivity to three antigens was increased in all cohorts tested: N-acetylglucosamine-1-phosphate transferase, gamma subunit (GNPTG), heterogeneous nuclear ribonucleoprotein A1-like 2 (HNRNPA1), and insulin-like growth factor binding protein 2 (IGFBP2).ConclusionsComprehensive sequential screening for autoantibodies reveals novel candidates for diagnostic markers in both seropositive and seronegative RA and suggests new fields of research into the pathogenesis of RA.

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