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Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.

Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.

Cluster Analysis↗

Toward functional analysis of protein interactome using "in vitro virus": in silico analyses of Fos/Jun interactors.

Our high-throughput in vitro virus (IVV) method for selection of protein-protein interactions (PPI) and complexes, based on a simple cell-free co-translation and selection followed by computational sequence data analysis, was previously used to identify 31 Fos and Jun interactors. Here, in silico analyses of biological function, localization and phenotype of these AP-1 (Fos/Jun) interactors were performed. The results suggest that Fos and Jun do not necessarily work together, but also interact separately with novel interactors, including products of disease-related genes. Fos showed transcription-related activities, while Jun interacted with motor-related and structural proteins. The reliability of the IVV selection for the Fos interactors was further confirmed by means of in vitro reciprocal prey and bait protein experiments and co-immunoprecipitation. Further study of these novel interactors may provide clues to new pathways or mechanisms of biological functions and diseases.

Animals↗

Modeling interactome: scale-free or geometric?

MOTIVATION: Networks have been used to model many real-world phenomena to better understand the phenomena and to guide experiments in order to predict their behavior. Since incorrect models lead to incorrect predictions, it is vital to have as accurate a model as possible. As a result, new techniques and models for analyzing and modeling real-world networks have recently been introduced. RESULTS: One example of large and complex networks involves protein-protein interaction (PPI) networks. We analyze PPI networks of yeast Saccharomyces cerevisiae and fruitfly Drosophila melanogaster using a newly introduced measure of local network structure as well as the standardly used measures of global network structure. We examine the fit of four different network models, including Erdos-Renyi, scale-free and geometric random network models, to these PPI networks with respect to the measures of local and global network structure. We demonstrate that the currently accepted scale-free model of PPI networks fails to fit the data in several respects and show that a random geometric model provides a much more accurate model of the PPI data. We hypothesize that only the noise in these networks is scale-free. CONCLUSIONS: We systematically evaluate how well-different network models fit the PPI networks. We show that the structure of PPI networks is better modeled by a geometric random graph than by a scale-free model. SUPPLEMENTARY INFORMATION: Supplementary information is available at http://www.cs.utoronto.ca/~juris/data/data/ppiGRG04/

Algorithms↗

The tYNA platform for comparative interactomics: a web tool for managing, comparing and mining multiple networks.

UNLABELLED: Biological processes involve complex networks of interactions between molecules. Various large-scale experiments and curation efforts have led to preliminary versions of complete cellular networks for a number of organisms. To grapple with these networks, we developed TopNet-like Yale Network Analyzer (tYNA), a Web system for managing, comparing and mining multiple networks, both directed and undirected. tYNA efficiently implements methods that have proven useful in network analysis, including identifying defective cliques, finding small network motifs (such as feed-forward loops), calculating global statistics (such as the clustering coefficient and eccentricity), and identifying hubs and bottlenecks. It also allows one to manage a large number of private and public networks using a flexible tagging system, to filter them based on a variety of criteria, and to visualize them through an interactive graphical interface. A number of commonly used biological datasets have been pre-loaded into tYNA, standardized and grouped into different categories. AVAILABILITY: The tYNA system can be accessed at http://networks.gersteinlab.org/tyna. The source code, JavaDoc API and WSDL can also be downloaded from the website. tYNA can also be accessed from the Cytoscape software using a plugin.

Computer Graphics↗

The interactome as a tree--an attempt to visualize the protein-protein interaction network in yeast.

The refinement and high-throughput of protein interaction detection methods offer us a protein-protein interaction network in yeast. The challenge coming along with the network is to find better ways to make it accessible for biological investigation. Visualization would be helpful for extraction of meaningful biological information from the network. However, traditional ways of visualizing the network are unsuitable because of the large number of proteins. Here, we provide a simple but information-rich approach for visualization which integrates topological and biological information. In our method, the topological information such as quasi-cliques or spoke-like modules of the network is extracted into a clustering tree, where biological information spanning from protein functional annotation to expression profile correlations can be annotated onto the representation of it. We have developed a software named PINC based on our approach. Compared with previous clustering methods, our clustering method ADJW performs well both in retaining a meaningful image of the protein interaction network as well as in enriching the image with biological information, therefore is more suitable in visualization of the network.

Algorithms↗

Decoding the Functional Interactome of Non-Model Organisms with PHILHARMONIC.

Despite the widespread availability of genome sequencing pipelines, many genes remain part of the genome's "dark matter," where existing inference tools cannot even begin to guess the biological function of their proteins from sequence alone. This challenge is especially pronounced in organisms that are highly evolutionarily distant from well-studied models, where homology-based methods break down. Here, we describe PHILHARMONIC, a computational method that combines deep learning-based de novo protein interaction network inference with robust unsupervised spectral clustering and remote homology to illuminate functional organization in any non-model organism. From only a sequenced proteome, we show PHILHARMONIC predicts protein functions, functional communities, and higher-order network structure with high accuracy. We validate its performance using experimental gene expression and pathway data in D. melanogaster, and we demonstrate its broad utility by analyzing temperature sensing and stress response pathways in the reef-building coral P. damicornis and its algal symbiont C. goreaui. PHILHARMONIC provides a general-purpose engine for functional discovery and biological hypothesis generation in non-model organisms, enabling systems-level insights across the full diversity of life.

Journal Article↗

A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach.

This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7-d-old barley (Hordeum vulgare) cv Himalaya seedlings using yeast two-hybrid screens complemented with 14-3-3 protein affinity purification and tandem mass spectrometry. Independent experiments for a subset of genes confirmed the yeast two-hybrid interactions, demonstrating a low false positive identification rate. These combined approaches resulted in the identification of more than 150 putative targets; 15% were previously reported to be 14-3-3 interactors, including, for example, Serpin, RF2A, WPK4 kinase, P-type proton-translocating adenosine triphosphatase, EF1A, glutamine synthetase, and invertases. The affinity purification resulted in 30 interactors, of which 44% function in metabolism, while the yeast two-hybrid screens identified 132 different proteins, with 35% of the proteins involved in signal transduction. A number of proteins have a well-described function in hormonal signaling, such as the auxin transport protein PIN1 and NPH3 and components of the brassinosteroid pathway, such as the receptor kinase BAK1 (OsPERK1) and BRI1-kinase domain-interacting protein 129. However, 14-3-3 interactions with these signal mediators have not been confirmed in the affinity purification. Confirmations of the 14-3-3 interaction with the three ABF-like transcription factors are shown using far western analysis. Also, a REPRESSION OF SHOOT GROWTH ortholog named RF2A was identified; these transcription factors play important roles in the abscisic acid and gibberellin pathways, respectively. We speculate that 14-3-3 proteins have a role in cross talk between these hormonal pathways. The specificity and complementary nature of both the affinity purification and the yeast two-hybrid approaches is discussed.

14-3-3 Proteins↗

Microarrays for the functional analysis of the chemical-kinase interactome.

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals for monitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC50 of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.

Biological Assay↗

Direct and heterologous approaches to identify the LET-756/FGF interactome.

BACKGROUND: Fibroblast growth factors (FGFs) are multifunctional proteins that play important roles in cell communication, proliferation and differentiation. However, many aspects of their activities are not well defined. LET-756, one of the two C. elegans FGFs, is expressed throughout development and is essential for worm development. It is both expressed in the nucleus and secreted. RESULTS: To identify nuclear factors associated with LET-756, we used three approaches. First, we screened a two-hybrid cDNA library derived from mixed stages worms and from a normalized library, using LET-756 as bait. This direct approach allowed the identification of several binding partners that play various roles in the nucleus/nucleolus, such as PAL-1, a transcription regulator, or RPS-16, a component of the small ribosomal subunit. The interactions were validated by co-immunoprecipitation and determination of their site of occurrence in mammalian cells. Second, because patterns of protein interactions may be conserved throughout species, we searched for orthologs of known mammalian interactors and measured binary interaction with these predicted candidates. We found KIN-3 and KIN-10, the orthologs of CK2alpha and CK2beta, as new partners of LET-756. Third, following the assumption that recognition motifs mediating protein interaction may be conserved between species, we screened a two-hybrid cDNA human library using LET-756 as bait. Among the few FGF partners detected was 14-3-3beta. In support of this interaction we showed that the two 14-3-3beta orthologous proteins, FTT-1 and FTT-2/PAR-5, interacted with LET-756. CONCLUSION: We have conducted the first extensive search for LET-756 interactors using a multi-directional approach and established the first interaction map of LET-756/FGF with other FGF binding proteins from other species. The interactors identified play various roles in developmental process or basic biochemical events such as ribosome biogenesis.

14-3-3 Proteins↗

Consolidating the set of known human protein-protein interactions in preparation for large-scale mapping of the human interactome.

BACKGROUND: Extensive protein interaction maps are being constructed for yeast, worm, and fly to ask how the proteins organize into pathways and systems, but no such genome-wide interaction map yet exists for the set of human proteins. To prepare for studies in humans, we wished to establish tests for the accuracy of future interaction assays and to consolidate the known interactions among human proteins. RESULTS: We established two tests of the accuracy of human protein interaction datasets and measured the relative accuracy of the available data. We then developed and applied natural language processing and literature-mining algorithms to recover from Medline abstracts 6,580 interactions among 3,737 human proteins. A three-part algorithm was used: first, human protein names were identified in Medline abstracts using a discriminator based on conditional random fields, then interactions were identified by the co-occurrence of protein names across the set of Medline abstracts, filtering the interactions with a Bayesian classifier to enrich for legitimate physical interactions. These mined interactions were combined with existing interaction data to obtain a network of 31,609 interactions among 7,748 human proteins, accurate to the same degree as the existing datasets. CONCLUSION: These interactions and the accuracy benchmarks will aid interpretation of current functional genomics data and provide a basis for determining the quality of future large-scale human protein interaction assays. Projecting from the approximately 15 interactions per protein in the best-sampled interaction set to the estimated 25,000 human genes implies more than 375,000 interactions in the complete human protein interaction network. This set therefore represents no more than 10% of the complete network.

Algorithms↗

Discovering the interactome, functions, and clinical relevance of enhancer RNAs in kidney renal clear cell carcinoma.

Enhancer RNA (eRNA) has emerged as a key player in cancer biology, influencing various aspects of tumor development and progression. In this study, we investigated the role of eRNAs in kidney renal clear cell carcinoma (KIRC), the most common subtype of renal cell carcinoma. Leveraging high-throughput sequencing data and bioinformatics analysis, we identified differentially expressed eRNAs in KIRC and constructed eRNA-centric regulatory networks. Our findings revealed that up-regulated eRNAs in KIRC potentially regulate immune response and hypoxia pathways, while down-regulated eRNAs may impact ion transport, cell cycle, and metabolism. Furthermore, we developed a diagnostic prediction model based on eRNA expression profiles, demonstrating its effectiveness in KIRC diagnosis. Finally, we elucidated the regulatory mechanism of an eRNA (ENSR00000305834) on the expression of SLC15A2, a potential prognostic biomarker in KIRC, through bioinformatics analysis and in vitro validation experiments. In summary, Our study highlights the clinical significance of eRNAs in KIRC and underscores their potential as therapeutic targets.

Carcinoma, Renal Cell↗

Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes.

Recent proteome-wide screening approaches have provided a wealth of information about interacting proteins in various organisms. To test for a potential association between protein connectivity and the amount of predicted structural disorder, the disorder propensities of proteins with various numbers of interacting partners from four eukaryotic organisms (Caenorhabditis elegans, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens) were investigated. The results of PONDR VL-XT disorder analysis show that for all four studied organisms, hub proteins, defined here as those that interact with > or = 10 partners, are significantly more disordered than end proteins, defined here as those that interact with just one partner. The proportion of predicted disordered residues, the average disorder score, and the number of predicted disordered regions of various lengths were higher overall in hubs than in ends. A binary classification of hubs and ends into ordered and disordered subclasses using the consensus prediction method showed a significant enrichment of wholly disordered proteins and a significant depletion of wholly ordered proteins in hubs relative to ends in worm, fly, and human. The functional annotation of yeast hubs and ends using GO categories and the correlation of these annotations with disorder predictions demonstrate that proteins with regulation, transcription, and development annotations are enriched in disorder, whereas proteins with catalytic activity, transport, and membrane localization annotations are depleted in disorder. The results of this study demonstrate that intrinsic structural disorder is a distinctive and common characteristic of eukaryotic hub proteins, and that disorder may serve as a determinant of protein interactivity.

Amino Acids↗

Proximity interactome of alphavirus replicase component nsP3 includes proviral host factors eIF4G and AHNAK.

All positive-strand RNA viruses replicate their genomes in association with modified intracellular membranes, inducing either membrane invaginations termed spherules, or double-membrane vesicles. Alphaviruses encode four non-structural proteins nsP1-nsP4, all of which are essential for RNA replication and spherule formation. To understand the host factors associated with the replication complex, we fused the efficient biotin ligase miniTurbo with Semliki Forest virus (SFV) nsP3, which is located on the cytoplasmic surface of the spherules. We characterized the proximal proteome of nsP3 in three cell lines, including cells unable to form stress granules, and identified >300 host proteins constituting the microenvironment of nsP3. These included all the nsPs, as well as several previously characterized nsP3 binding proteins. However, the majority of the identified interactors had no previously identified roles in alphavirus replication, including 39 of the top 50 interacting proteins. The most prominent biological processes involving the proximal proteins were nucleic acid metabolism, translational regulation, cytoskeletal rearrangement and membrane remodeling. siRNA silencing confirmed six novel proviral factors, USP10, AHNAK, eIF4G1, SH3GL1, XAB2 and ANKRD17, which are associated with distinct cellular functions. All of these except SH3GL1 were also important for the replication of chikungunya virus. We discovered that the small molecule 4E1RCat, which inhibits the interaction between the canonical translation initiation factors eIF4G and eIF4E, exhibits antiviral activity against SFV. Since the same molecule was previously found to inhibit coronaviruses, this suggest the possibility that translation initiation factors could be considered as targets for broadly acting antivirals.

Viral Nonstructural Proteins↗