A human protein-protein interaction network: a resource for annotating the proteome

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.     

Identification of connexin-interacting proteins: application of the yeast two-hybrid screen

Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy.     

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA)² (2–6), where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7, 8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction “hub” according to their binding affinity.Quantitative estimation of protein-protein interactions in vivo will require the amount of the complex to be directly measured or the level of reconstituted reporters to be directly proportional to the strength of the interactions. To achieve quantitative analysis of protein interactions in eukaryotic expression system, we have designed a yeast surface two-hybrid (YS2H) system that can express a pair of proteins, one protein as a fusion to a yeast cell wall protein, agglutinin, and the other in a secretory form. When two proteins interact in this system, they associate in the secretory pathway, and the prey that would otherwise be released into the media is captured on the surface by the bait. We have devised two different schemes to quantitatively estimate the affinity of two interacting molecules: flow cytometric detection of antibody binding to the epitope tags fused to the prey and the bait, and the GFP readout from the complementation of split GFP fragments fused to the prey and the bait. They are induced under a bi-directional promoter to promote a synchronized and comparable level of expression.Herein we demonstrate the quantitative nature of YS2H in predicting the affinity between two interacting proteins, particularly in the range of 100 pm to 10 μm. This feature allowed us to examine specific interactions between antigen and antibody, to identify hot spots of allosteric activation in integrins, and to isolate camelid heavy chain-only antibodies against botulinum neurotoxin as components of therapeutic agents to treat botulism (9). With the incorporation of PCA technique into the YS2H, our system may be developed into an in vivo tool to measure the kinetics of protein-protein interactions. Potential applications of YS2H include affinity maturation of antibodies, differentiation of weak to high affinity binders to the hub protein in interaction networks, and confirmation of hypothetical interacting pairs of proteins in a high throughput manner.     

蛋白质相互作用的研究方法

随着基因组测序工程数量的增加,未知功能蛋白质测序工作也呈现指数式增长。生物学进程主要是由蛋白质执行和控制的,因此,阐明未知或已知蛋白质的生物学功能和从细胞水平上确定细胞机制,已成为蛋白质组学研究的主要目标。目前,随着酵母[1]、果蝇[2]、线虫[3]相互作用图谱的相继完成,蛋白质相互作用研究方法也不断发展和完善。综述了当前研究蛋白质相互作用的主要技术方法,包括酵母双杂交技术,GSTpull-down技术,免疫共沉淀技术和串联亲和纯化技术等多种研究方法,分析了各种技术方法的优缺点以及各种方法的改进,在试验中可根据不同的要求和目的选择适宜的方法。     

Re-engineering a split-GFP reassembly screen to examine RING-domain interactions between BARD1 and BRCA1 mutants observed in cancer patients

Identification of protein-protein interactions is critical for understanding protein function and regulation. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2-hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly, also called Bimolecular Fluorescence Complementation (BiFC), is especially attractive because the GFP chromophore forms spontaneously on protein folding in virtually every cell type tested. However, cellular fluorescence evolves slowly in bacteria and fails to evolve at all for some interactions. We aimed to use split-GFP reassembly to examine the determinants of association for a heterodimeric four-helix bundle, and we chose the N-terminal RING domains of BARD1 and the tumor suppressor BRCA1 as our test system. The wild-type interaction failed to give fluorescence with the split sg100 GFP variant. We found that split folding-reporter GFP (a hybrid of EGFP and GFPuv) evolves fluorescence much faster (overnight) with associating peptides and also evolves fluorescence for the BRCA1/BARD1 wild-type pair. Six cancer-associated BRCA1 interface mutants were examined with the system, and only two resulted in a significant reduction in complex reassembly. These results are generally in accord with Y2H studies, but the differences highlight the utility of complementary approaches. The split frGFP system may also be generally useful for other proteins and cell types, as the split-Venus system has proven to be in mammalian cells.     

Integrating a functional proteomic approach into the target discovery process

Functional proteomics is a promising technique for the rational identification of novel therapeutic targets by elucidation of the function of newly identified proteins in disease-relevant cellular pathways. Of the recently described high-throughput approaches for analyzing protein-protein interactions, the yeast two-hybrid (Y2H) system has turned out to be one of the most suitable for genome-wide analysis. However, this system presents a challenging technical problem: the high prevalence of false positives and false negatives in datasets due to intrinsic limitations of the technology and the use of a high-throughput, genetic assay. We discuss here the different experimental strategies applied to Y2H assays, their general limitations and advantages. We also address the issue of the contribution of protein interaction mapping to functional biology, especially when combined with complementary genomic and proteomic analyses. Finally, we illustrate how the combination of protein interaction maps with relevant functional assays can provide biological support to large-scale protein interaction datasets and contribute to the identification and validation of potential therapeutic targets.     

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions. The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.     

Dissecting protein-protein interactions using directed evolution

Protein-protein interactions are essential for life. They are responsible for most cellular functions and when they go awry often lead to disease. Proteins are inherently complex. They are flexible macromolecules whose constituent amino acid components act in combinatorial and networked ways when they engage one another in binding interactions. It is just this complexity that allows them to conduct such a broad array of biological functions. Despite decades of intense study of the molecular basis of protein-protein interactions, key gaps in our understanding remain, hindering our ability to accurately predict the specificities and affinities of their interactions. Until recently, most protein-protein investigations have been probed experimentally at the single-amino acid level, making them, by definition, incapable of capturing the combinatorial nature of, and networked communications between, the numerous residues within and outside of the protein-protein interface. This aspect of protein-protein interactions, however, is emerging as a major driving force for protein affinity and specificity. Understanding a combinatorial process necessarily requires a combinatorial experimental tool. Much like the organisms in which they reside, proteins naturally evolve over time, through a combinatorial process of mutagenesis and selection, to functionally associate. Elucidating the process by which proteins have evolved may be one of the keys to deciphering the molecular rules that govern their interactions with one another. Directed evolution is a technique performed in the laboratory that mimics natural evolution on a tractable time scale that has been utilized widely to engineer proteins with novel capabilities, including altered binding properties. In this review, we discuss directed evolution as an emerging tool for dissecting protein-protein interactions.     

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact ^1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible^8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays¹⁰. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.Download video file.^{(47M, mov)}     

Global approaches to protein-protein interactions

The availability of complete, annotated genome sequences for a variety of eukaryotic organisms has paved the way for a paradigm shift in biomedical research from the 'one gene-one hypothesis' approach to more global, systematic strategies that analyse genes or proteins on a genome- and proteome-wide scale. One daunting task in the post-genome era is to determine how the complement of expressed cellular proteins - the proteome - is organised into functional, higher-order networks, by mapping all constitutive and dynamic protein-protein interactions. Traditionally, reductionist approaches have typically focused on a few, selected gene products and their interactions in a particular physiological context. In contrast, more holistic strategies aim at understanding complex biological systems, for example global protein-protein interaction networks on a cellular or organismal level. Several large-scale proteomics technologies have been developed to generate comprehensive, cellular protein-protein interaction maps.