Detecting protein-protein interactions by Far western blotting

Far western blotting (WB) was derived from the standard WB method to detect protein-protein interactions in vitro. In Far WB, proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with purified bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purification. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., fluorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Typically, 2-3 d are required to carry out the experiment.     

Imaging Beads-Retained Prey Assay for Rapid and Quantitative Protein-Protein Interaction

Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications.     

A novel approach for the identification of protein–protein interaction with integral membrane proteins

Protein–protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein–protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein–protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein–protein interactions with high specificity and selectivity, where other methods fail.     

Two-hybrid reporter vectors for gap repair cloning

Yeast two-hybrid analysis is a valuable approach to the discovery and characterization of protein interactions. We have developed vectors that can indicate the presence of an insert when used in two-hybrid bait and prey construction by gap repair cloning. The strategy uses a recombination cloning site flanked by sequences encoding the GAL4 activation and binding domains. After gap repair cloning in standard hosts carrying an ADE2 reporter gene, disruption of GAL4 by an insert can be identified by the development of red colony color, while empty vector plasmids produce white colonies. Function in yeast two-hybrid applications was initially validated using known interacting proteins in pair-wise analyses, and subsequently, the bait vectors were used in library screens with the mouse Mad212 and human Mccd1 proteins, identifying a number of putative new interactions for these proteins. These vectors should facilitate high-throughput yeast two-hybrid screens in which large numbers of bait and prey constructs may be required.     

应用酵母双杂交系统筛选橡胶延长因子REF的互作蛋白

利用酵母双杂交系统,以橡胶树(Hevea brasiliensis)橡胶延长因子基因REF的开放阅读框(ORF)构建无自激活性的诱饵表达载体pBD-GAL4-REF,并筛选以pAD-GAL4-2.1载体构建的橡胶树胶乳cDNA文库,对阳性克隆的cDNA插入片段进行测序及生物学功能分析。通过酵母双杂交筛选,共获得5种可能与REF互作的候选蛋白质,它们分别为与诱饵蛋白REF高度同源的REF家族成员、小橡胶粒子蛋白(SRPP)、翻译控制肿瘤蛋白(TCTP)、激发子响应蛋白和泛素耦联酶E2,这表明橡胶延长因子REF除了与自身高度同源蛋白质可能存在相互作用之外,还可能与TCTP和激发子响应蛋白等其它蛋白质发生相互作用。这些结果有助于揭示橡胶粒子的生物学功能。     

A novel approach for the identification of protein-protein interaction with integral membrane proteins

Protein-protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail.     

Construction of two recombination yeast two-hybrid vectors by in vitro recombination

Recombination-based restrictionless, ligation-independent cloning has been proven to be advantageous over restriction digestion and ligation cloning. To utilize the recombination cloning and previously constructed two-hybrid cDNA libraries, a new Gateway yeast two-hybrid bait vector, pEZY202, and a new prey vector, pEZY45, were constructed. The two-hybrid vectors were generated by in vitro recombination using a protocol that can be easily adapted for the conversion of other existing vectors. The new vectors were used to assay the interaction between the WW domain of PQBP1 (PQBPww) and the WW domain binding protein WBP11. Both PQBPww and WBP11 were cloned into a Gateway donor vector by in vitro recombination. They were then subcloned into pEZY45 and pEZY202, respectively, by in vitro recombination. The binding between PQBPww and WBP11 was reported in a two-hybrid experiment using the new vectors. The results of testing the new vectors in combination with the original vectors indicated that the new bait vector could be used to screen cDNA libraries that are constructed using the original prey vectors.     

A two-step two-hybrid system to identify functionally significant protein-protein interactions

The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system. Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest. In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein. Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait. Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.     

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein–protein interactions in mammalian cells

We recently reported a two-hybrid trap for detecting protein–protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait–prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the βc chain and its modifying enzyme to the GM-CSFRα chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFβ/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.     

Retrieval of granular bait by the Argentine ant (Hymenoptera: Formicidae): effect of clumped versus scattered dispersion patterns

Argentine ants, Linepithema humile (Mayr), use mass recruitment foraging, with clumped prey items being retrieved more efficiently than dispersed prey. However, in prior field experiments, granular baits, whether dispensed in containers or broadly scattered, had a similar impact on Argentine ant populations. In laboratory experiments, granular insecticide bait was encountered faster by Argentine ant workers and more granules were initially returned to the colony when the granules were scattered versus clumped. After 2 h, granules from both dispersion patterns were retrieved equally. Our results suggest that Argentine ant colonies adjust their foraging patterns to resources of different quality (prey versus bait). Also, foraging activity patterns for bait in the laboratory are consistent with prior field results demonstrating no efficacy advantages to discrete granular bait placements.     

Tyrosine phosphorylation-dependent yeast two-hybrid system for the identification of the SH2 domain-binding proteins.

In this paper, we established a modified yeast two-hybrid system, which is specialized for the detection of SH2 domain-binding proteins. The employment of the SH2 domain-tyrosine kinase fusion protein as bait allowed the efficient identification of SH2 domain-binding proteins. The general applicability of the system was tested using various combinations of SH2-kinase fusion bait and prey. The results indicate that the system specifically detected the previously reported in vivo interactions between the SH2 domains and their binding partners. In addition, using this system, we found the interaction between the adaptor protein, Lad, and the SH2 domain of Grb2 or PLC-gamma1. The binding of Lad to Grb2 was further confirmed in mammalian cells by a co-immunoprecipitation study. The conclusion is that the established tyrosine phosphorylation-dependent yeast two-hybrid system provides a novel and efficient way to define the SH2 domain-binding molecules.     

M-Track: detecting short-lived protein-protein interactions in vivo

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.     

Construction of a reading frame-independent yeast two-hybrid vector system for site-specific recombinational cloning and protein interaction screening

The yeast two-hybrid (Y2H) system is a powerful method to identify protein-protein inter-actions (PPI) in vivo, requiring minimal prior information of the putative interactors. The time and effort required for each experiment can be significantly reduced if the "bait" and the "prey" proteins are cloned into specific recombination-amenable two-hybrid vectors. We describe the construction of a reading frame-independent vector system for Y2H PPI studies. The described vector system knits together the advantages of site-specific recombination cloning with the Y2H system. The produced plasmids enable recombination-based cloning of genes or gene fragments in all possible reading frames into Y2H library vectors. Thus, Y2H screening libraries can be rapidly constructed and will present more amino termini in the correct reading frame. Additionally, advantageous for small-scale Y2H studies, there is no need to know the natural reading frame of the genes of interest, because the bait and prey genes can be transferred into the vectors by a single reaction and are present in all possible reading frames. Since the Y2H system per se is a positive selection system, only pairs of bait and prey genes harboring the correct reading frames will emerge. We tested the new vectors within the Y2H system and demonstrated full functionality without any undesired effects on the Y2H system itself. Besides the vector construction, we investigated the utility of the system for Y2H analysis and demonstrated clearly its practicability in genome-wide Y2H screenings and the advantage of using additional reading-frame Y2H cDNA libraries. We performed a series of genome-wide Y2H library screenings with the human vitamin D receptor protein (VDR) as bait. We investigated: (i) whether more protein interactors are found by using three instead of one reading-frame destination vectors; (ii) how much overlap between the different reading-frame libraries exists; and (iii) the rate of possible additional autoactivators. We conclude that our vectors deliver significantly more interactors and outperform a single reading-frame library. This new system could enable simple and fast large-scale PPI studies and the construction of high-quality screening libraries.     

Fishing with a Bait or Lure: A Brief Review of the Cognitive Issues

Bait fishing is a behaviour described in only 12 species of birds, seven of which belong to the family Ardeidae (herons), the remaining five are scattered among four other bird families. This behaviour is defined as having the following characteristics: (1) Objects placed by the bait fisher on the water are buoyant and within a radius at which the fisher can strike at prey. (2) The objects attract or distract the fisher’s prey, with the effect that the fisher enhances its chances of prey capture. A review of the literature indicates that bait items are both selected from and placed within the environment to achieve enhanced prey capture success. It is concluded that bait fishing is a real and distinctive behaviour. The evolutionary route to bait fishing has most likely been through an association between particular floating objects and the occurrence of fish prey. The repositioning of these floating objects and the collection of objects of similar character would have then been sufficient to achieve the bait fishing behaviour now seen. Bait fishing falls within a commonly used definition of tool use. However, it is argued that, as with tool use and tool making in general, this does not necessarily imply special cognitive ability. The rare occurrence of bait fishing both within and across species as could be an indication of cognitive constraint; but this remains undemonstrated. Alternatively, this rarity could be explained if fishing is rarely more profitable than alternative foraging tactics.     

Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods. Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t(1/2) ≤ 0.1 sec) with a low false positive rate. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters. The recombinant protein libraries are produced using a convenient and high-level mammalian expression system, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (β-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates.     

A Novel Genetic System Based on Zinc Finger Nucleases for the Identification of Interactions between Proteins In Vivo

Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein–protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research.     

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.