Yeast Two-Hybrid System for Dissecting the Rice MAPK Interactome
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An important step in the analysis of protein function is identification of the interaction partners of each protein. The two-hybrid system has been widely used to identify and explore protein-protein interactions. By using various two-hybrid systems, numerous protein interactions that regulate apoptosis signaling have been discovered that reveal unexpected functions of cancer-relevant proteins. Methods for performing two-hybrid experiments using either yeast or mammalian cells will be described in this chapter.
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The yeast two-hybrid assay has proved a powerful tool in identifying and characterising binary protein-protein interactions. Not only can it be used to map interacting protein domains, it can also be used to screen cDNA libraries with a desired bait to identify novel binding partners. A number of factors including ease of use, cost effectiveness and suitability for high throughput analysis have made yeast-two hybrid one of the assays of choice for defining protein-protein interaction networks or interactomes for a range of organisms. The focus of this review is on the definition of viral interactomes using the yeast two-hybrid assay and the relevance of such studies to our understanding of viral pathogenesis. Keywords: Yeast two-hybrid assay, interactome, virus, proteomics, drug targets
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To characterize a protein's function, it is often advantageous to identify other proteins with which it interacts. The yeast two-hybrid system is one of the most versatile methods available for detection and characterization of protein-protein interactions, and in the recent years it has become a mature and robust technology. A further improvement to this technique is the ability to examine and distinguish more than one interaction simultaneously. This is achieved in the Dual Bait, which has successfully been used to detect proteins and peptides that target specific motifs in larger proteins, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.
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Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.
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The yeast two-hybrid system is a sensitive method to detect intracellular interactions between proteins. It has been successfully employed in the identification of novel interactors of several proteins, including adhesion molecules (for a general review on the method, see refs. 1 and 2). In general, the high sensitivity of the yeast two-hybrid system allows the detection of weak and transient interactions that escape biochemical analysis. Howewer, in some limited cases, interactions found with biochemical methods have not been reproduced in the yeast assay, possibly because of the lack of some posttranslational modifications. More generally, the high sensitivity of the yeast interaction assay may lead to the disclosure of false-positive protein-protein interactions. For this reason, it is always necessary to confirm the yeast interaction data using other methods of study for protein binding.
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Protein fulfilling the their roles, one of important ways is through protein-protein interaction. In functional genomic era, identifying all of protein-protein interaction in proteome and mapping the protein interactions that have been attracting many scientists' attention , of which large-scale yeast two-hybrid system is one strategy of most widely used. In recent two years, ambitious projects have launched to examine all of the protein-protein interaction in Saccharomyces cer-evisiae using large-scale yeast two-hybrid system. Nevertheless, huge protein network is larger than that we predict and single yeast two-hybrid system cannot solve all the problems, which need be complemented by other wags.
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Abstract To understand how proteins function to control cellular processes, their interactions with other proteins must be identified and characterised. The yeast two‐hybrid system is a simple and efficient assay for protein interactions. In a yeast two‐hybrid assay, the two proteins to be tested are expressed in a yeast nucleus with each protein fused to one‐half of a transcription activator. If the two‐hybrid proteins interact, the transcription activator is reconstituted and turns on reporter genes that can be easily detected. This assay has been used to identify tens of thousands of protein interactions, to map protein interaction domains and to characterise mutant variants of proteins. A variety of related assays have been developed, all based on the ability of two interacting hybrid proteins to activate a reporter system. These assays along with the original yeast two‐hybrid assay contribute to the characterisation of the protein interactions – or protein interactome – for humans and a wide range of other organisms. Key Concepts The function of most proteins involves interacting with one or more other proteins. A binary interaction is a direct physical interaction between two proteins. Understanding a protein's function requires charting its binary interactions. The interactome is all of the protein interactions for a particular cell or an entire organism. Two‐hybrid assays detect binary protein interactions by expressing the two test proteins in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produce a detectable signal. In a yeast two‐hybrid assay, the two proteins to be tested for interaction are fused to the two halves of a transcription factor in yeast. Two‐hybrid assays, like all protein interaction assays, can produce false positives, which are interactions that are detected in the assay even though they do not occur under normal conditions in vivo . Two‐hybrid and other protein interaction assays can also result in missed interactions or false negatives. Use of multiple different protein interaction assays can reduce the number of false negatives and provide cross‐validation to rule out false positives.
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Abstract Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two hybrid (qYY2H) system that not only enables discovery of specific protein-protein interactions, but also efficient, quantitative estimation of their binding affinities ( K D ). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semi-empirical framework for estimating the K D of monovalent bait-prey interactions, using measurements of the apparent K D of yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.
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Abstract To understand how proteins function to control cellular processes, their interactions with other proteins must be identified and characterised. The yeast two‐hybrid system is a simple and efficient assay for protein–protein interactions. In a yeast two‐hybrid assay, two proteins are expressed in a yeast nucleus with each protein fused to one‐half of a transcription activator. If the two hybrid proteins interact, the transcription activator is reconstituted and turns on easily detectable reporter genes. This assay has been used to identify tens of thousands of protein interactions, to map protein interaction domains and to characterise mutant variants of proteins. A variety of related assays have been developed, all based on the ability of two interacting hybrid proteins to activate a reporter system. These assays along with the original two‐hybrid assay are contributing to the characterisation of the protein interactions – or protein interactome – for humans and several model organisms. Key Concepts: The role that most proteins play in cells involves interacting with one or more proteins. A binary interaction is a physical interaction between two proteins. Understanding a protein's function requires charting its binary interactions. Interactome is a term used to refer to all of the protein interactions for a particular cell or an entire organism. Two‐hybrid assays are assays for binary protein interactions, where two test proteins are expressed in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produces a detectable signal. In a yeast two‐hybrid assay, the two proteins to be tested for interaction are fused to the two halves of a transcription factor in yeast, which activates reporter genes if the proteins interact. In a protein complementation assay, the two proteins are fused to separate halves of a reporter protein like an enzyme, which will be reconstituted if the two halves are brought into close proximity via the protein–protein interaction. False positives are interactions that are detected in the assay even though they do not occur under normal conditions in vivo . Protein interaction assays can also result in missed interactions or false negatives. Use of multiple different protein interaction assays can reduce the number of false negatives and provide cross‐validation to rule out false positives.
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