Proteinprotein interaction 1

Protein–protein interaction

The horseshoe shaped ribonuclease inhibitor (shown as wireframe)forms a protein–protein interaction with the ribonuclease protein.

The contacts between the two proteins are shown as colouredpatches.

Protein–protein interactions occur when two or moreproteins bind together, often to carry out theirbiological function. Many of the most importantmolecular processes in the cell such as DNA replicationare carried out by large molecular machines that arebuilt from a large number of protein componentsorganised by their protein–protein interactions. Proteininteractions have been studied from the perspectives ofbiochemistry, quantum chemistry, molecular dynamics,chemical biology, signal transduction and othermetabolic or genetic/epigenetic networks. Indeed,protein–protein interactions are at the core of the entireinteractomics system of any living cell.

Interactions between proteins are important for themajority of biological functions. For example, signalsfrom the exterior of a cell are mediated to the inside ofthat cell by protein–protein interactions of the signalingmolecules. This process, called signal transduction,plays a fundamental role in many biological processesand in many diseases (e.g. cancers). Proteins mightinteract for a long time to form part of a protein complex, a protein may be carrying another protein (for example,from cytoplasm to nucleus or vice versa in the case of the nuclear pore importins), or a protein may interact brieflywith another protein just to modify it (for example, a protein kinase will add a phosphate to a target protein). Thismodification of proteins can itself change protein–protein interactions. For example, some proteins with SH2domains only bind to other proteins when they are phosphorylated on the amino acid tyrosine while bromodomainsspecifically recognise acetylated lysines. In conclusion, protein–protein interactions are of central importance forvirtually every process in a living cell. Information about these interactions improves our understanding of diseasesand can provide the basis for new therapeutic approaches.

Methods to investigate protein–protein interactionsAs protein–protein interactions are so important there are a multitude of methods to detect them.[1] Each of theapproaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of themethod. A high sensitivity means that many of the interactions that occur in reality are detected by the screen. Ahigh specificity indicates that most of the interactions detected by the screen are also occurring in reality. Methodssuch as yeast two-hybrid screening can be used to detect novel protein–protein interactions.

StructureThe structures of many protein complexes have been unlocked by the technique of X-ray crystallography.[2] Whereas many high throughput techniques to investigate protein interactions can only tell you which protein interacts with which other proteins. Molecular structure can give fine details about which specific parts are interacting and what kinds of chemical bonds mediate that interaction. One of the earliest protein structures to be solved was that of haemoglobin by Perutz and colleagues, which is a complex of four proteins: two alpha chains and two beta chains.[3]

As the number of structures available for protein complexes grew researchers began to investigate the underlying

Proteinprotein interaction 2

principles of protein-protein interactions. Based on just three structures of the insulin dimer, trypsin-pancreatictrypsin inhibitor complex and oxyhaemoglobin, Cyrus Chothia and Joel Janin found that between 1,130 and 1,720Angstroms2 of surface area was removed from contact with water indicating that hydrophobicity was the majorfactor stabilising protein-protein interactions.[4] Later studies refined the buried surface area of the majority ofinteractions to be 1,600±350 Angstroms2. However, much larger interaction interfaces were observed that wereassociated with large changes in conformation of one of the interaction partners.[2]

Visualization of networks

Network visualisation of the human interactome whereeach point represents a protein and each blue line

between them is an interaction.

Visualization of protein–protein interaction networks is a popularapplication of scientific visualization techniques.[5] Althoughprotein interaction diagrams are common in textbooks, diagramsof whole cell protein interaction networks were not as commonsince the level of complexity made them difficult to generate. Oneexample of a manually produced molecular interaction map is KurtKohn's 1999 map of cell cycle control.[6] Drawing on Kohn's map,in 2000 Schwikowski, Uetz, and Fields published a paper onprotein–protein interactions in yeast, linking together 1,548interacting proteins determined by two-hybrid testing. They used alayered graph drawing method to find an initial placement of thenodes and then improved the layout using a force-basedalgorithm.[7][8][9] The Cytoscape software is a widely usedapplication to visualise protein-protein interaction networks.

Database collectionsMethods for identifying interacting proteins have defined hundreds of thousands of interactions. These interactionsare collected together in specialised biological databases that allow the interactions to be assembled and studiedfurther. The first of these databases was DIP, the database of interacting proteins.[10] Since that time a large numberof further database collections have been created such as BioGRID, STRING and ConsensusPathDB.

References[1] Phizicky EM, Fields S (March 1995). "Protein-protein interactions: methods for detection and analysis". Microbiol. Rev. 59 (1): 94–123.

PMC 239356. PMID 7708014.[2] Janin J, Chothia C (September 1990). "The structure of protein-protein recognition sites". J. Biol. Chem. 265 (27): 16027–30.

PMID 2204619.[3] PERUTZ MF, ROSSMANN MG, CULLIS AF, MUIRHEAD H, WILL G, NORTH AC (February 1960). "Structure of haemoglobin: a

three-dimensional Fourier synthesis at 5.5-A. resolution, obtained by X-ray analysis". Nature 185 (4711): 416–22. PMID 18990801.[4] Chothia C, Janin J (August 1975). "Principles of protein-protein recognition". Nature 256 (5520): 705–8. PMID 1153006.[5] De Las Rivas J, Fontanillo C (June 2010). "Protein-protein interactions essentials: key concepts to building and analyzing interactome

networks". PLoS Comput. Biol. 6 (6): e1000807. doi:10.1371/journal.pcbi.1000807. PMC 2891586. PMID 20589078.[6] Kurt W. Kohn (August 1, 1999). "Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems". Molecular

Biology of the Cell 10 (8): 2703–2734. PMC 25504. PMID 10436023.[7] Benno Schwikowski1, Peter Uetz, and Stanley Fields (2000). "A network of protein−protein interactions in yeast" (http:/ / www. nature. com/

nbt/ journal/ v18/ n12/ full/ nbt1200_1257. html). Nature Biotechnology 18 (12): 1257–1261. doi:10.1038/82360. PMID 11101803. .[8] Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex

characterization and proteome exploration. Nat Biotechnol. 17:1030-2. Rigaut, G; Shevchenko, A; Rutz, B; Wilm, M; Mann, M; Séraphin, B(1999). "A generic protein purification method for protein complex characterization and proteome exploration.". Nature Biotechnology 17(10): 1030–2. doi:10.1038/13732. PMID 10504710.

[9] Prieto C, De Las Rivas J (2006). APID: Agile Protein Interaction DataAnalyzer. Nucleic Acids Res. 34:W298-302. Prieto, C; De Las Rivas, J (2006). "APID: Agile Protein Interaction DataAnalyzer.". Nucleic Acids Research 34 (Web Server issue): W298–302. doi:10.1093/nar/gkl128.

Protein–protein interaction

screening

The screening of protein–protein interactions refers to the identification of protein interactions with high-throughput screening methods such as computer- and/or robot-assisted plate reading, flow cytometry analyzing.

The interactions between proteins are central to virtually every process in a living cell. Information about these interactions improves understanding of diseases and can provide the basis for new therapeutic approaches.

Methods to screen protein–protein interactions

Though there are many methods to detect protein–protein interactions, the

majority of these methods—such as Co-immunoprecipitation, Fluorescence

resonance energy transfer (FRET) and dual polarisation interferometry—are not

screening approaches.

Ex vivo or in vivo methods

Bimolecular Fluorescence Complementation (BiFC) is a new technique for observing the interactions of proteins. Combining it with other new techniques DERB can enable the screening of protein–protein interactions and their modulators.

The yeast two-hybrid screen investigates the interaction between

artificial fusion proteins inside the nucleus of yeast. This approach can identify the binding partners of a protein without bias. However, the method has a notoriously high false-positive rate, which makes it necessary to verify the identified interactions co-immunoprecipitation.

In-vitro methods

The Tandem affinity purification (TAP) method allows the high-throughput identification of proteins interactions. In contrast with the Y2H approach, the accuracy of the method can be compared to those of small-scale experiments (Collins et al., 2007) and the interactions are detected within the correct cellular environment as by co-immunoprecipitation. However, the TAP tag method requires two successive steps of protein purification, and thus can not readily detect transient protein–protein interactions. Recent genome-wide TAP experiments were performed by Krogan et al., 2006 and Gavin et al., 2006, providing updated protein interaction data for yeast organisms.

Chemical crosslinking is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. Common crosslinkers for this application include the non-cleavable [NHS-ester] crosslinker, [bis-sulfosuccinimidyl suberate] (BS3); a cleavable version of BS3, [dithiobis(sulfosuccinimidyl propionate)](DTSSP); and the [imidoester] crosslinker [dimethyl dithiobispropionimidate] (DTBP) that is popular for fixing interactions in ChIP assays.

Two-hybrid screening 1

Two-hybrid screening

Overview of two-hybrid assay, checking for interactions between two proteins,called here Bait and Prey.A. Gal4 transcription factor gene produces two domain

protein (BD and AD), which is essential for transcription of the reporter gene(LacZ).B,C. Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey.

None of them is usually sufficient to initiate the transcription (of the reportergene) alone.D. When both fusion proteins are produced and Bait part of the firstinteract with Prey part of the second, transcription of the reporter gene occurs.

Two-hybrid screening (also known as yeasttwo-hybrid system or Y2H) is a molecularbiology technique used to discoverprotein–protein interactions[1] andprotein–DNA interactions[2][3] by testing forphysical interactions (such as binding)between two proteins or a single protein and aDNA molecule, respectively.

The premise behind the test is the activationof downstream reporter gene(s) by thebinding of a transcription factor onto anupstream activating sequence (UAS). Fortwo-hybrid screening, the transcription factoris split into two separate fragments, called thebinding domain (BD) and activating domain(AD). The BD is the domain responsible forbinding to the UAS and the AD is the domainresponsible for the activation oftranscription.[1][2] The Y2H is thus aprotein-fragment complementation assay.

History

Pioneered by Stanley Fields and Song in1989, the technique was originally designedto detect protein–protein interactions usingthe GAL4 transcriptional activator of theyeast Saccharomyces cerevisiae. The GAL4protein activated transcription of a proteininvolved in galactose utilization, which formed the basis of selection.[4] Since then, the same principle has beenadapted to describe many alternative methods including some that detect protein–DNA interactions, DNA-DNAinteractions and use Escherichia coli instead of yeast.[3]

Basic premise

The key to the two-hybrid screen is that in most eukaryotic transcription factors, the activating and binding domainsare modular and can function in proximity to each other without direct binding.[5] This means that even though thetranscription factor is split into two fragments, it can still activate transcription when the two fragments are indirectlyconnected.

The most common screening approach is the yeast two-hybrid assay.[6] This system often utilizes a geneticallyengineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) islacking. When grown on media that lacks these nutrients, the yeast fail to survive. This mutant yeast strain can bemade to incorporate foreign DNA in the form of plasmids. In yeast two-hybrid screening, separate bait and preyplasmids are simultaneously introduced into the mutant yeast strain.

Two-hybrid screening 2

Plasmids are engineered to produce a protein product in which the DNA-binding domain (BD) fragment is fusedonto a protein while another plasmid is engineered to produce a protein product in which the activation domain (AD)fragment is fused onto another protein. The protein fused to the BD may be referred to as the bait protein, and istypically a known protein the investigator is using to identify new binding partners. The protein fused to the AD maybe referred to as the prey protein and can be either a single known protein or a library of known or unknownproteins. In this context, a library may consist of a collection of protein-encoding sequences that represent all theproteins expressed in a particular organism or tissue, or may be generated by synthesising random DNAsequences.[3] Regardless of the source, they are subsequently incorporated into the protein-encoding sequence of aplasmid, which is then transfected into the cells chosen for the screening method.[3] This technique, when using alibrary, assumes that each cell is transfected with no more than a single plasmid and that, therefore, each cellultimately expresses no more than a single member from the protein library.If the bait and prey proteins interact (i.e., bind), then the AD and BD of the transcription factor are indirectlyconnected, bringing the AD in proximity to the transcription start site and transcription of reporter gene(s) can occur.If the two proteins do not interact, there is no transcription of the reporter gene. In this way, a successful interactionbetween the fused protein is linked to a change in the cell phenotype.[1]

The challenge of separating cells that express proteins that happen to interact with their counterpart fusion proteinsfrom those that do not, is addressed in the following section.

Fixed domainsIn any study, some of the protein domains, those under investigation, will be varied according to the goals of thestudy whereas other domains, those that are not themselves being investigated, will be kept constant. For example ina two-hybrid study to select DNA-binding domains, the DNA-binding domain, BD, will be varied whilst the twointeracting proteins, the bait and prey, must be kept constant to maintain a strong binding between the BD and AD.There are a number of domains from which to choose the BD, bait and prey and AD, if these are to remain constant.In protein–protein interaction investigations, the BD may be chosen from any of many strong DNA-binding domainssuch as Zif268.[2] A frequent choice of bait and prey domains are residues 263–352 of yeast Gal11P with a N342Vmutation[2] and residues 58–97 of yeast Gal4,[2] respectively. These domains can be used in both yeast- andbacterial-based selection techniques and are known to bind together strongly.[1][2]

The AD chosen must be able to activate transcription of the reporter gene, using the cell's own transcriptionmachinery. Thus, the variety of ADs available for use in yeast-based techniques may not be suited to use in theirbacterial-based analogues. The herpes simplex virus-derived AD, VP16 and yeast Gal4 AD have been used withsuccess in yeast[1] whilst a portion of the α-subunit of E. coli RNA polymerase has been utilised in E. coli-basedmethods.[2][3]

Whilst powerfully activating domains may allow greater sensitivity towards weaker interactions, conversely, aweaker AD may provide greater stringency.

Construction of expression plasmidsA number of engineered genetic sequences must be incorporated into the host cell to perform two-hybrid analysis orone of its derivative techniques. The considerations and methods used in the construction and delivery of thesesequences differ according to the needs of the assay and the organism chosen as the experimental background.There are two broad categories of hybrid library: random libraries and cDNA-based libraries. A cDNA library is constituted by the cDNA produced through reverse transcription of mRNA collected from specific cells of types of cell. This library can be ligated into a construct so that it is attached to the BD or AD being used in the assay.[1] A random library uses lengths of DNA of random sequence in place of these cDNA sections. A number of methods exist for the production of these random sequences, including cassette mutagenesis.[2] Regardless of the source of the

Two-hybrid screening 3

DNA library, it is ligated into the appropriate place in the relevant plasmid/phagemid using the appropriaterestriction endonucleases.[2]

E. coli-specific considerationsBy placing the hybrid proteins under the control of IPTG-inducible lac promoters, they are expressed only on mediasupplemented with IPTG. Further, by including different antibiotic resistance genes in each genetic construct, thegrowth of non-transformed cells is easily prevented through culture on media containing the correspondingantibiotics. This is particularly important for counter selection methods in which a lack of interaction is needed forcell survival.[2]

The reporter gene may be inserted into the E. coli genome by first inserting it into an episome, a type of plasmid withthe ability to incorporate itself into the bacterial cell genome[2] with a copy number of approximately one per cell.[7]

The hybrid expression phagemids can be electroporated into E. coli XL-1 Blue cells which after amplification andinfection with VCS-M13 helper phage, will yield a stock of library phage. These phage will each contain onesingle-stranded member of the phagemid library.[2]

Recovery of protein informationOnce the selection has been performed, the primary structure of the proteins which display the appropriatecharacteristics must be determined. This is achieved by retrieval of the protein-encoding sequences (as originallyinserted) from the cells showing the appropriate phenotype.

E. coli

The phagemid used to transform E. coli cells may be "rescued" from the selected cells by infecting them withVCS-M13 helper phage. The resulting phage particles that are produced contain the single-stranded phagemids andare used to infect XL-1 Blue cells.[2] The double-stranded phagemids are subsequently collected from these XL-1Blue cells, essentially reversing the process used to produce the original library phage. Finally, the DNA sequencesare determined through dideoxy sequencing.[2]

Controlling sensitivityThe Escherichia coli-derived Tet-R repressor can be used in line with a conventional reporter gene and can becontrolled by tetracycline or doxicycline (Tet-R inhibitors). Thus the expression of Tet-R is controlled by thestandard two-hybrid system but the Tet-R in turn controls (represses) the expression of a previously mentionedreporter such as HIS3, through its Tet-R promoter. Tetracycline or its derivatives can then be used to regulate thesensitivity of a system utilising Tet-R.[1]

Sensitivity may also be controlled by varying the dependency of the cells on their reporter genes. For example, thiseffected by altering the concentration of histidine in the growth medium for his3-dependent cells and altering theconcentration of streptomycin for aadA dependent cells.[2][3] Selection-gene-dependency may also be controlled byapplying an inhibitor of the selection gene at a suitable concentration. 3-Amino-1,2,4-triazole (3-AT) for example, isa competitive inhibitor of the HIS3-gene product and may be used to titrate the minimum level of HIS3 expressionrequired for growth on histidine-deficient media.[2]

Sensitivity may also be modulated by varying the number of operator sequences in the reporter DNA.

Two-hybrid screening 4

Non-fusion proteinsA third, non-fusion protein may be co-expressed with two fusion proteins. Depending on the investigation, the thirdprotein may modify one of the fusion proteins or mediate or interfere with their interaction.[1]

Co-expression of the third protein may be necessary for modification or activation of one or both of the fusionproteins. For example S. cerevisiae possesses no endogenous tyrosine kinase. If an investigation involves a proteinthat requires tyrosine phosphorylation, the kinase must be supplied in the form of a tyrosine kinase gene.[1]

The non-fusion protein may mediate the interaction by binding both fusion proteins simultaneously, as in the case ofligand-dependent receptor dimerization.[1]

For a protein with an interacting partner, its functional homology to other proteins may be assessed by supplying thethird protein in non-fusion form, which then may or may not compete with the fusion-protein for its binding partner.Binding between the third protein and the other fusion protein will interrupt the formation of the reporter expressionactivation complex and thus reduce reporter expression, leading to the distinguishing change in phenotype.[1]

Split-ubiquitin yeast two-hybridOne limitation of classic yeast two-hybrid screens is that they are limited to soluble proteins. It is thereforeimpossible to use them to study the protein–protein interactions between insoluble integral membrane proteins. Thesplit-ubiquitin system provides a method for overcoming this limitation.[8] In the split-ubiquitin system, two integralmembrane proteins to be studied are fused to two different ubiquitin moieties: a C-terminal ubiquitin moiety ("Cub",residues 35–76) and an N-terminal ubiquitin moiety ("Nub", residues 1–34). These fused proteins are called the baitand prey, respectively. In addition to being fused to an integral membrane protein, the Cub moiety is also fused to atranscription factor (TF) that can be cleaved off by ubiquitin specific proteases. Upon bait–prey interaction, Nub andCub-moieties assemble, reconstituting the split-ubiquitin. The reconstituted split-ubiquitin molecule is recognized byubiquitin specific proteases, which cleave off the reporter protein, allowing it to induce the transcription of reportergenes.

One-, three- and one-two-hybrid variants

One-hybridThe one-hybrid variation of this technique is designed to investigate protein–DNA interactions and uses a singlefusion protein in which the AD is linked directly to the binding domain. The binding domain in this case however isnot necessarily of fixed sequence as in two-hybrid protein–protein analysis but may be constituted by a library. Thislibrary can be selected against the desired target sequence, which is inserted in the promoter region of the reportergene construct. In a positive-selection system, a binding domain that successfully binds the UAS and allowstranscription is thus selected.[1]

Note that selection of DNA-binding domains is not necessarily performed using a one-hybrid system, but may alsobe performed using a two-hybrid system in which the binding domain is varied and the bait and prey proteins arekept constant.[2][3]

Two-hybrid screening 5

Three-hybrid

Overview of three-hybrid assay.

RNA-protein interactions have beeninvestigated through a three-hybridvariation of the two-hybrid technique.In this case, a hybrid RNA moleculeserves to adjoin together the twoprotein fusion domains—which are notintended to interact with each other butrather the intermediary RNA molecule(through their RNA-bindingdomains).[1] Techniques involvingnon-fusion proteins that perform a similar function, as described in the 'non-fusion proteins' section above, may alsobe referred to as three-hybrid methods.

One-two-hybridSimultaneous use of the one- and two-hybrid methods (that is, simultaneous protein–protein and protein–DNAinteraction) is known as a one-two-hybrid approach and expected to increase the stringency of the screen.[1]

Host organismAlthough theoretically, any living cell might be used as the background to a two-hybrid analysis, there are practicalconsiderations that dictate which is chosen. The chosen cell line should be relatively cheap and easy to culture andsufficiently robust to withstand application of the investigative methods and reagents.[1]

YeastS. cerevisiae was the model organism used during the two-hybrid technique's inception. It has several characteristicsthat make it a robust organism to host the interaction, including the ability to form tertiary protein structures, neutralinternal pH, enhanced ability to form disulfide bonds and reduced-state glutathione among other cytosolic bufferfactors, to maintain a hospitable internal environment.[1] The yeast model can be manipulated through non-moleculartechniques and its complete genome sequence is known.[1] Yeast systems are tolerant of diverse culture conditionsand harsh chemicals that could not be applied to mammalian tissue cultures.[1]

A number of yeast strains have been created specifically for Y2H screens, e.g. Y187[9] and AH109,[10] bothproduced by Clontech.Proteins from as small as eight to as large as 750 amino acids have been studied using yeast.[1]

E. coli

E. coli-based methods have several characteristics that may make them preferable to yeast-based homologues. Thehigher transformation efficiency and faster rate of growth lends E. coli to the use of larger libraries (in excess of108).[2] A low false positive rate of approximately 3x10−8, the absence of requirement for a nuclear localisationsignal to be included in the protein sequence and the ability to study proteins that would be toxic to yeast may alsobe major factors to consider when choosing an experimental background organism.[2]

It may be of note that the methylation activity of certain E. coli DNA methyltransferase proteins may interfere withsome DNA-binding protein selections. If this is anticipated, the use of an E. coli strain that is defective for aparticular methyltransferase may be an obvious solution.[2]

Two-hybrid screening 6

Applications

Determination of sequences crucial for interactionBy changing specific amino acids by mutating the corresponding DNA base-pairs in the plasmids used, theimportance of those amino acid residues in maintaining the interaction can be determined.[1]

After using bacterial cell-based method to select DNA-binding proteins, it is necessary to check the specificity ofthese domains as there is a limit to the extent to which the bacterial cell genome can act as a sink for domains withan affinity for other sequences (or indeed, a general affinity for DNA).[2]

Drug and poison discoveryProtein–protein signalling interactions pose suitable therapeutic targets due to their specificity and pervasiveness.The random drug discovery approach uses compound banks that comprise random chemical structures, and requiresa high-throughput method to test these structures in their intended target.[1]

The cell chosen for the investigation can be specifically engineered to mirror the molecular aspect that theinvestigator intends to study and then used to identify new human or animal therapeutics or anti-pest agents.[1]

Determination of protein functionBy determination of the interaction partners of unknown proteins, the possible functions of these new proteins maybe inferred.[1] This can be done using a single known protein against a library of unknown proteins or conversely, byselecting from a library of known proteins using a single protein of unknown function.[1]

Zinc finger protein selectionTo select zinc finger proteins (ZFPs) for protein engineering, methods adapted from the two-hybrid screeningtechnique have been used with success.[2][3] A ZFP is itself a DNA-binding protein used in the construction ofcustom DNA-binding domains that bind to a desired DNA sequence.[11]

By using a selection gene with the desired target sequence included in the UAS, and randomising the relevant aminoacid sequences to produce a ZFP library, cells that host a DNA-ZFP interaction with the required characteristics canbe selected. Each ZFP typically recognises only 3–4 base pairs, so to prevent recognition of sites outside the UAS,the randomised ZFP is engineered into a 'scaffold' consisting of another two ZFPs of constant sequence. The UAS isthus designed to include the target sequence of the constant scaffold in addition to the sequence for which a ZFP isselected.[2][3]

A number of other DNA-binding domains may also be investigated using this system.[2]

Strengths•• Two-hybrid screens are low-tech; they can be carried out in any lab without sophisticated equipment.•• Two-hybrid screens can provide an important first hint for the identification of interaction partners.•• The assay is scalable, which makes it possible to screen for interactions among many proteins. Furthermore, it can

be automated, and by using robots many proteins can be screened against thousands of potentially interactingproteins in a relatively short time.

• Yeast two-hybrid data can be of similar quality to data generated by the alternative approach of coaffinitypurification followed by mass spectrometry (AP/MS).[12]

Two-hybrid screening 7

Weaknesses• The main criticism applied to the yeast two-hybrid screen of protein–protein interactions is the possibility of a

high number of false positive (and false negative) identifications. The exact rate of false positive results is notknown, but earlier estimates were as high as 70%.[13] The reason for this high error rate lies in the characteristicsof the screen:

•• Certain assay variants overexpress the fusion proteins which may cause unnatural protein concentrations that leadto unspecific (false) positives.

•• The hybrid proteins are fusion proteins; that is, the fused parts may inhibit certain interactions, especially if aninteraction takes place at the N-terminus of a test protein (where the DNA-binding or activation domain istypically attached).

• An interaction may not happen in yeast, the typical host organism for Y2H. For instance, if a bacterial protein istested in yeast, it may lack a chaperone for proper folding that is only present in its bacterial host. Moreover, amammalian protein is sometimes not correctly modified in yeast (e.g., missing phosphorylation), which can alsolead to false results.

•• The Y2H takes place in the nucleus. If test proteins are not localized to the nucleus (because they have otherlocalization signals) two interacting proteins may be found to be non-interacting.

•• Some proteins might specifically interact when they are co-expressed in the yeast, although in reality they arenever present in the same cell at the same time. However, in most cases it cannot be ruled out that such proteinsare indeed expressed in certain cells or under certain circumstances.

Each of these points alone can give rise to false results. Due to the combined effects of all error sources yeasttwo-hybrid have to be interpreted with caution. The probability of generating false positives means that allinteractions should be confirmed by a high confidence assay, for example co-immunoprecipitation of the endogenousproteins, which is difficult for large scale protein–protein interaction data. Alternatively, Y2H data can be verifiedusing multiple Y2H variants[14] or bioinformatics techniques. The latter test whether interacting proteins areexpressed at the same time, share some common features (such as gene ontology annotations or certain networktopologies), have homologous interactions in other species.[15]

References[1] Young K (1998). "Yeast two-hybrid: so many interactions, (in) so little time." (http:/ / www. biolreprod. org/ cgi/ reprint/ 58/ 2/ 302). Biol

Reprod 58 (2): 302–11. doi:10.1095/biolreprod58.2.302. PMID 9475380. .[2] Joung J, Ramm E, Pabo C (2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions" (http:/

/ www. pnas. org/ cgi/ content/ full/ 97/ 13/ 7382). Proc. Natl. Acad. Sci. U.S.A. 97 (13): 7382–7. doi:10.1073/pnas.110149297. PMC 16554.PMID 10852947. .

[3] Hurt J, Thibodeau S, Hirsh A, Pabo C, Joung J (2003). "Highly specific zinc finger proteins obtained by directed domain shuffling andcell-based selection" (http:/ / www. pnas. org/ cgi/ content/ full/ 100/ 21/ 12271). Proc. Natl. Acad. Sci. U.S.A. 100 (21): 12271–6.doi:10.1073/pnas.2135381100. PMC 218748. PMID 14527993. .

[4] Fields S, Song O (1989). "A novel genetic system to detect protein-protein interactions" (http:/ / www. nature. com/ nature/ journal/ v340/n6230/ abs/ 340245a0. html;jsessionid=2F9E056F57E5547D17D2D6658C9B0437) (abstract). Nature 340 (6230): 245–6.Bibcode 1989Natur.340..245F. doi:10.1038/340245a0. PMID 2547163. . Abstract is free; full-text article is not.

[5] Verschure P, Visser A, Rots M (2006). "Step out of the groove: epigenetic gene control systems and engineered transcription factors". AdvGenet 56: 163–204. doi:10.1016/S0065-2660(06)56005-5. PMID 16735158.

[6] Gietz R.D., Triggs-Raine Barbara, Robbins Anne, Graham Kevin, Woods Robin (1997). "Identification of proteins that interact with a proteinof interest: Applications of the yeast two-hybrid system" (http:/ / www. springerlink. com/ content/ t5w6u8667583641p/?p=163f5ecf5cff441b940a429da9d83187& pi=0). Mol Cel Biochem 172 (1–2): 67–79. doi:10.1023/A:1006859319926. PMID 9278233. .

[7] Whipple F (1998). "Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli". Nucleic Acids Res 26 (16):3700–6. doi:10.1093/nar/26.16.3700. PMC 147751. PMID 9685485.

[8] Stagljar I, Korostensky C, Johnsson N, te Heesen S (April 1998). "A genetic system based on split-ubiquitin for the analysis of interactionsbetween membrane proteins in vivo" (http:/ / www. pnas. org/ cgi/ pmidlookup?view=long& pmid=9560251). Proc. Natl. Acad. Sci. U.S.A. 95(9): 5187–92. doi:10.1073/pnas.95.9.5187. PMC 20236. PMID 9560251. .

[9] Fromont-Racine, Micheline; Rain, Jean-Christophe, Legrain, Pierre (1997). "Toward a functional analysis of the yeast genome throughexhaustive two-hybrid screens". Nature Genetics 16 (3): 277–282. doi:10.1038/ng0797-277.

Two-hybrid screening 8

[10] Lu, L; Horstmann, H, Ng, C, Hong, W (2001 Dec). "Regulation of Golgi structure and function by ARF-like protein 1 (Arl1).". Journal ofCell Science 114 (Pt 24): 4543–55. PMID 11792819.

[11] Gommans W, Haisma H, Rots M (2005). "Engineering zinc finger protein transcription factors: therapeutic relevance of switchingendogenous gene expression on or off at command" (http:/ / www. rug. nl/ farmacie/ onderzoek/ basiseenheden/ therapeuticgenemodulation/publicaties/ publicaties2004/ !find?path=/ farmacie/ onderzoek/ basisEenheden/ THErapeuticGeneModulation/ publicaties/ publicaties2004/2005_6. pdf& params=as=pdf). J Mol Biol 354 (3): 507–19. doi:10.1016/j.jmb.2005.06.082. PMID 16253273. .

[12] Haiyuan Yu et al. (2008). "High-Quality Binary Protein Interaction Map of the Yeast Interactome Network". Science 322 (5898): 104–110.doi:10.1126/science.1158684. PMC 2746753. PMID 18719252.

[13] Deane C, Salwiński Ł, Xenarios I, Eisenberg D (2002). "Protein interactions: two methods for assessment of the reliability of highthroughput observations" (http:/ / www. mcponline. org/ cgi/ content/ full/ 1/ 5/ 349). Mol Cell Proteomics 1 (5): 349–56.doi:10.1074/mcp.M100037-MCP200. PMID 12118076. .

[14] Chen, Y. C.; Rajagopala, S. V.; Stellberger, T.; Uetz, P. (2010). "Exhaustive benchmarking of the yeast two-hybrid system". NatureMethods 7 (9): 667–668; author 668 668. doi:10.1038/nmeth0910-667. PMID 20805792.

[15] Koegl, M.; Uetz, P. (2008). "Improving yeast two-hybrid screening systems". Briefings in Functional Genomics and Proteomics 6 (4):302–312. doi:10.1093/bfgp/elm035. PMID 18218650.

External links• Complete Yeast Two-Hybrid Protocol (http:/ / www. singerinstruments. com/ index. php?option=com_content&

task=view& id=181& Itemid=980)• Detail on sister technique two-hybrid system (http:/ / www. biochem. arizona. edu/ classes/ bioc568/

two-hybrid_system. htm)• Science Creative Quarterly's overview of the yeast two hybrid system (http:/ / www. scq. ubc. ca/ ?p=246)• Gateway-Compatible Yeast One-Hybrid Screens (http:/ / www. cshprotocols. org/ cgi/ content/ full/ 2006/ 28/

pdb. prot4590)• Video animation of the Yeast Two-Hybrid System (http:/ / www. sumanasinc. com/ webcontent/ animations/

content/ yeasttwohybrid. html)• Two-Hybrid+System+Techniques (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=&

term=Two-Hybrid+ System+ Techniques) at the US National Library of Medicine Medical Subject Headings(MeSH)

Article Sources and Contributors 9

Article Sources and ContributorsTwo-hybrid screening  Source: http://en.wikipedia.org/w/index.php?oldid=508304163  Contributors: A.bit, Alansohn, Alboyle, Anna K., Arcadian, Ariliand, Boku wa kage, Bubbachuck,DChetkovich, DabMachine, Dai mingjie, Danshil, Dekimasu, Drdaveng, Eahd201, Element16, Fixthatspelling, Flyguy649, Gregjohnso, Ilia Kr., Jebus989, Jeffw245, Jesse V., John Broughton,Jopept1, Katieh5584, Lotje, Luna Santin, Medibio, Miguel Andrade, Narayanese, O RLY?, Pbogomiakov, Peteruetz, Pixie, Plindenbaum, RDBrown, Rich Farmbrough, Rjwilmsi,RogerDodger88, Safflle, Seans Potato Business, Serephine, Several Times, Sintaku, Tassedethe, TestPilot, Tide rolls, Tinz, Tobycat, Tony1, Trey314159, Whosasking, Zeyaraun, 70 anonymousedits

Image Sources, Licenses and ContributorsImage:Two hybrid assay.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Two_hybrid_assay.svg  License: Creative Commons Attribution-Sharealike 3.0,2.5,2.0,1.0  Contributors:AnnaImage:Three-hybrid-system.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Three-hybrid-system.svg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: Ilia Kr.

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