Protein-protein interaction

From DrugPedia: A Wikipedia for Drug discovery

Jump to: navigation, search

Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks.

The interactions between proteins are important for many biological functions. For example, signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signaling molecules. This process, called signal transduction, plays a fundamental role in many biological processes and in many diseases (e.g. cancer). Proteins might interact 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 briefly with another protein just to modify it (for example, a protein kinase will add a phosphate to a target protein). This modification of proteins can itself change protein-protein interactions. For example, some proteins with SH2 domains only bind to other proteins when they are phosphorylated on the amino acid tyrosine. In conclusion, protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches.


Contents

[edit] Methods to investigate protein-protein interactions

As protein-protein interactions are so important there are a multitude of methods to detect them. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur in reality are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are also occurring in reality.

  • Co-immunoprecipitation is considered to be the gold standard assay for protein-protein interactions, especially when it is performed with endogenous (not overexpressed and not tagged) proteins. The protein of interest is isolated with a specific antibody. Interaction partners which stick to this protein are subsequently identified by western blotting. Interactions detected by this approach are considered to be real. However, this method can only verify interactions between suspected interaction partners. Thus, it is not a screening approach.
  • Bimolecular Fluorescence Complementation (BiFC) is a new technique in observing the interactions of proteins. Combining with other new techniques, this method can be used to screen protein-protein interactions and their modulators.
  • Fluorescence resonance energy transfer (FRET) is a common technique when observing the interactions of only two different proteins.
  • Pull-down assays are a common variation of immunoprecipitation and are used identically, although this approach is more amenable to an initial screen for interacting proteins.
  • Label transfer can be used for screening or confirmation of protein interactions and can provide information about the interface where the interaction takes place. Label transfer can also detect weak or transient interactions that are difficult to capture using other in vitro detection strategies. In a label transfer reaction, a known protein is tagged with a detectable label. The label is then passed to an interacting protein, which can then be identified by the presence of the label.
  • The yeast two-hybrid screen investigates the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify binding partners of a protein in an unbiased manner. However, the method has a notorious high false-positive rate which makes it necessary to verify the identified interactions by co-immunoprecipitation.
  • In-vivo crosslinking of protein complexes using photo-reactive amino acid analogs was introduced in 2005 by researchers from the Max Planck Institute In this method, cells are grown with photoreactive diazirine analogs to leucine and methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog.
  • Tandem affinity purification (TAP) method allows high throughput identification of proteins interactions. In contrast to Y2H approach 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 consequently it 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 organism.
  • 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.
  • Quantitative immunoprecipitation combined with knock-down (QUICK) relies on co-immunoprecipitation, quantitative mass spectrometry (SILAC) and RNA interference (RNAi). This method detects interactions among endogenous non-tagged proteins. Thus, it has the same high confidence as co-immunoprecipitation. However, this method also depends on the availability of suitable antibodies.
  • Dual Polarisation Interferometry (DPI) can be used to measure protein-protein interactions. DPI provides real-time, high-resolution measurements of molecular size, density and mass. While tagging is not necessary, one of the protein species must be immobilized on the surface of a waveguide.
  • Protein-protein docking, the prediction of protein-protein interaction based on the three-dimensional protein structures only is not satisfactory.
  • Static Light Scattering (SLS) measures changes in the Rayleigh scattering of protein complexes in solution and can non-destructively characterize both weak and strong interactions without tagging or immobilization of the protein. The measurement consists of mixing a series of aliquots of different concentrations or compositions with the anylate, measuring the effect of the changes in light scattering as a result of the interaction, and fitting the correlated light scattering changes with concentration to a model. Weak, non-specific interactions are typically characterized via the second virial coefficient. This type of analysis can determine the equilibrium association constant for associated complexes.
  • Chemical crosslinking followed by high mass MALDI mass spectrometry can be used to analyze intact protein interactions in place before trying to isolate/identify interacting proteins. This method detects interactions among non-tagged proteins and is available from CovalX.
  • SPINE (Strep-protein interaction experiment) uses a combination of reversible crosslinking with formaldehyde and an incorporation of an affinity tag to detect interaction partners in vivo.
  • Surface plasmon resonance can be used to measure protein-protein interaction.
  • With Fluorescence correlation spectroscopy, one protein is labeled with a fluorescent dye and the other is left unlabeled. The two proteins are then mixed and the data outputs the fraction of the labeled protein that is unbound and bound to the other protein, allowing you to get a measure of KD and binding affinity. You can also take time-course measurements to characterize binding kinetics. FCS also tells you the size of the formed complexes so you can measure the stoichiometry of binding.

[edit] Protein-protein interaction network visualization

Visualization of protein-protein interaction networks is a popular application of scientific visualization techniques. Although protein interaction diagrams are common in textbooks, diagrams of whole cell protein interaction networks were not as common since the level of complexity made them difficult to generate. One example of a manually produced molecular interaction map is Kurt Kohn's 1999 map of cell cycle control.

Drawing on Kohn's map, in 2000 Schwikowski, Uetz, and Fields published a paper on protein-protein interactions in yeast, linking together 1,548 interacting proteins determined by two-hybrid testing. They used a force-directed (Sugiyama) graph drawing algorithm to automatically generate an image of their network.

An experimental view of Kurt Kohn's 1999 map gmap. Image was merged via gimp 2.2.17 and then uploaded to maplib.net


[edit] Protein-protein interaction databases

[edit] Interaction network software

  • APID Agile Protein Interaction DataAnalyzer is an interactive bioinformatics web tool to explore and analyze in a unified and comparative platform main currently known information about protein–protein interactions.
  • APID2NET: unified interactome graphic analyzer is an open access tool, included in Cytoscape, that allows to surf unified interactome data by quering APID server and facilitates dynamic analysis of the protein-protein interaction networks.
  • Cytoscape is an open source bioinformatics software platform for visualizing molecular interaction networks and integrating these interactions with gene expression profiles and other state data.
  • NAViGaTOR
  • NetPro Is a comprehensive fully hand-curated knowledgebase of Protein-Protein, Protein-Small molecules DNA and RNA interactions.
  • Osprey
  • PA 800 Protein Characterization System uses Capillary Electrophoreses technology to determine a protein's molecular weight, resolve differences in iso electric point, generate high-resolution peptide maps and carbohydrate profiles, and provide front-end separation and introduction of these proteins to mass spectrometry.
  • VisANT
  • yEd, graph editor.
  • Graphviz


[edit] Original Source

This article was originally posted in Wikipedia

Retrieved from "http://277988.efsst.group/drugpedia/index.php/Protein-protein_interaction"