An interactome network refers to all the protein-protein interactions which take place inside a cell. Such interactome maps have been created for yeast, worms, flies, and are beginning to be created for humans.
In yeast, the size of the interactome is estimated to be approximately 28,000 protein-protein interactions, while the number of interactions in humans is estimated to be around 650,000.
Different methods used to map and analyse the interactome are discussed below.
Fluorescence Cross-Correlation Spectroscopy
Fluorescence correlation spectroscopy or FCS measures the thermodynamic fluctuations in the fluorescence intensity of tagged proteins. Using confocal microscope, the dynamics of how fluorescently-tagged protein molecules move though a small focal volume are quantified. The diffusion properties of the tagged proteins depend on their concentration and interactions with other molecules. In fluorescence cross correlation microscopy, two fluorescently labeled proteins are visualised. If these proteins are associated with each other, they move in a synchronized manner. This leads to similar fluctuations in their fluorescent signals over time. Thus, quantitative FCCS can measure protein-protein interactions to generate quantitative interactome maps.
Genome, Proteome and Now, the Interactome
Bimolecular Complementation Methods
Protein-protein complementation assay (PCA)
This method is also used to study protein-protein interactions. In this method, a fluorescent protein is split in to two, and then fused to the N-or C-terminals of two potential interacting proteins. If the proteins interact, the two units combine, leading to fluorescence which can then be visualised.
This method is a variant of PCA where GFP is split into two parts and fused to potentially interacting peptides. The interaction of peptides leads to assembly of functional GFP molecules which then fluoresce.
This is another variant of PCA where, instead of fluorescent proteins, luciferase is used. Again the luciferase is fragmented into two to visualise interactions. An important property of this method is that the association of the luciferase fragments is irreversible. Thus, this method can be used to study both interaction and dissociation of proteins inside a cell.
FRET or Forster Energy Resonance Transfer is a method based upon the distance between two fluorophores. In this method, if located close enough, an excited fluorophore molecule (donor) transfers its energy to another fluorophore molecule (acceptor). This leads to a fluorescence emission in the acceptor molecule. The measurements of the excitation and emission wavelengths can be used to measure interactions between two molecules as FRET can occur only when the donor or acceptor molecules are in very close proximity or in direct interaction with each other. The limitation of this method is that studying protein-protein interaction to form a interactome map requires tagging the proteins of interest with the appropriate acceptor and donor fluorophores. This requires creating genetic fusion proteins.
BRET or bioluminescence-based resonance energy transfer involves oxidation of a substrate by luciferase enzyme. The oxidized substrate then emits light at 395 nm which is captured by an acceptor GFP molecule leading to GFP fluorescence. As there is no need for external light source in BRET, there is less background noise, autoflourescence, and light scattering.
Luciferase-Based Co-Immunoprecipitation Methods
This method is also used to detect protein-protein interactions to generate the interactome map. This method is less laborious and time-consuming than the conventional co-immunoprecipitation methods. In this method, the acceptor and donor proteins are fused with FLAG and Renilla constructs. Any interactions or associations can be detected using the luciferase enzymatic assay.
Using this method, a large number of donor/acceptor or interacting partners can be detected. This is in contrast to the conventional co-immunoprecipitation methods where only a single interaction can be determined. This advantage is critical while constructing interactome maps which involve thousands of interactions. However, one disadvantage of this method is that the FLAG-tagged protein cannot be quantified which may generate false negative results.
Proximity Ligation Assays
In this method, antibodies are attached to short single-stranded DNA oligonucleotides or PLA probes. These antibodies then bind to protein inside cells. The proximity or interaction of two proteins can lead to ligation of DNA molecules which can then be amplified using a polymerase chain reaction. The fluorescent probes then bind to these amplified DNA regions which then acts as a marker to show interacting proteins.
Thus, different methods can be used to map interactions in a cell which are then used to form the interactome.