Proteins-networks alteration of protein interactions. Thus, in order to

Proteins-networks are involved in the vast majority of cellular functions, a new challenge is to better understand the organization and functions of these protein networks. Protein-protein interactions thus play an essential role in the functioning of the cell by regulating growth, proliferation, differentiation and cell death, for instance. A lot of diverse mechanisms, such as regulation and control of DNA replication, RNA transcription, protein translation and signal transduction include protein-complexes. Moreover, the development of various diseases such as neurodegenerative disorders and cancers are known to be attributed to the alteration of protein interactions. Thus, in order to better understand the different mechanisms that regulate life, a global understanding at the level of protein network complexes is important.

To detect and analyse protein-protein interactions, many genetic, biochemical, biophysical, and computational approaches have been developed. Different types of information can be obtained, depending on the method used.

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Biochemical methods are based on the detection of an interaction between a target protein and a protein of interest. Each of these methods has its sensitivity and specificity. High sensitivity means that most existing interactions will be detected. High specificity means that most of the detected interactions exist in the cell. The use of one of these methods depends on the type of result sought. One in vitro biochemical method is the affinity chromatography. It is a chromatographic technique that allows separation of a compound using biological interactions between a specific ligand and its substrate, in this case the molecule to be isolated. The co-immunoprecipitation is the main method of studying protein interactions since it is possible to use the endogenous proteins, labelled with a tag and overexpressed it. From the cell lysate, the protein of interest is immune-precipitated with a specific antibody bound to protein A or G beads. The conditions are non-denaturing and all proteins interact with the protein of interest will also be precipitated. After washing, the protein complexes are denatured and the interaction partners are analysed on denaturing gel (SDS-PAGE) followed by a western blot specific for the supposed target protein. The specificity of the technique is high, but it is necessary to have at least 2 antibodies available.

 

Another in vitro precipitation method is the pull-down approach, here the produced protein of interest is recombinantly fused to a tag, like GST, that will allow its affinity purification on a corresponding matrix. The purified target protein or protein lysate is incubated with the protein of interest bound to the matrix. After washing, the complexes formed are analysed by immunoblotting to verify the presence of the target protein.

 

Fluorescent energy transfer techniques are methods of detecting protein interactions in vivo. Two proteins, one fused to a donor fluorophore and the other for an acceptor fluorophore, are transfected into the cell. When both proteins interact, there is fluorescence emission. These techniques allow the real-time study of the dynamics of association and dissociation of the complex in living cells. But, the results obtained can be distorted because of the heterogeneous expression, or by a random co-localization of both proteins.

 

The double hybrid is another in vivo method of studying protein-protein interactions. The double hybrid technique is an artificial system for the demonstration of protein interactions. It allows to see interactions that usually do not work by co-immunoprecipitation. The double hybrid uses hybrid proteins, called target protein and bait protein. The interaction of these two hybrid proteins is detected by the appearance of a simple phenotype that can be analysed, like the synthesis of luminol.

 

Another method of interaction of protein complexes is based on the physical properties of proteins and in particular the application of mass spectrometry to study protein-protein interactions. This method allows to obtain additional information about the complex structure but also to make screens of cellular proteomes. However, these techniques are still difficult to apply and the necessary equipment is expensive.

 

Many more methods exist, which are not all listed here. Each of these methods has its advantages and disadvantages in detecting protein interactions.