2.2.3 Protein-protein interaction networks

Proteins play a major role in the majority of cellular processes.

Catalysts, storage, transport, signalling, cellular structure, immune system, cell growth are main examples of their importance but there are many more.

All cellular building blocks (DNA, RNA, proteins, lipids, carbohydrates) interact with each other but the protein interactions are particularly important for their functions.

Proteins are the main actuators in the cell and the basic way the have to carry out their functions is to interact.

The concept of interaction is built in the role of proteins much more than for instance in DNA, which main function is information storage.

A protein-protein interaction (PPI)(De Las Rivas and Fontanillo 2010) obviously occurs when at least two proteins interact.

This interaction requires some kind of molecular docking event, has a cellular purpose and occurs in a specific biological background or context.

Generic interactions such as the ones that occur when a protein is being made, folded or degraded should be excluded.

PPIs can be static, for instance when they form part of a protein complex such as ATP synthase or even a ribosome, or they can be temporary or transient, for example in signalling interactions or activation of gene expression via transcription factors.

Detecting PPIs can be achieved using a variety of experimental methods, all of them high-throughput techniques.

The main three used nowadays to detect PPIs are yeast two-hydrid (Y2H)(Brückner et al.

2009; Suter, Kittanakom, and Stagljar 2008), bimolecular fluorescent complementation (BiFC)(Kerppola 2006, 2008) and affinity purification coupled to mass spectrometry (AP-MS)(Berggård, Linse, and James 2007).

Figure 2.10 shows the PPIN of Saccharomyces cerevisiae detected by Y2H and AP-MS methods (from Yu et al. 2008).

In Chapter 3, data from the literature obtained through Y2H and BiFC is used to build the PPI networks (PPIN), hence their molecular mechanisms are explained further next.

In Y2H method, one protein is fused to a DNA-binding domain and the other is fused to an activation domain.

If both proteins interact the fused proteins work as a transcription factor that express some reporter gene.

GAL4-binding domain is the most commonly used system.

BiFC is based on the union of fluorescent protein subunits that are attached to elements of the same macromolecular complex.

Proteins being tested are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in vivo.

Interaction of these proteins pushes the fluorescent fragments closer, allowing the reporter protein to reform in its original structure and release its fluorescent signal.

These methods produce an immense amount of PPIs data, which is stored in online databases.

The most common are STRING (Franceschini et al. 2013), IntAct (Kerrien et al. 2012) and BioGRID (Stark et al. 2006) although there are some more.

In general, information about PPIs is spread in more databases than metabolic information which is centred in two or three main sources.

Therefore it is

Figure 2.10. PPIN of Saccharomyces cerevisiae detected by (a) Y2H and (b) AP-MS methods (from Yu et al. 2008).

very frequent to find mayor discrepancies and contradictory information regarding PPIs even in well studied organisms such as Escherichia coli or Saccharomyces cerevisiae.

This issue is dealt with in detail in Chapter 3.

PPINs are formed by thousands of proteins interacting with each other.

They are involved in practically every aspect of the cell’s life.

A lot of attention has been given to these networks in the past decade, both for relatively simple organisms (Rajagopala et al. 2014; Uetz et al. 2000; Yu et al. 2008) and more complex (Hegele et al.
2012; Uetz et al. 2006).

This system level proteomic analysis highlights very useful information such as discovery of functional modules or motifs, prediction of protein function (the guilt by association principle) and comparative analysis among species.