STUDY OF PROTEIN INTERACTIONS

One of the most effective approaches for proteomic investigation of protein interactions is direct analysis of multiprotein complexes using a combination of traditional techniques from biochemistry, molecular biology, and innovative proteomic methodology. Use of biochemical approaches followed by proteomic analysis can be applied not only to identification of the proteins involved in one-to-one interactions, but also to analysis of large complexes consisting of many proteins. Three main techniques are used for studying protein interactions: immunoprecipitation-based methods, the yeast two-hybrid system and protein microarrays.

Immunoprecipitation-based methods

With Immunoprecipitation (IP), the protein of interest is isolated from a complex mixture, such as a cell lysate, using an analyte-specific reagent (ASR; e.g., antibody) along with its interacting partners (Figure 9). Alternatively, the protein of interest can be fused to a high affinity tag and isolated from the complex mixture by using the appropriate capture reagent.

The use of IP allows endogenous proteins to be isolated without the need for cDNAs encoding the protein of interest or the need to express the fusion construct; however, for high-throughput applications this would require a specific ASR for every protein of interest, whereas tag-based affinity purifications using a single isolation chemistry can be applied to all proteins. To identify the binding partners, the proteins that associate with the index protein are often separated by gel electrophoresis and can be probed either with specific reagents on Western blots (if their identities are suspected and the corresponding reagents are available), or they can be digested before or after separation by specific proteases followed by analysis on a mass spectrometer [64].

This approach has the potential of capturing natural protein complexes and does not require previous knowledge on the interacting proteins or cloning of their genes. However, because all the proteins co-purify together, this method cannot determine which proteins are in direct contact with one another.

Figure 9. Immunoprecipitation. Proteins of interest (rectangle) can be isolated from complex biological sample by using antibodies specific to the protein or by modifying the protein with a tag (triangle). The protein of interest and its binding partners can then be separated based on charge, size or isoelectric point, and detected using antibodies specific to the interacting partners or by using mass spectrometry.

Yeast two-hybrid system

In contrast to IP-based method, which is primarily an in vitro biochemical method, the Yeast two-hybrid system (YTH) detects interactions in vivo in yeast cells. This is accomplished by measuring the signal from reporter genes, whose transcription is induced when their cognate transcription factors are reconstituted, by bringing together two functional halves through the interaction of the linked proteins [65]. In the two-hybrid assay (Figure 10), two fusion proteins are created: the protein of interest (X), which is constructed to have a DNA binding domain (BD) attached to its N-terminus, and its potential binding partner (Y), which is fused to an activation domain (AD). If protein X interacts with protein Y, the binding of these two will form an intact and functional transcriptional activator. This newly formed transcriptional activator will then go on to transcribe a reporter gene, which is simply a gene whose protein product can be easily detected and measured. In this way, the amount of the reporter produced can be used as a measure of interaction between our protein of interest and its potential partner.

A variety of reporter systems have been adapted to the YTH system, usually expressing enzymes that either produce metabolites to support growth or induce color changes in specific substrates. The YTH systems specifically measure binary interactions, although the interactions must occur in yeast, and specifically in the context of the yeast nucleus, which may lead to false negatives, especially for some mammalian proteins. To address this, similar approaches, called mammalian two-hybrid systems, have been developed that

Protein complex in a

biological sample Isolate complex

Detect binding partners

140kDa

120kDa

80kDa

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reconstitute active domains of reporter proteins, such as functional enzymes, ubiquitin, fluorescent proteins, and others to demonstrate the presence of an interaction [66-67].

Mammalian two-hybrid systems have been successfully used for monitoring protein interactions in the cytosol as well as among membrane-bound proteins, but challenges associated with the high-throughput introduction of DNA into mammalian cells, and the need for high-quality libraries of genes to test, have limited their widespread adoption [68].

Figure 10. The principle of the yeast two-hybrid system. Two plasmids are constructed, the bait-encoding protein X fused to the C-terminus of a transcription factor DNA-binding domain (BD) and the prey-encoding protein Y fused to an activation domain (AD). Alternatively, the prey can consist of proteins encoded by an expression library. Each plasmid is introduced into an appropriate yeast strain either by co-transformation, sequential transformation, or by yeast mating. Only if proteins X and Y physically interact with one another are the BD and AD brought together to reconstitute a functionally active transcription factor that binds to upstream specific activation sequences (UAS) in the promoters of the reporter genes, and to activate their expression.

Protein microarrays

The idea of protein microarray is not new. In fact, the basics and theoretical considerations of protein microarrays were introduced in the 1980's by Ekins and colleagues [69-70]. There is considerable benefit to be gained from microarray technology [71]. First, in principle thousands of proteins can be spotted on a single slide or similar support, enabling researchers to interrogate simultaneously the function of many different proteins with minimal sample consumption. Second, hundreds or even thousands of copies of an array can be fabricated in parallel, enabling the same proteins to be probed repeatedly with many different molecules under numerous conditions [72]. Protein microarrays are finding their way into

both expression and functional proteomics [73]. In expression proteomics, protein-detecting microarrays comprise several different affinity reagents, such as antibodies, arrayed at high spatial density on a solid support. Each agent (or ‘probe’) captures its target protein from a complex mixture (e.g., serum or cell lysate), and the captured proteins are subsequently detected and quantified. Protein microarrays also provide a well-controlled, in vitro, way in which to study protein function on a genome-wide basis. For this application, sample proteins (or peptides), rather than affinity reagents, are arrayed on a solid support and then probed with compounds of interest (Figure 11).

Figure 11. Functional protein chips are constructed by immobilizing large numbers of purified proteins on a solid surface. Protein chips have an enormous potential in assaying for a wide range of biochemical activities (e.g., protein-protein, protein-nucleic acid and enzyme-substrate interactions), as well as drug and drug target identification.

With this strategy, new substrates or ligands of known enzymes (or receptors or drugs), as well as disease related post-translational modifications, can be identified. An advantage of studying protein function in an array format is that the investigator can control the conditions of the experiment. This includes factors such as pH, temperature, ionic strength and the presence or absence of cofactors, as well as the modification states of the proteins under investigation. Furthermore, protein microarrays can be used to study the interaction of proteins with non-protein molecules, including nucleic acids, lipids and small organic compounds.

An extension of these methodologies is tissue microarray technology (TMA) – a high-throughput technique that allows the rapid visualization of one or a few molecular targets (proteins or nucleic acids) in hundreds or thousands of tissue specimens at a time. Arrays are generated by robotically spotting small cylinders (~0.6 mm³) of tissue derived from

individual paraffin-embedded specimens onto a slide. Proteins and nucleic acids are usually identified by immunohistochemistry and fluorescence in situ hybridization, respectively. The TMA technical principle is therefore similar to that of the direct assay version of antibody microarrays, with the fundamental difference that TMA provides investigators with precious information on target distribution at both the cell level (e.g., cytoplasm vs nucleus) and the tissue level (e.g., stromal vs tumor cells). Most applications of TMA can be found in the field of cancer research [74].