Interactive proteomics research technologies: recent applications and advances

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Proteins rarely exert their function alone. They normally function in multiprotein complexes that play central roles in all biological functions. Thus, it is not surprising that the investigation of protein–protein interactions on a global scale, of so-called interactomes, has become crucial to modern molecular biology. Dissecting partners in protein complexes gives insight into their molecular function and can help in understanding disease-related mechanisms, ultimately resulting in better drug target definition. A variety of methods exist to unravel protein interaction circuitries and recently, significant progress has been made in adapting these tools for the generation of large-scale interaction datasets. Here, we present an overview of the latest advances and applications of interactive proteomics research technologies.

Introduction

Proteins are critical components of all cells, and interactions between proteins are essential for most cellular functions. Fundamental processes such as gene transcription, cell cycle control, signal transduction or regulatory processes depend on the correct function of protein complexes. One of the major milestones in the ‘post-genomic’ era is to functionally annotate the wealth of accumulated genomic data; hence, techniques to map protein–protein interactions (PPIs) have become pivotal to the analysis of proteins, providing insight into their function [1, 2]. Perturbations in the formation or the crosstalk of proteins organized within complexes can often lead to cellular dysfunction, and ultimately result in disease. Therefore, a thorough understanding of PPIs has become important to understand the pathophysiology of diseases and, as a consequence, to define new drug targets [3]. Today, improved technologies allowing for the high-throughput (HTP) study of PPIs have led to the generation of large-scale PPI networks for several model organisms. In general, strategies to analyze PPIs comprise both genetic and biochemical approaches [1]. Biochemical methods (such as co-immunoprecipitations and affinity purifications), explore protein interactomes by directly working with proteins in order to determine the composition of complexes, whereas genetic methods indirectly determine PPIs on the basis of reconstitution of reporters upon interaction. Among these genetic assays, the classical yeast two-hybrid (Y2H) system has become the most widely used method to assess both individual PPIs and global protein maps, and has for example been adapted and applied to study the human interactome [4]. To date, many variants of the Y2H and other techniques have emerged, most of them aiming to improve the throughput and to study PPIs in their natural environment. In this paper we will review new developments in these PPI technologies, and provide an overview of recent important applications of these methods in biomedical research. We specifically focus on genetic approaches, as mass spectrometry-based biochemical methods will be reviewed elsewhere in this issue.

Section snippets

Protein-fragment complementation assay (PCA)

In PCA, two proteins of interest are fused to complementary fragments of a reporter (e.g. a fluorescent protein or an enzyme). These fragments, if brought into close proximity via an interaction between two proteins, are capable of proper folding and assembly, thereby reconstituting reporter activity. Note that the key to the success of this method is that the reporter fragments must not be capable of folding together spontaneously unless they are brought into close proximity via an

Membrane yeast two-hybrid (MYTH)

The membrane yeast two-hybrid (MYTH) assay represents a powerful, PCA-related variant of the traditional Y2H methodology. Unlike conventional Y2H methods, MYTH does not require that interactions occur in the nucleus, thereby allowing the use of full-length membrane proteins, in their natural context of the cellular membrane [11, 12]. The MYTH system is based on the principle of ‘split-ubiquitin’, the observation that the highly conserved protein ubiquitin can be split into two stable moieties,

Resonance-energy transfer (RET) systems

RET technologies allow for monitoring interactions in real time, thus allowing the study of dynamic processes in vivo [17, 18]. Classical techniques are based on the detection of resonance-energy transfer (RET) between fluorescent (FRET) or bioluminescent (BRET) proteins fused to interacting proteins. The principle of RET is that non-radiative energy transfer between an excited donor and an acceptor protein permits the study of spatial relationships. In FRET, classical donor and acceptor

LUMIER (luminescence-based mammalian interactome mapping)

Although mammalian cells are less amenable to HTP technologies, LUMIER is an automated method applicable to 96-well formats. Figure 1e illustrates the general principles of this method. LUMIER was originally developed to map the TGF-β interactome; key members of the TGF-β pathway were fused to Renilla luciferase, co-immunoprecipitated with 518 FLAG-tagged library prey proteins and subsequently assayed for luciferase activity [28]. Detected interactions revealed novel connections with other

The mammalian protein–protein interaction trap (MAPPIT) and its variants

In the mammalian protein–protein interaction trap (MAPPIT) the PPIs take place in the cytosol of intact mammalian cells. Due to the fact that the sites of assay readout (nucleus) and interaction (cytosol) are spatially separated, this system has an additional control mechanism, in conjunction with its ligand-dependency. This system is based on the type I cytokine signaling pathway; upon ligand-binding, type I cytokine receptor subunits cluster, resulting in trans-phosphorylation and activation

Phage display

Phage display is a powerful method to identify and optimize polypeptides with novel functions. Polypeptides are displayed on the surface of bacteriophages, achieved by creating fusions of the polypeptide with a phage coat protein. Upon phage assembly, the resulting fusion genes are incorporated into phage particles and the encoded polypeptide is then displayed on the surface, establishing a physical linkage between the phenotype and genotype. Phage libraries, which can consist of artificial or

Protein microarrays

Protein microarray technologies are based upon the use of planar or bead-based surfaces displaying immobilized proteins [43, 44, 45] (see Figure 1h). They allow for the parallel analysis of a large number of samples, and have a range of clinical and research applications. While protein array technologies come in many forms, most can generally be assigned to one of two major categories; functional arrays, used to probe the activities of a particular protein or agent of interest against a host of

Concluding remarks and perspectives

Obtaining a proper understanding of PPIs and the subcellular context in which proteins are involved is fundamental to gaining an understanding of their function. Most HTP interaction data available today are still based on the classical Y2H, due to the robustness and affordability of this methodology. Although it has recently been shown that Y2H networks provide high-quality binary interaction information [49], this method has intrinsic limitations, such as its constriction of interactions to

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The Stagljar lab is supported by grants from the Canadian Foundation for Innovation (CFI), the Canadian Institute for Health Research (CIHR), the Canadian Cancer Society Research Institute (CCSRI), the Heart and Stroke Foundation, the Cystic Fibrosis Foundation, the Ontario Genomics Institute and Novartis. J.P. is a recipient of an FWF-Erwin-Schrödinger postdoctoral fellowship.

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