Proteomics in context

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The genome-sequencing project has brought us an encyclopaedia of the molecular parts of living systems. The next step is to understand the contextual meaning of the ‘words’ of the book of life, which essentially means to elucidate the rules of interaction between the proteins. In fact, recent efforts to map the network of interactions at a genome-scale using high-throughput two-hybrid systems and in vitro protein interaction studies have quickly provided a glimpse of the wiring diagram of regulatory networks. However, information about the cellular context of signaling events, such as the subcellular location of the interaction, still requires old-fashioned, ‘pre-genomic’ approaches centred around one particular protein at a time.Now, Remy and Michnick[1xVisualization of biochemical networks in living cells. Remy, I. and Michnick, S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7678–7683Crossref | PubMed | Scopus (75)See all References[1] report an efficient experimental approach that allows the parallel detection and localization of biologically relevant protein–protein interactions within living mammalian cells. They use a method developed in their laboratory called protein-fragment complementation in which two rationally dissected fragments of dihydrofolate reductase (DHFR), a common selection marker, are each fused to a protein or peptide of interest. Because the DHFR fragments are unable to fold independently, cell survival in nucleotide-free media relies on the ability of the fused proteins to interact, thereby positioning the two DHFR fragments in close proximity so that folding and reconstitution of enzyme activity can proceed. The insulin and growth factor receptor tyrosine kinase (RTK)-mediated translation initiation pathway and an overlapping pathway controlled by the serine–threonine kinase FK506-binding protein FRAP were chosen as test cases. The authors tested 148 combinations of 35 different proteins in the RTK–FRAP signal transduction pathways and identified 14 interaction partners, five of which had not been demonstrated previously.Following this initial survival screen, a fluorescence read-out assay allowed them to gather additional information regarding the nature and context of the identified interactions. Reassembled DHFR binds with high affinity to fluorescein-conjugated methotrexate in a 1:1 complex, which is retained in cells. The fluorescence signal provides a quantitative measure of the number of molecules of folded DHFR and, therefore, the number of interacting protein complexes. At the same time, visualization of the cells using fluorescence microscopy pinpoints the subcellular location of the interaction. To probe the position of a particular protein–protein interaction within the higher-order organization of the signaling network, the authors established a pharmacological profile for each combination of protein partners. Protein interactions and localization were monitored by fluorescent DHFR following perturbation with an array of specific small-molecule inhibitors or stimulators. By examining the response profiles the authors were able to assign a position within the signaling network to newly discovered interactions. For example, they identify a novel interaction between FRAP and PDK1 that co-localizes with the known interaction between FRAP and PKB, suggesting a point of crosstalk between the RTK and FRAP signaling pathways.This pharmacological profiling based on the protein-fragment complementation system can be applied to larger, more complex biochemical networks. It will enable us to combine the efficiency of broad-scale genomics screening with traditional, information-rich cell biology approaches to obtain insights into the collective function and cellular organization of gene products.