Proteome-scale Binary Interactomics in Human Cells

Abstract

Because proteins are the main mediators of most cellular processes they are also prime therapeutic targets. Identifying physical links among proteins and between drugs and their protein targets is essential in order to understand the mechanisms through which both proteins themselves and the molecules they are targeted with act. Thus, there is a strong need for sensitive methods that enable mapping out these biomolecular interactions. Here we present a robust and sensitive approach to screen proteome-scale collections of proteins for binding to proteins or small molecules using the well validated MAPPIT (Mammalian Protein-Protein Interaction Trap) and MASPIT (Mammalian Small Molecule-Protein Interaction Trap) assays. Using high-density reverse transfected cell microarrays, a close to proteome-wide collection of human ORF clones can be screened for interactors at high throughput. The versatility of the platform is demonstrated through several examples. With MAPPIT, we screened a 15k ORF library for binding partners of RNF41, an E3 ubiquitin protein ligase implicated in receptor sorting, identifying known and novel interacting proteins. The potential related to the fact that MAPPIT operates in living human cells is illustrated in a screen where the protein collection is scanned for interactions with the glucocorticoid receptor (GR) in its unliganded versus dexamethasone-induced activated state. Several proteins were identified the interaction of which is modulated upon ligand binding to the GR, including a number of previously reported GR interactors. Finally, the screening technology also enables detecting small molecule target proteins, which in many drug discovery programs represents an important hurdle. We show the efficiency of MASPITbased target profiling through screening with tamoxifen, a first-line breast cancer drug, and reversine, an investigational drug with interesting dedifferentiation and antitumor activity. In both cases, cell microarray screens yielded known and new potential drug targets highlighting the utility of the technology beyond fundamental biology. Molecular & Cellular

The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic

“…These approaches have collectively and systematically characterized the proteins within specific membrane compartments, as well as provided insights into the plethora of genes and proteins that function and regulate organelles and their trafficking routes. Other proteome‐scale assays to systematically understand the interactions between proteins and small molecules also provide important information for drug discovery for membrane receptors, such as by the use of a receptor‐based system which detects specific pairs of protein‐protein or protein‐drug interactions . Collectively, these provide a wealth of information to form hypotheses for further reductionist approaches, which will without a doubt reveal further insight into the mechanism and regulation of specific processes.…”

Discovering cellular protein‐protein interactions: Technological strategies and opportunities

“…However, some technologies are more easily adapted for high‐throughput studies than others. Y2H is one of the technologies that is probably most amendable to high‐throughput applications, but other technologies have also introduced advancements facilitating large set‐ups, for example, MAPPIT . Many co‐complex methods have the inherent advantage to easily enable the evaluation of all potential interactors of a certain bait protein in a certain cell type, but are much more difficult to scale up to the evaluation of very large numbers of baits.…”

“…In a benchmarking study that compares five binary technologies, MAPPIT seems one of the most accurately performing ones . Moreover, MAPPIT allows screening in array format, and reverse MAPPIT (Figure ) allows to detect potential disruptors of PPIs by screening with a positive read‐out . This positive read‐out in reverse MAPPIT is made possible by coupling the prey to a JAK‐STAT signaling inhibitor domain, and by coupling the bait to the non‐mutated cytokine receptor.…”

“…In a benchmarking study that compares five binary technologies, MAPPIT seems one of the most accurately performing ones. 59 Moreover, MAPPIT allows screening in array format, [60][61][62] (Figure 3), which is a technology that is similar to Y3H but based on MAPPIT and thus operates in a mammalian background. 64 In MASPIT, the small molecule of interest is fused to methotrexate, and the bait of MAPPIT is replaced by dihydrofolate reductase that binds this methotrexate moiety.…”

“…Y2H is one of the technologies that is probably most amendable to high-throughput applications, but other technologies have also introduced advancements facilitating large set-ups, for example, MAPPIT. 60,62 Many cocomplex methods have the inherent advantage to easily enable the evaluation of all potential interactors of a certain bait protein in a certain cell type, but are much more difficult to scale up to the evaluation of very large numbers of baits. Furthermore, some technologies like the coelution approaches are only useful at large scale, while others already standardly need extensive normalization and optimization at smaller scales, like AVEXIS.…”