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Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells

Abstract

Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor–based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53.

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Figure 1: Schematic view of the forward MAPPIT and reverse MAPPIT principles.
Figure 2: Inhibition of signaling via a chimeric SOCS3-CIS construct is lost upon coexpression of a competitor SOCS protein.
Figure 3: Analysis of the interaction between FKBP12 and ALK4.
Figure 4: Structure-function analysis using ReverseMAPPIT.
Figure 5: Dose-dependent disruption of the p53-MDM2 interaction by Nutlin-3 as evaluated by reverse MAPPIT.

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Acknowledgements

We wish to acknowledge R. Devos for the pGEX-PTP-1B vector; K. Kas for critical reading of the manuscript, M. Goethals for peptide synthesis and D. Defeau for technical assistance. This project was supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen; GBOU 010090), Ghent University (UGent; GOA 12051401) and the valorisation fund of the Flanders Interuniversity Institute for Biotechnology (VIB). S.E. is a Postdoctoral Fellow of the Fund for Scientific Research-Flanders (FWO-V).

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Correspondence to Jan Tavernier.

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Supplementary Fig. 1

Dose-dependent disruption of the p53/MDM2 interaction: comparison of different configurations. (PDF 975 kb)

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Eyckerman, S., Lemmens, I., Catteeuw, D. et al. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells. Nat Methods 2, 427–433 (2005). https://doi.org/10.1038/nmeth760

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