title: A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.
abstract: Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.
authors.affiliation: Edinburgh Cancer Research Centre, Cell Signalling Unit, University of Edinburgh, EH4 2XR, United Kingdom; Department of Radiation Oncology and Biology, University of Oxford, OX3 7DQ, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Nicholson
authors.affiliation: Proteomics Core Facility, University of Geneva, Switzerland.
authors.forename: Alex
authors.initials: A
authors.lastname: Scherl
authors.affiliation: Edinburgh Cancer Research Centre, Cell Signalling Unit, University of Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Luke
authors.initials: L
authors.lastname: Way
authors.affiliation: Edinburgh Centre for Chemical Biology, University of Edinburgh, EH9 3JG, United Kingdom.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Blackburn
authors.affiliation: Edinburgh Centre for Chemical Biology, University of Edinburgh, EH9 3JG, United Kingdom.
authors.forename: Malcolm D
authors.initials: MD
authors.lastname: Walkinshaw
authors.affiliation: Edinburgh Cancer Research Centre, Cell Signalling Unit, University of Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Ball
authors.affiliation: Edinburgh Cancer Research Centre, Cell Signalling Unit, University of Edinburgh, EH4 2XR, United Kingdom. Electronic address: Ted.Hupp@ed.ac.uk.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D016384:Consensus Sequence; D021983:Cyclophilins / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D061986:MCF-7 Cells; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D007093:Imidazoles; D010879:Piperazines; C081786:cyclophilin B; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D021983:Cyclophilins
keywords: CypB; Interactome; Linear motifs; MDM2; Proteomics