title: A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time.
abstract: Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.
authors.affiliation: Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Yao-Cheng
authors.initials: YC
authors.lastname: Li
authors.affiliation: Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Luo Wei
authors.initials: LW
authors.lastname: Rodewald
authors.affiliation: Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Hoppmann
authors.affiliation: Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Ee Tsin
authors.initials: ET
authors.lastname: Wong
authors.affiliation: Sanofi Tucson Innovation Center, 2090 East Innovation Park Drive, Oro Valley, AZ 85755, USA.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Lebreton
authors.affiliation: Sanofi Tucson Innovation Center, 2090 East Innovation Park Drive, Oro Valley, AZ 85755, USA.
authors.forename: Pavel
authors.initials: P
authors.lastname: Safar
authors.affiliation: Sanofi Tucson Innovation Center, 2090 East Innovation Park Drive, Oro Valley, AZ 85755, USA.
authors.forename: Marcel
authors.initials: M
authors.lastname: Patek
authors.affiliation: Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Sanofi Tucson Innovation Center, 2090 East Innovation Park Drive, Oro Valley, AZ 85755, USA.
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Wertman
authors.affiliation: Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address: wahl@salk.edu.
authors.forename: Geoffrey M
authors.initials: GM
authors.lastname: Wahl
mesh_terms: D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D045522:Recombinases; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D049409:Luciferases, Firefly; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2