title: Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.
abstract: Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Xiu-Lei
authors.initials: XL
authors.lastname: Mo
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Ivanov
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA Department of Dermatology, XiangYa Hospital, Central South University, Changsha 410008, China.
authors.forename: Rina
authors.initials: R
authors.lastname: Su
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Havel
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Zenggang
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Hematology and Medical Oncology and Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.
authors.forename: Fadlo R
authors.initials: FR
authors.lastname: Khuri
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA hfu@emory.edu dyuhong@emory.edu.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA Department of Hematology and Medical Oncology and Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA hfu@emory.edu dyuhong@emory.edu.
authors.forename: Haian
authors.initials: H
authors.lastname: Fu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005453:Fluorescence; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008904:Miniaturization; D010879:Piperazines / Q000494:pharmacology; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D007093:Imidazoles; D010879:Piperazines; D054852:Small Molecule Libraries; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D008156:Luciferases; D051736:Proto-Oncogene Proteins c-mdm2
keywords: Hippo signaling pathway; NanoLuc luciferase; bioluminescence resonance energy transfer; protein–protein interaction