Format

Send to

Choose Destination
See comment in PubMed Commons below
J Mol Cell Biol. 2016 Jun;8(3):271-81. doi: 10.1093/jmcb/mjv064. Epub 2015 Nov 16.

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.

Author information

1
Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
2
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.
3
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.
4
Department of Hematology and Medical Oncology and Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.
5
Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA hfu@emory.edu dyuhong@emory.edu.
6
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.

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.

KEYWORDS:

Hippo signaling pathway; NanoLuc luciferase; bioluminescence resonance energy transfer; protein–protein interaction

PMID:
26578655
PMCID:
PMC4937889
DOI:
10.1093/jmcb/mjv064
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center