MP02:Fluorescence and bioluminescence genetically encoded probes for imaging protein conformations and interactions in living cells and animals

From LabAutopedia

Jump to: navigation, search

Muhammad Awais1, Yoshio Umezawa2, Takeaki Ozawa2, Moritoshi Sato3, Robert Sutton1
1Liverpool NIHR Pancreas Biomedical Research Unit, Royal Liverpool and Broadgreen University Hospitals NHS Trust, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, The University of Liverpool, Liverpool, UK . 2Department of Chemistry, The University of Tokyo, Tokyo, Japan. 3Graduate School of Arts and Science, The University of Tokyo, Tokyo, Japan

Nuclear receptors (NRs) are well-established therapeutic targets for the treatment of several diseases including cancer, diabetes, and inflammation and drugs development is focused on the ligands that either promote or block the NRs activities. We developed genetically encoded fluorescent probes for steroid (estrogen, androgen, progesterone, and glucocorticoid receptors)[1-5] and non-steroid (peroxisome proliferator-activated receptor)[6] NRs to visualize the activities of their ligands, in real-time, in the physiological environment of single living cells. The principle is based on the ligand-induced conformational change(s) in the NR ligand-binding domain (NR LBD) to interact with the coregulator protein, coactivator/corepressor, in intact living cells. The LBD of each NR was connected to the peptide sequence from the NR-interacting coactivator or corepressor protein through a flexible linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent proteins (CFP and YFP, donor and acceptor fluorophore, respectively) in such a way that the excitation and emission spectra of these fluorescent proteins are suitable for fluorescence resonance energy transfer from CFP to YFP. We also developed multicolor bioluminescent probes based on the principle of the protein-fragment complementation (PFC) assay for analysis of protein interactions in live cells and mice. The sensitivity and signal-to-background ratio of the PFC assay were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase [7,8]. Our probes are capable of distinguishing, rapidly and conveniently, ligands of different potencies for the NRs. Moreover, the discrimination among agonists, antagonists, and selective NR modulators is possible using the present methods. The molecular details of a ligand binding to a NR and subsequently induced conformational changes to recruit coregulator protein(s) are important features to understand the ligand pharmacology in the living body. Therefore, our probes would be useful in the development of drugs that can be used against a broad range of diseases related to NRs.
1. Awais, M. JALA 2010, JALA 2010, 15, 396-404.
2. Awais, M.; Sato, M.; Lee, X.; Umezawa, Y. Angew. Chem. Int. Ed. 2006, 45, 2707-2712.
3. Awais, M.; Sato, M.; Sasaki, K.; Umezawa Y. Anal. Chem. 2004, 76, 2181-2186.
4. Awais, M.; Sato, M.; Umezawa Y. ChemBioChem. 2007, 8, 737-743.
5. Awais, M.; Sato, M.; Umezawa Y. Steroids 2007, 72, 949-954.
6. Awais, M.; Sato, M.; Umezawa Y. Biosensors and Bioelectronics 2007, 22, 2564-2569.
7. Kim, S. B.; Awais, M.; Sato, M.; Umezawa Y.; Tao, H. Analytical Chemistry 2007, 79, 1874-1880.
8. Hida, N.; Awais, M.; Taeuchi, M.; Ueno, N.; Tashiro, M.; Takagi, C.; Singh, T.; Hayashi, M.; Ohmiya, Y.; Ozawa, T. PLoS ONE 2009, 4, e5868.

Click [+] for other articles on 
LabAutomation2011 Posters(48 P, 2 F)
The Market Place for Lab Automation & Screening  The Market Place