The broad, long-term objective of this application is the development of a general method to reversibly regulate the stability (and thus function) of any protein of interest using synthetic, bioavailable small molecules. This application describes basic biomedical research that, if successful, would enable widespread advances in the diagnosis and treatment of human disease by facilitating fundamental biological studies as well as new animal models of human disease. It is known that fusion of an unstable protein domain to a second protein creates a chimeric protein that is unstable in cells and degraded by the proteasome. The goal of this application is to create an unstable protein domain that is conditionally stabilized when bound to a high-affinity ligand. For use in mice, the unstable domain would be fused to a protein of interest in the context of a knock- in mouse, and the stabilizing ligand would be constitutively administered until such time as it was desired to interrogate the role of the protein of interest. Withdrawal of the ligand would cause rapid degradation of the protein of interest. Readministering the ligand would stabilize the chimeric protein to normal levels.
The specific aims of this application incorporate both rational design and diversity- based screening techniques to identify mutants of the FKBP protein that are unstable in mammalian cells but stable when bound to a cell-permeable, high-affinity ligand. New ligands that possess desirable pharmacological properties will be developed, and this method will be validated using several proteins of known scientific and therapeutic importance. Relevance: The goal of this research is to develop a general new method to rapidly and reversibly inactivate a specific protein in either cultured human cells or in mice. This methodology would enable the development of many new models for human diseases (e.g., cultured cells or knock-out mice), and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM073046-01A1
Application #
7038441
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Jones, Warren
Project Start
2006-01-01
Project End
2009-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
1
Fiscal Year
2006
Total Cost
$258,562
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Miyazaki, Yusuke; Mizumoto, Kota; Dey, Gautam et al. (2016) A method to rapidly create protein aggregates in living cells. Nat Commun 7:11689
Navarro, Raul; Chen, Ling-Chun; Rakhit, Rishi et al. (2016) A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore. ACS Chem Biol 11:2101-4
Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W et al. (2015) Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells. Elife 4:
Collins, Sean R; Yang, Hee Won; Bonger, Kimberly M et al. (2015) Using light to shape chemical gradients for parallel and automated analysis of chemotaxis. Mol Syst Biol 11:804
Bonger, Kimberly M; Rakhit, Rishi; Payumo, Alexander Y et al. (2014) General method for regulating protein stability with light. ACS Chem Biol 9:111-5
Rakhit, Rishi; Navarro, Raul; Wandless, Thomas J (2014) Chemical biology strategies for posttranslational control of protein function. Chem Biol 21:1238-52
Sellmyer, Mark A; Bronsart, Laura; Imoto, Hiroshi et al. (2013) Visualizing cellular interactions with a generalized proximity reporter. Proc Natl Acad Sci U S A 110:8567-72
Chu, Bernard W; Kovary, Kyle M; Guillaume, Johan et al. (2013) The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates. J Biol Chem 288:34575-87
Cho, Ukrae; Zimmerman, Stephanie M; Chen, Ling-chun et al. (2013) Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule. PLoS One 8:e72393
Watanabe, Kaori; Al-Bassam, Sarmad; Miyazaki, Yusuke et al. (2012) Networks of polarized actin filaments in the axon initial segment provide a mechanism for sorting axonal and dendritic proteins. Cell Rep 2:1546-53

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