The broad, long-term objective of this research program is to develop general technology to conditionally regulate protein function at the level of the protein molecules rather than by targeting the DNA or mRNA precursors that encode a protein-of-interest. This technology is highly specific for the targeted protein and provides rapid and tunable control of protein function using cell-permeable small molecules. The goal is to engineer small protein domains called destabilizing domains that are rapidly and conditionally degraded when expressed in mammalian cells. The instability of a destabilizing domain is faithfully conferred to other proteins fused to these small domains. A cell-permeable ligand that binds tightly to the destabilizing domains regulates their stability. The genetic fusion of the destabilizing domain to the gene-of-interest ensures specificity, and the attendant small-molecule control confers speed, reversibility and dose-dependence to this method. The reagents developed to date involve domains that are unstable in the absence of ligand and stabilized when ligand binds.
The specific aims of this proposal are designed to deliver complementary new domains that are destabilized rather than stabilized by the addition of the cell-permeable ligand. A mechanistic understanding of how these domains are recognized and degraded in mammalian cells will make this technology more useful to users. These studies may also reveal general mechanisms that cells use to recognize and degrade unfolded or misfolded proteins, and these mechanisms are likely relevant to important human diseases.

Public Health Relevance

The goal of this research program is to develop novel methods to rapidly and reversibly regulate the stability of specific proteins in eukaryotic cells and living mammals. This methodology would enable the development of many new models for human diseases, 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 #
5R01GM073046-07
Application #
8267691
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Gerratana, Barbara
Project Start
2006-01-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
7
Fiscal Year
2012
Total Cost
$315,049
Indirect Cost
$117,049
Name
Stanford University
Department
Biology
Type
Schools of Medicine
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
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
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
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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