This proposal seeks to develop a novel method for controlling the activity of engineered proteins within cells. The use of chemical dimerizing agents such as rapalog have been very valuable as a means of activating cell surface receptors or disabling proteins in a cell by crosslinking them. We seek to create a Chemically-Induced Dimerization agent (CID) that is cleaved by light, thereby making the dimerization rapidly reversible. Such a molecule needs to lack endogenous binding partners in cells, be readily membrane permeant, have no toxic side effects, and be efficiently cleaved by light of a wavelength that will not cause cellular damage or interfere with standard imaging methods. We propose to characterize a candidate photocleavable CID to determine its kinetics and concentration dependence and thereby validate its suitability for work in cells in culture. We propose methods to optimize its affinity and efficacy, and we propose to test the reagent in a "split kinesin" assay of axonal transport to determine if it can be used to rapidly uncouple a cargo from its anterograde motor in a live neuron.

Public Health Relevance

Inactivating a particular protein has been a major means of probing its functional importance in the nervous system. Classical means of doing so include the use of mutants, RNAi, or blocking drugs but these methods have their drawbacks. Mutants may have early developmental defects and RNAi can only slowly bring about the depletion of a protein from a cell. For many proteins, no drugs are available to selectively inhibit them. We are proposing to develop and validate a chemical method to selectively activate or inhibit a protein. It uses a dimerizing agent that can either 1) hold two halves of a protein together or 2) tie one protein to another. This dimerizing agent is designed to be lysed by illumination at 405 nm so that the protein state can be rapidly changed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS087582-01
Application #
8684027
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Talley, Edmund M
Project Start
2014-02-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$263,000
Indirect Cost
$113,000
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115