Development and Characterization of a Novel Protein Stability Probe
type: CreativeWork
description: As a regulator of gene transcription, (MYC) modulates cell proliferation, growth, and metabolism. The deregulation of c-MYC (MYC) drives over 50% of cancers, making it a promising therapeutic target. Unfortunately, traditional approaches to target MYC have been unsuccessful. MYC turnover is tightly regulated, with a half-life of just 30 minutes in non-transformed cells, however only one pathway has been well-characterized as regulating MYC stability. Current strategies to measure protein half-life are low-throughput or prone to false readouts, and have hampered the discovery of novel regulatory pathways for MYC that could be targeted therapeutically. To overcome this barrier, we developed a protein stability probe, consisting of c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus), and a high-content confocal screening pipeline to identify regulators of MYC using automated image analysis. This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. The MYC-Venus probe was piloted by screening a kinase inhibitor library to identify known and novel kinases that regulate MYC stability. The Venus probe was also validated with another short half-life protein, MCL-1. This validated stability probe was expanded to a cell system that models MYC-driven human breast cancer, in order to further characterize the functionality of the MYC-Venus stability probe. A 438-compound library was screened, and compounds that either increase or decrease MYC-Venus levels with minimal cytotoxicity were identified. Compounds that increased MYC-Venus levels can be attributed to an artefact of the CMV promoter used to express MYC-Venus, demonstrating that the Venus probe can also detect changes in protein levels. While compounds that decreased MYC-Venus levels were also identified, none modulated MYC stability at the timepoints tested. The utility of the MYC-Venus probe was further demonstrated by adapting an existing assay to quantify change…
abstract: As a regulator of gene transcription, (MYC) modulates cell proliferation, growth, and metabolism. The deregulation of c-MYC (MYC) drives over 50% of cancers, making it a promising therapeutic target. Unfortunately, traditional approaches to target MYC have been unsuccessful. MYC turnover is tightly regulated, with a half-life of just 30 minutes in non-transformed cells, however only one pathway has been well-characterized as regulating MYC stability. Current strategies to measure protein half-life are low-throughput or prone to false readouts, and have hampered the discovery of novel regulatory pathways for MYC that could be targeted therapeutically. To overcome this barrier, we developed a protein stability probe, consisting of c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus), and a high-content confocal screening pipeline to identify regulators of MYC using automated image analysis. This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. The MYC-Venus probe was piloted by screening a kinase inhibitor library to identify known and novel kinases that regulate MYC stability. The Venus probe was also validated with another short half-life protein, MCL-1. This validated stability probe was expanded to a cell system that models MYC-driven human breast cancer, in order to further characterize the functionality of the MYC-Venus stability probe. A 438-compound library was screened, and compounds that either increase or decrease MYC-Venus levels with minimal cytotoxicity were identified. Compounds that increased MYC-Venus levels can be attributed to an artefact of the CMV promoter used to express MYC-Venus, demonstrating that the Venus probe can also detect changes in protein levels. While compounds that decreased MYC-Venus levels were also identified, none modulated MYC stability at the timepoints tested. The utility of the MYC-Venus probe was further demonstrated by adapting an existing assay to quantify change…
author: Hickman, Katherine Ashley
license: http://creativecommons.org/licenses/by-nc/4.0/
date published: 2021-03
date modified: 2026-07-27T21:28:20Z
provider: University of Toronto
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Toronto

PropertyValue
property ID: Handle
value: http://hdl.handle.net/1807/104966