{"id":{"repo_id":"toronto-retro","oai_identifier":"oai:utoronto.scholaris.ca:1807/104966"},"canonical_url":"https://search.dev.ndltd.org/etd/toronto-retro/oai:utoronto.scholaris.ca:1807/104966","repository":{"repo_id":"toronto-retro","name":"University of Toronto","base_url":"https://utoronto.scholaris.ca/server/oai/request"},"display":{"title":"Development and Characterization of a Novel Protein Stability Probe","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 changes in protein stability through confocal imaging, increasing the number of compounds that can be validated for future screens. The development and characterization of the Venus stability probe lays the foundation for future work to identify novel regulators of short half-life proteins, a field that has been hampered by a lack of screening tools.","abstract_html":"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 changes in protein stability through confocal imaging, increasing the number of compounds that can be validated for future screens. The development and characterization of the Venus stability probe lays the foundation for future work to identify novel regulators of short half-life proteins, a field that has been hampered by a lack of screening tools.","abstract_has_math":false,"creators":["Hickman, Katherine Ashley"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Medical Biophysics","school":null,"contributors":[],"advisors":["Andrews, David"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-03","date_published":"2021-03","updated_at":"2026-07-27T21:28:20Z","subjects":[],"languages":[],"rights":["Attribution-NonCommercial 4.0 International"],"rights_urls":["http://creativecommons.org/licenses/by-nc/4.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1807/104966","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Andrews, David"]},{"key":"dc:contributor.department","label":"Department","values":["Medical Biophysics"]},{"key":"dc:creator","label":"Author","values":["Hickman, Katherine Ashley"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2021-03"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2021-03-15T14:36:57Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2021-03-15T14:36:57Z"]},{"key":"dc:date.issued","label":"Date","values":["2021-03"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["Attribution-NonCommercial 4.0 International"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://creativecommons.org/licenses/by-nc/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1807/104966"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 changes in protein stability through confocal imaging, increasing the number of compounds that can be validated for future screens. The development and characterization of the Venus stability probe lays the foundation for future work to identify novel regulators of short half-life proteins, a field that has been hampered by a lack of screening tools."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Development and Characterization of a Novel Protein Stability Probe"]}]}],"canonical_facts":{"dc:contributor.advisor":["Andrews, David"],"dc:contributor.department":["Medical Biophysics"],"dc:creator":["Hickman, Katherine Ashley"],"dc:date":["2021-03"],"dc:date.accessioned":["2021-03-15T14:36:57Z"],"dc:date.available":["2021-03-15T14:36:57Z"],"dc:date.issued":["2021-03"],"dc:description.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 changes in protein stability through confocal imaging, increasing the number of compounds that can be validated for future screens. The development and characterization of the Venus stability probe lays the foundation for future work to identify novel regulators of short half-life proteins, a field that has been hampered by a lack of screening tools."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1807/104966"],"dc:rights":["Attribution-NonCommercial 4.0 International"],"dc:rights.uri":["http://creativecommons.org/licenses/by-nc/4.0/"],"dc:title":["Development and Characterization of a Novel Protein Stability Probe"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:28:20Z"}