Investigating the mammalian heat-shock induced ubiquitination response
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Rose, Amalia
issued: 2018-03-07T16:53:07Z
issued: 2018
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: Aggregation of misfolded proteins in the cell is often indicative of a failing protein quality control system, whose responsibility is to mediate either the refolding or degradation of these aberrant proteins. Heat-shock (HS), which causes proteins to misfold, leads to a marked increase of the ubiquitination of proteins that display poor solubility in the cell. The E3 ubiquitin ligase Rsp5 has previously been shown to be the major ubiquitin ligase that targets cytosolic misfolded proteins upon HS in S. cerevisiae. Its closest mammalian orthologue, Nedd4-1, was also found to be responsible for an increased ubiquitination upon HS in HeLa and MEF cells, however further work is needed to better characterize this response. Such characterization included looking at which proteins get ubiquitinated upon HS to then determine whether they share common characteristics such as cellular localization, and if they are newly synthesized versus pre-existing. Using diGly enrichment coupled with mass spectrometry, we identified over 300 proteins that were further ubiquitinated upon HS. An important portion of these proteins are localized in the nucleus, whereas proteins associated to the mitochondria are markedly under-represented. As well, proteins involved in processes such as pre-mRNA processing, cell cycle and SUMOylation were found to be enriched. Using a pulse-SILAC approach, we found that a large portion of proteins ubiquitinated after HS were pre-existing, whereas some were newly synthesized including ribosomal proteins. We also investigated the potential role of other Nedd4 family members in the HS ubiquitination response in Hek293 cells. Using shRNA, we found that the knockdown of the Itch E3, but not of Nedd4-1, caused a decrease of the HS-induced ubiquitination response in these cells. Accordingly, we also showed a reduced cell viability upon Itch knocked down under HS.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/64750
note: INVESTIGATING THE MAMMALIAN HEAT-SHOCK INDUCED UBIQUITINATION RESPONSE by AMALIA ROSE B.Sc., The University of British Columbia (2015) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2018 © Amalia Rose, 2018 ii Abstract Aggregation of misfolded proteins in the cell is often indicative of a failing protein quality control system, whose responsibility is to mediate either the refolding or degradation of these aberrant proteins. Heat-shock (HS), which causes proteins to misfold, leads to a marked increase of the ubiquitination of proteins that display poor solubility in the cell. The E3 ubiquitin ligase Rsp5 has previously been shown to be the major ubiquitin ligase that targets cytosolic misfolded proteins upon HS in S. cerevisiae. Its closest mammalian orthologue, Nedd4-1, was also found to be responsible for increased ubiquitination upon HS in HeLa and MEF cells, however further work is needed to better characterize this response. Such characterization included looking at which proteins get ubiquitinated upon HS to then determine whether they share common characteristics such as cellular localization, and if they are newly synthesized versus pre-existing. Using diGly enrichment coupled with mass spectrometry, we identified over 300 proteins that were further ubiquitinated upon HS. An important portion of these proteins are localized in the nucleus, whereas proteins associated to the mitochondria are markedly under-represented. As well, proteins involved in processes such as pre-mRNA processing, cell cycle and SUMOylation were found to be enriched. Using a pulse-SILAC approach, we found that a large portion of proteins ubiquitinated after HS were pre-existing, whereas some were newly synthesized including ribosomal proteins. We also investigated the potential role of other Nedd4…
has type: Thesis/Dissertation
date issued: 2018-05
is shown at: 10.14288/1.0364158
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/64750