Salmonella Typhimurium based genome-wide CRISPR/Cas9 knockout screen to study p16 degradation
department or school: Medicine, Faculty of
department or school: Medical Genetics, Department of
data provider: DSpace
degree campus: UBCV
creator: Dawo, Sebastian
issued: 2022-03-31T21:35:14Z
issued: 2022
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: The state of a cell and cell function is ultimately characterized by the expression of specific proteins. The equilibrium of a “healthy” amount of protein is maintained by the balance between newly generated proteins via translation and the degradation. Proteins half-life in eukaryotic cells can span from minutes to several days and are tightly controlled by the selective degradation of proteins via the ubiquitin-proteasome system. Finding specific genes that are involved in the degradation of a protein of interest harbours many applications for translational medicine, but also enables a better understanding of basic cellular biology. To enable such screens, we have established a FACS-based genome-wide CRISPR/Cas9 knockout screening platform, based on protein delivery via the type three secretion system (T3SS) of an avirulent strain of Salmonella typhimurium. We discovered that the delivered proteins are dynamically degraded following transfer, therefore this protein delivery system offers a suitable and unique approach to study protein degradation. As a proof of concept, we studied the poorly understood degradation of the key tumor suppressor protein p16, which additionally is lysine-free, contradicting a longstanding concept that canonical ubiquitin is conjugated to a lysine residue. The screen yielded several promising candidate genes including genes of the Torsin family, the Sec62/63 complex, and COPS6. Validation of the candidate genes uncovered THAP1 as a potential general regulator of protein degradation. We anticipate this method will offer novel mechanistic insights into the degradation of intracellularly delivered proteins and might uncover new pathways of protein homeostasis.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/81074
note: SALMONELLA TYPHIMURIUM BASED GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREEN TO STUDY p16 DEGRADATION by Sebastian Dawo B.Sc., The University of Applied Sciences Weihenstephan-Triesdorf, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2022 © Sebastian Dawo, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: SALMONELLA TYPHIMURIUM BASED GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREEN TO STUDY p16 DEGRADATION submitted by Sebastian Dawo in partial fulfillment of the requirements for the degree of Master of Science in Medical Genetics Examining Committee: Prof. Josef Penninger, Medical Genetics, UBC Supervisor Prof. Philip Hieter, Medical Genetics, UBC Supervisory Committee Member Prof. Peter Stirling, Medical Genetics, UBC Additional Examiner Additional Supervisory Committee Members: Prof. Christopher Overall, Centre for Blood Research, UBC Supervisory Committee Member iii Abstract The state of a cell and cell function is ultimately characterized by the expression of specific proteins. The equilibrium of a “healthy” amount of protein is maintained by the balance between newly generated proteins via translation and the degradation. Proteins half-life in eukaryotic cells can span from minutes to several days and are tightly controlled by the selective degradation of proteins via the ubiquitin-proteasome system. Finding specific genes that are involved in the degradation of a protein of interest harbours many applications for translational medicine, but also enables a better understanding of basic cellular biology. To enable such screens, we have established a FACS-based genome-wide CRISPR/Cas9 knockout screening platform, based on protein delivery via the type three secretion system (T3SS) of an avi…
has type: Thesis/Dissertation
date issued: 2022-05
is shown at: 10.14288/1.0411664
language: eng
degree discipline: Medical Genetics
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
contributor: Penninger, Josef
type: Text
identifier URI: http://hdl.handle.net/2429/81074