Development of Peptide Based E3 Ligase Reporter

Public Deposited

Downloadable Content

Download PDF
Request Version for Screen Reader
Last Modified
  • March 21, 2019
Creator
  • Woss, Gregery
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • This dissertation describes the development of a new analytical tool capable of analyzing E3 ligase activity, a critical element in the ubiquitin proteasome system. This was accomplished through the development of a novel class of peptide-based fluorescent E3 ligase reporters which are intended to be analyzed by capillary electrophoresis (CE). These E3 ligase reporters incorporate a degradation signal, or degron, which is recognized and ubiquitinated by an E3 ligase, as well as a fluorescent tag to facilitate detection by laser induced fluorescence (LIF). In order to identify potent degrons, a library of E3 ligase reporters incorporating degrons from various sources was synthesized. These reporters were characterized in cell lysates, then optimized to produce substrates that would be efficiently ubiquitinated in cells. In addition to working with minimal degron sequences, an expanded sub-library of reporters based around the degron isolated from p53 was characterized in order to identify a compact degron element and investigate its ubiquitination kinetics and specificity. In order to circumvent peptidase-mediated degradation, a traditional weakness of peptide reporters, investigations were also carried out to develop novel degradation-resistant E3 ligase reporters incorporating β-hairpin secondary structures. Serendipitously, a β-hairpin that was both peptidase-resistant and functioned as an E3 ligase reporter was identified. Finally, electrophoretic separation conditions for the analysis of E3 ligase peptide reporters were identified, and ubiquitination of E3 ligase reporters in cell lysates was observed by CE-LIF. This dissertation lays the groundwork for future development of next generation peptide-based E3 ligase reporters.
Date of publication
Keyword
DOI
Resource type
Rights statement
  • In Copyright
Advisor
  • Wightman, R. Mark
  • Jorgenson, James
  • Allbritton, Nancy
  • Waters, Marcey
  • Lockett, Matthew
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
Language

Relations

Parents:

This work has no parents.

Items

Thumbnail Title Date Uploaded Visibility Actions
file details Woss_unc_0153D_16875.pdf 2019-04-09 Public Download
file details PREMIS_Events_Metadata_0_3768a2c6-60aa-4a9e-8ed3-3f39a0301919.txt 2019-04-09 Public Download
file details original_metadata_file_3768a2c6-60aa-4a9e-8ed3-3f39a0301919.xml 2019-04-09 Public Download