title: A comparative analysis of the ubiquitination kinetics of multiple degrons to identify an ideal targeting sequence for a proteasome reporter.
abstract: The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Melvin
authors.forename: Gregery S
authors.initials: GS
authors.lastname: Woss
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Park
authors.forename: Lukas D
authors.initials: LD
authors.lastname: Dumberger
authors.forename: Marcey L
authors.initials: ML
authors.lastname: Waters
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D020298:U937 Cells; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology
chemical_list: D046988:Proteasome Endopeptidase Complex