title: Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.
abstract: The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington's disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
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: Marcey L
authors.initials: ML
authors.lastname: Waters
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059010:Single-Cell Analysis; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D051176:beta Catenin / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D051176:beta Catenin; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex