A high throughput screening, fluorescence polarization assay that can monitor the sequential conjugation/deconjugation states of ubiquitin

Abstract: Protein ubiquitination is an essential part of the regulation of a wide variety of biological processes. Although ubiquitination is commonly associated with proteasomal degradation, roles in immune signaling and cell cycle regulation have also been identified. The diversity of regulation is made possible by the fact that mono- and distinct forms of poly-ubiquitination have separate regulatory roles. As expected for such a vital process, dysregulation of ubiquitination is associated with numerous diseases including cancer and auto-immune disorders. Therefore, research efforts have sought ways to correct dysregulation and more recently have sought to harness the protein degradation abilities of ubiquitination with protein targeting chimeric molecules (PROTACs). These efforts have been hindered by a paucity of tools to look at all stages of the protein ubiquitination cycle. Here we describe an HTS assay, using fluorescently labeled ubiquitin (Ub) to monitor the sequential conjugation and deconjugation states of Ub based on changes in fluorescence polarization. The results show that 5 different stages of the ubiquitin cycle can be observed and further that individual stages can be monitored to screen for compounds that alter the function of enzymes involved in the ubiquitin cycle.