A Luciferase Fusion Protein Library to Identify & Monitor Ubiquitylation Targets

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Regulated proteolysis plays a critical role in cellular homeostasis. Many proteins are targeted for destruction by polyubiquitylation, which flags them for proteasomal degradation. Polyubiquitylation in humans involves the sequential action of the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. There are estimated to be hundreds of potential E3s and hence hundreds (if not thousands) of potential ubiquitylation targets in the human proteome. Fusion proteins consisting of an E3 substrate and a bioluminescent (or fluorescent) protein are often recognized by the corresponding E3 and targeted for degradation. Such reporters can then be used to monitor signals that influence the stability of the substrate moiety. For example, p27-Luciferase and HIF-luciferase fusion proteins have been used to monitor cdk2 activity and oxygen availability, respectively, in intact cells. Phosphorylation of p27 by cdk2 generates a binding site for an E3 containing Skp2 and prolyl hydroxylation of HIF, which requires oxygen and 2- oxoglutarate, generates a binding site for an E3 containing pVHL. In specific aim 1we will shuttle -12,000 open reading frames (ORFs) into an ORF-luciferase fusion vector. We will ask what % of these ORFs behave as though they are polyubiquitylated based on 1) accumulation at the restrictive temperature in ts20 cells, which harbor a temperature-sensitive E1 allele and 2) accumulation in the presence of a proteasomal inhibitor. This aim should yield a library of ORF-luciferase fusion proteins that is highly enriched for E3 substrates. This library would be a resource for the discovery of novel substrates for E3s of interest (including E3s linked to disease) and for the discovery of reporters that could be used to monitor various molecular pathways and their responses to pharmacological agents. Inspecific aim 2 and 3 we will use this library to search for novel pVHL substrates (aim 2) or novel reporters for small molecule hydroxylase inhibitors (aim 3). Reisolation of HIF in aims 2 and 3 would constitute proof of concept with respect to the potential utility of this approach.