Targeted degradation of proteins by affinity peptide conjugated ubiquitin (APCU)

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Targeted degradation of proteins by affinity peptide conjugated ubiquitin (APCU) Protein degradation possesses many unique properties that cannot be covered by DNA/RNA manipulation tools. To understand the mechanism of biological processes, and to achieve desirable therapeutic effects, techniques designed to induce protein degradation are in critical needs. However, very few options are currently available for such purposes. Also, the existing tools have known limitations such as difficulty for adaptation or bell-shaped dose-response curve, or vulnerability for a negative regulation by the de- ubiquitination system. To expand the option for tools, and to overcome limitations of current methods, we propose to establish a new approach to induce protein degradation by conjugating a ubiquitin moiety with a molecule of high affinity to a target protein. For a proof or concept, peptides will first be used as the affinity molecule. This prototype is thus called affinity peptide conjugated ubiquitin (APCU). In our preliminary study, we designed a prototype APCU molecule with a peptide targeting MCL1, an oncoprotein that is over-expressed in many types of cancer. We found that this molecule can specifically reduce the level of MCL1 protein by promoting its degradation. In the proposed study, we will determine the action mechanism of the APCU approach and the optimal conditions for designing this types of molecules. We will also determine the dose response curve of the MCL1-targeting APCU and its sensitivity of de-ubiquitination system. Furthermore, to prepare for a wide usage and future in vivo applications, we will test the application of APCU on several other proteins and explore the feasibility of forming an APCU molecule with a conditional conjugation strategy. This proposed study, if secedes, will provide a highly valuable tool to specifically degrade a protein for mechanistic investigations and for potential therapeutic usages.