The long-term goal of this project is to develop a versatile tool to deplete specific proteins from the cytosol and nucleus of eukaryotic cells rapidl and effectively. The project will test the feasibility of a strategy based on proteasome adaptors, which we call degradons, built on a recombinant antibody platform. The proteasome adaptors or degradons will target proteins for destruction by the cellular Ubiquitin-Proteasome-System (UPS). Degradons should be useful as a research tool by allowing the researcher to remove specific proteins from cells to determine their function. They could also serve as a therapeutic approach to deplete toxic proteins such as oncogene products from cells. Degradons represents a new strategy to control cellular protein concentrations that is complementary to RNAi technology and could be used independently or in combination with RNAi approaches. Where RNAi interferes with protein synthesis, degradons induce protein destruction. Thus, degradons are not limited by natural protein turnover rates and can be specific for post-translationally modified forms of proteins. Degradon design builds on the detailed mechanistic understanding of proteasome biochemistry that has been developed over the past ten years and the maturation of experimental strategies to develop high affinity and specificity interaction agents, most importantly antibody technology. The degradon strategy is inspired by natural components of the UPS, the UbL-UBA proteins that serve as substrate adaptors, as well as viral proteins and oncogenes that act by subverting the UPS to purge tumor suppressor proteins from cells. Degradons will consist of two interaction domains, a proteasome binding domain derived from natural substrate receptors, viral proteins or oncogenes, and a target recognition domain, mostly derived from a recombinant antibody reagent. The two domains will be linked by a flexible, degradation-resistant arm. Degradons will position the bound target optimally for destruction but themselves escapes proteolysis to act catalytically. Degradons will be delivered to cells using methods developed for antibody-based drugs. We will test degradon design on three target proteins representing major classes of regulatory proteins and important oncogens. They are the kinase Bcr-Abl, the transcription factor Myc, and the ubiquitin ligase Mdm2, which controls p53 concentrations. Sets of systematically engineered adaptors will first be characterized biochemically in an in vitro degradation system and the best designs will then be optimized in mammalian culture cells and cancer cell lines. Degradons will be tested by themselves in comparison to RNAi and combination of RNAi and degradons will explore synergistic effects between both methods of protein depletion. Finally, delivery of degradons as a protein drug by receptor-mediated uptake will be tested. If degradons show promise as a protein depletion agent, future experiments will test their effectiveness in primary cell cultures and animal models.
We propose to develop a versatile technology platform to rapidly deplete specific proteins from cells by shunting them into the cell's own protein degradation machinery, the ubiquitin proteasome system. The technology is designed to serve as a research tool, for therapeutic target validation, and, in principle, as a molecularly-targeted therapy approach. It is based on chimeric proteasome adaptors, we call degradons, which consist of two parts: (i) a recognition element composed of an affinity domain evolved or designed to bind to an oncogenic target, and (ii) a proteasome-binding carrier element, which feeds the target directly into the degradation machinery.
