Designing proteasome adaptors to deplete specific proteins from cells
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Cellular protein levels are governed by their rates of synthesis and degradation, and both processes are intricately regulated. One way to study the role of a protein in the cell is to artificially deplete it and observe the effects. The most common methodology for depleting proteins inhibits expression of the target through RNA interference. However, this technique acts at the protein synthesis level and cannot be used to study long-lived proteins or post-translational modifications. Thus, a complementary approach that acts on the proteins themselves would be useful. One method is to target a protein to the cell’s degradation machinery, the Ubiquitin Proteasome System (UPS). Proteins are targeted to the proteasome by the covalent attachment of ubiquitin molecules, which are recognized by the proteasome. The substrate is then translocated into the proteasome’s proteolytic core and degraded into short peptides, while the ubiquitin molecules are cleaved off and recycled. Recently, methods have been developed to deplete proteins by inducing their ubiquitination, which accelerates their degradation by the proteasome. Ubiquitin is a signaling molecule for numerous cellular pathways other than proteolysis, however, and inducing ubiquitination does not always lead to degradation. Therefore, I have developed a system to degrade specific proteins in cells using chimeric adaptors that shuttle proteins directly to the proteasome without the need for ubiquitination. I have shown that this system can be successfully applied to several proteins in vitro and that the adaptors can induce degradation of model and endogenous proteins in cells.