Developing Inducible Degradation Systems to Control Intracellular Protein Concentration using Proteasome Adaptors

The ubiquitin-proteasome system (UPS) controls the concentrations of hundreds of proteins through their regulated degradation and therefore affects numerous cellular processes including cell cycle control, signal transduction, and gene expression. The degradation signal, or degron, that targets proteins to the proteasome has two distinct components: a proteasome-binding tag and an initiation region. The proteasome binding tag is typically a chain of ubiquitin molecules attached to the protein substrate. The proteasome binds the tag and then engages the substrate at the initiation region. Although both components of the degron are usually located on the same polypeptide, I have shown that they can function to target a protein for degradation in vitro when separated onto two different subunits of a protein complex. Thus the ubiquitin degradation signal can act in trans. I have adapted this trans targeting mechanism to construct a broadly applicable inducible degradation system to control the degradation of individual protein in vivo.;The rapid removal of proteins from the cell can be used as a tool to study protein function and as a therapeutic strategy. The most common way to study protein function is to deplete the protein and observe the cellular response. Traditional methodologies involve disrupting the expression of the gene at the DNA level or manipulating mRNA levels by RNAi. However, these techniques are only effective against newly synthesized proteins, thereby leaving previously existing and stable proteins untouched. I have developed a novel technique that utilizes the ubiquitin proteasome system by rapidly inducing the degradation of specific proteins in mammalian cells in a ubiquitin-independent manner. I have expanded my system to be broadly applicable in multicellular organisms. This research tool can be used as a therapeutic strategy to rapidly remove harmful or detrimental proteins from human cells.