TARGETING SCF SUBSTRATES TO THE PROTEASOME

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DESCRIPTION (provided by applicant): The ubiquitin-mediated protein degradation by the proteasome has been only recently recognized as critical for cellular signaling in cell growth and proliferation. Since then, perturbations of the ubiquitin-mediated proteolysis have been implicated in multiple aspects of the pathogenesis of cancer. This makes the proteasome an attractive target for possible therapeutical intervention. The long-term goal of the proposed work is to understand the molecular mechanisms by which the proteasome recruits substrates and initiates their destruction. It is proposed to address this goal by biochemical dissection of protein degradation in vitro, using purified substrates and components of the SCF ubiquitin ligase pathway of yeast S. cerevisiae, which were discovered and characterized by the principal investigator's group. This pathway is conserved and controls degradation of major Gi cell cycle regulatory proteins and signaling molecules in organisms from yeast to humans. The knowledge obtained with yeast is therefore directly relevant to the understanding of SCF-mediated proteolysis in human cells. In the current application, it is proposed to uncover features of the proteasome that could serve as targets for pharmacological regulation of its activity at the steps of substrate recognition and processing for degradation, but not the degradation itself. This knowledge will be of considerable value for development of novel strategies for targeting the proteasome in cancer. In this proposal there are two specific aims: (1) Identify the mechanism by which SCF ubiquitin ligase associates with the proteasome and define its role in targeting substrates for degradation. It was observed that SCF targets selected proteins for degradation in two possible ways: (1) by promoting substrate ubiquitination and (2) by facilitating its direct contact with the proteasome. Defining the role of SCF binding to the proteasome in protein turnover requires isolation of SCF mutants that cannot bind the proteasome while maintaining the ubiquitin ligase activity. To isolate and characterize such mutants, an in vitro system with purified proteins has been developed that provides the investigator with a unique opportunity to address the protein-protein interactions required for SCF/proteasome binding. With these reagents the ubiquitin and the SCF-mediated degradation of natural SCF substrates both in vitro and in vivo, including defining the precise requirements for substrate recognition will be investigated. (2) Characterize the substrate unfolding step and its role in the release of the non-ubiquitinated subunits of substrate complexes. In the SCF pathway, the substrate polypeptide is only one component of a tightly bound multi-protein complex that is targeted to the proteasome. It is proposed to investigate the role of substrate unfolding as a potential discriminatory step in substrate selection. This includes: (1) establishing a reliable substrate-unfolding assay with purified proteasomes, (2) identification of the proteasome subunits that play a role in substrate unfolding using purified proteasome mutants, and (3) defining whether these subunits play a role in the release of the non-ubiquitinated components of substrate complexes.