Abstract 2148 "Chain Branching by NEDD4 Family Ubiquitin Ligases Regulates Protein Degradation"

Ubiquitin chains are post-translational signals that regulate the stability, activity, and localization of eukaryotic proteins in a variety of ways. Recent studies indicate that ubiquitin chains can be branched and that branched chains impact the proteins they are attached to through mechanisms that are distinct from those of unbranched chains. Despite evidence that branched chains are abundant in cells and have important physiological roles, our understanding of these modifications has been limited by a lack of robust methods to study them. Using a recently developed middle-down mass spectrometry approach in combination with a debranching assay based on the proteasome-associated deubiquitinase UCH37, we report here that the NEDD4 family ubiquitin ligases assemble branched ubiquitin chains in vitro. Multiple members of this family of ubiquitin ligases, including Rsp5 from S. cerevisiae and several human orthologs, form branched K48/K63 chains, as demonstrated through autoubiquitination and substrate ubiquitination assays. We also provide evidence to suggest that chain branching regulates the stability of NEDD4 family substrates by affecting the rate of proteasomal degradation. These results establish a new biochemical activity for a well-characterized family of ubiquitin ligases and provide a potential mechanism to explain how K63-specific enzymes redirect their chain forming capabilities to promote substrate degradation. This work was supported by the Undergraduate Research Award from the American Society for Biochemistry and Molecular Biology (ASBMB) and the Research Innovation and Scholarly Excellence (RISE) grant from the University of Tampa.