How ubiquitin-carrying enzymes contribute to ubiquitin ligase specificity

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In recent years novel drugs have been invented that induce proximity between a disease-causing protein and an enzyme called a ubiquitin ligase that promotes the destruction of the problematic protein. This new drug modality is feeding a billion dollar per year industry push to employ ubiquitin ligases to treat various human diseases. Most of these efforts have been utilizing a family of enzymes called the Cullin-RING ligases (CRLs). With some 200 members in humans, the CRLs collectively control approximately 20 % of ubiquitin-dependent protein degradation in cells. As such, an appreciation for how these enzymes are regulated is of considerable interest to a very wide audience. Human CRLs are known to partner with at least 7 additional enzymes, which we refer to as ubiquitin-carrying enzymes (UCEs), that are required to promote CRL-dependent protein substrate degradation. The control of CRLs is believed to be determined predominantly through their reversible modification with a protein called NEDD8. Furthermore, it also was believed that UCEs act promiscuously towards CRLs which would preclude CRL regulation at the level of CRL-UCE interaction. However, CRL-UCE specificity is strongly implied by the recent structure of an active CRL. Preliminary results from our R01 grant indicate that CRL-UCE specificity endows these pairs with exceptionally rapid rates of ubiquitin transfer, potentially providing an additional layer of control of CRL function beyond neddylation. In consideration of these observations, this administrative supplement seeks to obtain a stopped-flow instrument to develop biophysical protein-protein interaction assays to measure the kinetics of formation of the protein complexes between UCEs and CRLs. The results generated from these studies will help explain on a mechanistic level how CRL-UCE specificity is achieved.