Mechanisms of Substrate Recognition by the Cul3-based E3 Ligase

Cul3-based E3 ligase is responsible for regulating a variety of cellular pathways, many of which are known to have profound effects on the proper function of multicellular organisms. Although progress over the past years has been truly impressive, our understanding of the mechanisms of E2 recruitment and selection by the BCR complex and all the roles that Cul3 plays on kidneys remains in its infancy. To explore these aspects, this dissertation aims to analyze the Cul3 complex using two different approaches: (1) We used the powerful tool of chimeric analysis to map the essential domain binding characteristics of Cul3 taking advantage of the fact that the well-characterized cullin family members exist with non-redundant functions. We hypothesized that besides the substrate recruitment role, Cul3 substrate selection subunits must also be involved in E2 selection since the E2 is responsible for determining the branching pattern of ubiquitin. From this analysis, we characterized a unique role for the substrate adaptor subunits. (2) We performed a quantitative proteomics analysis utilizing a newly created kidney cell line with Cul3 deleted to identify potential new degradation substrates in kidneys. We hypothesized that the phenotypic difference between Cul3 knockouts in kidneys and known Cul3 substrate knockouts in kidneys imply other important substrates exist. This analysis identified both known substrates, validating the study, and new and novel substrates that remain to be characterized