Lindsay Czuba Scope: COBRE in Pharmaceutical Research and Innovation

The human apical sodium-dependent bile acid transporter (hASBT) is critical for gut-liver signaling and bile acid flux, yet it is unknown how it is dynamically regulated to rapidly adapt to regulatory signals. Prior work suggests that basal expression of hASBT is regulated in part at the posttranslational level and involves tyrosine phosphorylation. In contrast, ubiquitination and subsequent proteasomal degradation is implicated in the destabilization of hASBT. In obesity, hASBT stability in the membrane is increased but the mechanism is unknown. Currently, there is a fundamental gap in the knowledge of regarding which residues are involved in the ubiquitination of hASBT and the mechanisms by which they become modified. The goal of this research is to identify the degrons that signal for hASBT ubiquitin-mediated degradation and provide preliminary insight into how that process is mediated at the cellular level and to develop chemical biology approaches that can monitor these transient interactions. We hypothesize that hASBT membrane expression can be indirectly modulated by interfering with the ubiquitination-proteasome system. This overarching hypothesis will be tested in the following Specific Aims: 1) to develop and assess novel fluorescent transport assays for the rapid assessment of ASBT function and expression; 2) to identify lysine ubiquitination sites on ASBT and characterize their impact on the functional regulation and modulation of hASBT interactions; and 3) to identify the mechanisms that regulate ASBT’s ubiquitination status and stability using small molecules and genetic knockdown. The approach is innovative and meets the unmet need due to 1) the integration of modern advances in chemical biology with transporter biology needs, and 2) represents a paradigm shift in thinking of bile acid transport in the gut-liver axis through the emphasis of modulating cellular and molecular dynamics. Successful completion of this project is anticipated to elucidate the degron and interactome the promotes the destabilization of hASBT. This is significant as it will facilitate the identification of novel drug targets for modulating hASBT function and expression.