Czuba Project

PROJECT SUMMARY – PROJECT 8 (CZUBA) The human apical sodium-dependent bile acid transporter (ASBT) 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 ASBT is regulated in part at the post-translational level and involves tyrosine phosphorylation. In contrast, ubiquitination and subsequent proteasomal degradation are implicated in the destabilization of ASBT. In obesity, ASBT membrane expression is increased, but the mechanism is unknown. Currently, there is a critical gap regarding which residues are involved in the ubiquitination of ASBT and the mechanisms by which they become modified. The goal of this research is to identify the degrons that signal ASBT ubiquitin-mediated degradation, provide insight into how that process is mediated at the cellular level, and develop chemical biology approaches that can monitor these transient interactions. We hypothesize that ASBT’s ubiquitination, surface expression and stability is determined by discrete proteoforms which facilitate regulatory protein-protein interactions. This overarching hypothesis will be tested in the following Specific Aims: 1) develop and assess novel fluorescent transport assays for the rapid assessment of ASBT function and expression; 2) identify lysine ubiquitination sites on ASBT and characterize their impact on the functional regulation and modulation of ASBT interactions; and 3) identify the mechanisms that regulate ASBT’s ubiquitination status and stability using small molecules and genetic knockdown. The approach is innovative and timely due to the integration of modern advances in chemical biology with transporter biology needs, and represents a paradigm shift for studies of bile acid transport in the gut-liver axis through the emphasis of modulating cellular and molecular dynamics. Successful completion of this project will elucidate the degron and interactome that promotes the destabilization of ASBT. This is significant as it will facilitate the identification of novel drug targets for modulating ASBT function and expression. Integrated CTCB core support and mentorship will facilitate this project in a number of key ways. Specifically, the Bio Core will support assay development and optimization (Aims 1 and 3) and the Chem Core will synthesize a series of novel fluorescent bile acids (Aim 1 and 3) and assist in ‘click’ chemistry optimization (Aim 1). This will be augmented by a CTCB mentorship team with extensive expertise in membrane transport, membrane receptors, pharmaceutical science, and drug development.