Abstract 526: Molecular Characterization Of Sitosterolemia-associated Mutants Of ABCG5

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Background: The proteostasis network is essential for the maintenance of cellular integrity by ensuring the correct folding of newly synthesized proteins. However, the high fidelity of this system contributes to diseases such as Cystic Fibrosis (CF, ABCC7), Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3, ABCB4), and Sitosterolemia (ABCG5/ABCG8). Interference with proteostasis and allowing sub-optimally folded ATP-Binding Cassette (ABC) transporters to transit to the cell surface partially restores function, with clinical benefit for CF and PFIC3. We sought to delineate the molecular underpinnings of Sitosterolemia, an autosomal recessive form of Familial Hypercholesterolemia (FH), characterized by accumulation of phytosterols in the plasma and tissues. ABCG5 ABCG8 form an obligate heterodimer at the surface of the liver and small intestine, and mediates sterol transport into bile and the intestinal lumen. Case studies reveal 57 loss of function and over 40 missense mutations of ABCG5 associated with Sitosterolemia. Recent pharmaceutics show promise with rescue of mutations in CF and PFIC3 with small molecule chaperones and potentiators. Through our analysis of ABCG5 missense mutations causing Sitosterolemia, we anticipate rescue by these compounds as well. Methods: We established a classification system for missense mutations of ABCG5, similar to CF and PFIC3. Mutations were generated by site-directed mutagenesis, and confirmed through Sanger sequencing. Native and mutant ABCG5 were co-transfected with native ABCG8 into human hepatocytes and evaluated for protein abundance and trafficking beyond the endoplasmic reticulum (ER), by SDS-PAGE and immunoblot analysis. Results: Nine mutants of ABCG5 have been generated. Co-expression of ABCG5 with ABCG8 i n vitro demonstrated I68N, A98G, E146Q, and R419P successfully traffic while R284S, T305R, R389H, R419H, and N437K were arrested within the proteostasis network. Conclusions: Similar to CF and PFIC3, Sitosterolemia-associated mutations result in compromised heterodimer formation and trafficking to the apical surface. Small molecule chaperones and potentiators may partially rescue the mutants and provide clinical benefit for Sitosterolemia.