Structural and Functional Studies of ATP Binding Cassette Transporter ABCG1
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2021
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Doctoral Thesis
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yes
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Abstract
The reverse cholesterol transport pathway regulates sterol homeostasis in the human body and prevents lipid accumulation in cells. Several ATP binding cassette (ABC) transporters are involved in this pathway, including ABCG1, which in tandem with ABCA1 removes cholesterol from peripheral tissues. ABCG1 dysfunction has been linked to the pathogenesis of several metabolic diseases, such as atherosclerosis and diabetes. However, its function and transport mechanism are poorly understood. The transporter is closely related to the liver sterol transporter ABCG5/G8 and multidrug transporter ABCG2, structures of which have been obtained in recent years. While studies on ABCG2 have revealed the mechanism behind drug transport, studies on ABCG5/G8 have left unanswered questions on sterol transport across cell membranes. Furthermore, we still lack insight into the structural basis determining substrate diversity exhibited among ABC G-subfamily transporters. Therefore, we set out to structurally and functionally characterise ABCG1 to obtain answers to these questions.
In this thesis, I demonstrate structural and functional approaches applied to study ABCG1 mediated sterol transport. In chapter 2, I describe the identification of viable constructs for studies of ABCG1, as well as the optimisation of expression and purification protocols. Construct screening revealed significant stability differences between isoforms, as well as the effect of C and N-terminal tags on protein expression. In chapter 3, I focus on the functional characterisation of ABCG1, establishing a reconstitution procedure of the protein in liposomes and conducting in vitro ATP hydrolysis assays. I compared the activity of ABCG1 in different environments and tested the effect of different chemical compounds on the functional activity of ABCG1, revealing benzamil as a potent ABCG1 inhibitor.
In chapter 4, I describe the structural studies of ABCG1 using single particle cryo-EM. I describe different strategies to obtain a sample for structural studies. Our studies resulted in a 4 Å structure of a catalytically inactive mutant of ABCG1 in an inward-open conformation, revealing the binding cavity and putative sterol translocation pathway. The structure and its comparison with ABCG2 elucidated the basis for substrate diversity between ABCG1 and ABCG2. Comparison with ABCG5/G8 revealed significant structural differences, despite a similar functional role. Finally, the gained structural and functional insights allowed to propose a mechanism for ABCG1 mediated sterol transport.
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ETH Zurich
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03652 - Locher, Kaspar / Locher, Kaspar