Browsing Theses and Dissertations School of Pharmacy by Author "Claro da Silva, Tatiana"
UNDERSTANDING STRUCTURE-FUNCTION RELATIONSHIPS AND PROTEIN STABILITY OF THE HUMAN APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER (ASBT, SLC10A2)Claro da Silva, Tatiana; Swaan, Peter W. (2011)The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) is renowned as the major bile acid transporter in the intestine. It utilizes cellular sodium gradient to actively concentrate bile acids in the enterocytes, thereby playing a key role in the enterohepatic circulation of bile acids (EHC). ASBT is a promising target for prodrug approaches aiming at improving drug bioavailability, and for drugs to treat hypercholesterolaemia, since cholesterol metabolism is induced upon bile acid depletion. Moreover, its contributing role in drug-drug interactions is rapidly emerging. Topologically, the human (hASBT) is a glycoprotein that spans the membrane bilayer seven times, oriented with an extracellular N-terminus and a cytoplasmic C-terminus (Nexo/Ccyt). Despite its physiological and pharmacological relevance, ASBT remains to be fully characterized at the molecular level. Here, we summarize advances made by our group and others in the quest to understand ASBT’s structure-function relationships and its complex mechanism of bile acid transport. We also report our novel findings regarding protein regions relevant for function and protein stability in the hASBT. Our observations indicate that residues located at the transmembrane 1 (TM1) play a pivotal role in hASBT function, and that Gly50, placed at the interface of TM1 with the intracellular loop 1 (IL1), is critical for hASBT stability. Expanding our studies to IL1, we identified a cluster of amino acids comprising Cys51 – Lys57, which are likely involved in hASBT protein stability, whereas residues downstream Lys57 appear to be relevant for transport. We have demonstrated that successful mapping of regions implicated in hASBT’s transport cycle can be achieved with a combination of sitedirected mutagenesis, bile acid uptake and kinetics, sodium-activation assays and the substituted-cysteine accessibility method. Moreover, inhibition of the proteasome with MG132, and of prolyl-peptidyl isomerases with cyclosporine A and FK506, are valuable approaches to reveal the contribution of specific amino acids to hASBT stability. Finally, we integrate our data to propose an overall schematic of hASBT transport, which will contribute to a better understanding on ASBT physiology and, potentially, on other proteins in the SLC10 family.