The apical sodium-dependent bile acid transporter (ASBT) plays a key role in the enterohepatic recycling of bile salts, cholesterol homeostasis, and serves as a molecular target for hypercholesterolemic agents and pharmaceutical prodrug strategies. Despite its clinical significance, ASBT is poorly characterized at the molecular level. The proposed research will focus on the structural biology of ASBT. Using a novel approach that combines molecular and computational biology our long-term goal is to delineate the three-dimensional structure, ligand-binding domains, and cellular transport mechanism of ASBT. The following specific aims will be addressed: 1) To determine amino acids in critical protein domains that participate in substrate and sodium binding and translocation;we will use a combination of site-directed thiol modification, second-site suppressor mutagenesis and kinetic analysis of key mutants to address the hypothesis that amino acids lining the hydrophilic cleft of ASBT participate in substrate translocation. 2) To determine the structural organization and helical packing of ASBT transmembrane domains;here, we will use bifunctional chemical cross-linking reagents to study helical proximity and orientation in double cysteine mutant constructs;furthermore, we aim to determine the organization of ASBT in functional multimeric states. 3) To employ molecular dynamics simulations to refine the homology model of ASBT and probe conformations and conformational changes associated with the transport process;these studies will use the distance constraints obtained in aim 2 to refine our homology model of ASBT protein, which will be simulated in a fully solvated lipid bilayer system. Information gained by these studies will significantly increase our understanding of the structural interactions that drive bile acid transport and further our structural knowledge of solute carrier proteins in general. Additionally, it may aid future development of specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.
Bile acids play an invaluable role in the intestinal absorption of food-derived lipids and lipid-soluble vitamins and drugs. The human bile acid pool is efficiently conserved through recirculation by bile acid transporters expressed in the distal ileum and the liver. Fecal loss of bile acids is compensated by de novo synthesis in the liver from its precursor, cholesterol;thus, bile acid transporters play an intricate role in cholesterol catabolism and they may be used as a target for anti-hypercholesterolemic drugs. Furthermore, the intestinal bile acid transporter, ASBT, may be exploited as a drug delivery target for poorly permeable therapeutics. The present proposal builds upon our previous work that helped us identify key residues for ASBT that play a role in drug- protein interactions;this, in turn allowed the development of a functional three- dimensional model for human ASBT which can be used in the rational design of novel therapeutics aimed at lowering plasma cholesterol levels or prodrugs designed for enhanced intestinal permeability.
|Welch, Matthew A; KÃ¶ck, Kathleen; Urban, Thomas J et al. (2015) Toward predicting drug-induced liver injury: parallel computational approaches to identify multidrug resistance protein 4 and bile salt export pump inhibitors. Drug Metab Dispos 43:725-34|
|Lynch, Caitlin; Pan, Yongmei; Li, Linhao et al. (2014) Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes. Toxicol Appl Pharmacol 279:33-42|
|Moore, Robyn H; Chothe, Paresh; Swaan, Peter W (2013) Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2. Biochemistry 52:5117-24|
|Lynch, Caitlin; Pan, Yongmei; Li, Linhao et al. (2013) Identification of novel activators of constitutive androstane receptor from FDA-approved drugs by integrated computational and biological approaches. Pharm Res 30:489-501|
|Claro da Silva, Tatiana; Polli, James E; Swaan, Peter W (2013) The solute carrier family 10 (SLC10): beyond bile acid transport. Mol Aspects Med 34:252-69|
|Sabit, Hairat; Mallajosyula, Sairam S; MacKerell Jr, Alexander D et al. (2013) Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation. J Biol Chem 288:32394-404|
|GonzÃ¡lez, Pablo M; Hussainzada, Naissan; Swaan, Peter W et al. (2012) Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation. Pharm Res 29:1821-31|
|Ho, Horace T B; Pan, Yongmei; Cui, Zhiyi et al. (2011) Molecular analysis and structure-activity relationship modeling of the substrate/inhibitor interaction site of plasma membrane monoamine transporter. J Pharmacol Exp Ther 339:376-85|
|Bolusani, Swetha; Young, Blake A; Cole, Nicola A et al. (2011) Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues. J Biol Chem 286:5166-74|
|Mason, Cifford W; Buhimschi, Irina A; Buhimschi, Catalin S et al. (2011) ATP-binding cassette transporter expression in human placenta as a function of pregnancy condition. Drug Metab Dispos 39:1000-7|
Showing the most recent 10 out of 35 publications