Structural Biology of the Apical Bile Acid Transporter.

描述（由申请人提供）：顶钠依赖性胆汁酸转运蛋白（ASBT）在胆汁盐的肠肝循环、胆固醇稳态中起关键作用，并且是高胆固醇血症药物和药物前药策略的分子靶点。尽管有临床意义，但ASBT在分子水平上的特征却很差。拟开展ASBT的结构生物学研究。利用结合分子生物学和计算生物学的新方法，我们的长期目标是描绘ASBT的三维结构、配体结合域和细胞运输机制。以下具体目标将解决：1)确定参与底物和钠结合和易位的关键蛋白质结构域的氨基酸；我们将结合位点定向硫醇修饰、第二位点抑制突变和关键突变体的动力学分析来解决ASBT亲水性裂缝内的氨基酸参与底物易位的假设。2)确定ASBT跨膜结构域的结构组织和螺旋排列；在这里，我们将使用双功能化学交联试剂来研究双半胱氨酸突变体结构的螺旋接近性和取向；此外，我们的目标是确定ASBT在功能多聚态的组织。3)利用分子动力学模拟技术完善ASBT的同源性模型，探索与输运过程相关的构象和构象变化；这些研究将使用目标2中获得的距离约束来完善我们的ASBT蛋白同源性模型，该模型将在完全溶剂化的脂质双分子层系统中进行模拟。这些研究获得的信息将大大增加我们对驱动胆汁酸运输的结构相互作用的理解，并进一步了解溶质载体蛋白的结构。此外，它可能有助于未来开发针对高胆固醇血症和相关心血管疾病的特定治疗策略。公共卫生相关性：胆汁酸在肠道吸收食物来源的脂质、脂溶性维生素和药物方面发挥着无价的作用。人体胆汁酸库通过回肠远端和肝脏表达的胆汁酸转运体的再循环有效地保存。粪便中胆汁酸的损失通过肝脏中胆汁酸前体胆固醇的重新合成得到补偿；因此，胆汁酸转运体在胆固醇分解代谢中起着复杂的作用，它们可能被用作抗高胆固醇药物的靶点。此外，肠胆汁酸转运体ASBT可能被用作低渗透性治疗的药物递送靶点。目前的建议建立在我们以前的工作，帮助我们确定ASBT的关键残基，在药物-蛋白质相互作用中发挥作用；这反过来又使人类ASBT的功能三维模型得以发展，该模型可用于合理设计旨在降低血浆胆固醇水平的新疗法或设计用于增强肠道通透性的前药。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.