Structural Biology of the Apical Bile Acid Transporter.

描述(申请人提供)：心尖部钠依赖胆汁酸转运体(ASBT)在胆盐的肠-肝循环、胆固醇稳态中发挥关键作用，并作为高胆固醇药物和药物前药策略的分子靶点。尽管ASBT具有临床意义，但它在分子水平上的特征很差。拟议的研究将集中在ASBT的结构生物学上。利用一种结合了分子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、配体结合结构域和细胞转运机制。具体目标如下：1)确定参与底物和钠结合和转运的关键蛋白质结构域中的氨基酸；我们将结合定点硫醇修饰、第二位点抑制物突变和关键突变体的动力学分析来解决ASBT亲水性裂隙内氨基酸参与底物转运的假说。2)确定ASBT跨膜结构域的结构组织和螺旋堆积；在这里，我们将使用双功能化学交联剂来研究双半胱氨酸突变结构中的螺旋接近和定向；此外，我们的目的是确定ASBT在功能多聚体状态下的组织结构。3)利用分子动力学模拟来改进ASBT的同源模型，并探索与转运过程相关的构象和构象变化；这些研究将使用在目标2中获得的距离约束来改进我们的ASBT蛋白的同源模型，该模型将在完全溶解的脂类双层体系中模拟。通过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，并进一步加深我们对溶质载体蛋白的一般结构知识。此外，它可能有助于未来针对高胆固醇血症和相关心血管疾病的特定治疗策略的开发。与公共健康相关：胆汁酸在食物来源的脂肪、脂溶维生素和药物的肠道吸收中发挥着宝贵的作用。在回肠末端和肝脏中表达的胆汁酸转运体通过再循环有效地保存了人的胆汁酸池。粪便中胆汁酸的损失通过肝脏中胆汁酸前体胆固醇的从头合成来补偿；因此，胆汁酸转运体在胆固醇分解代谢中扮演着复杂的角色，它们可能被用作抗高胆固醇血症药物的靶点。此外，肠道胆汁酸转运体ASBT可能被用作渗透性差的治疗药物的给药靶点。本提案建立在我们之前工作的基础上，这些工作帮助我们确定了在药物-蛋白质相互作用中发挥作用的ASBT的关键残基；这反过来又允许开发出人类ASBT的功能性三维模型，该模型可用于旨在降低血浆胆固醇水平的新疗法的合理设计或旨在提高肠道通透性的前药设计。

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.