Structural Biology of the Apical Bile Acid Transporter.

顶端胆汁酸转运蛋白的结构生物学。

• 批准号: 7583574
• 负责人: PETER W SWAAN
• 金额: $ 31.2万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7583574
• 关键词: ASBT protein; Address; Amino Acids; Apical; Area; Award; Bile Acids; Binding; Biochemical; Biological Availability; Cardiovascular Diseases; Carrier Proteins; Catabolism; Chemicals; Cholesterol; Cholesterol Homeostasis; Class; Classification; Cleaved cell; Computational Molecular Biology; Cross-Linking Reagents; Crystallization; Cysteine; Data; Development; Distal; Drug Delivery Systems; Food; Future; Generations; Goals; Helix (Snails); Homeostasis; Homology Modeling; Human; Intestinal Absorption; Intestines; Kinetics; Knowledge; Ligand Binding Domain; Lipid Bilayers; Lipids; Liver; Membrane Proteins; Methods; Modeling; Modification; Molecular; Molecular Conformation; Molecular Target; Mutagenesis; Nutrient; Oral; Pathway interactions; Peptides; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Plasma; Play; Prodrugs; Proline; Protein Region; Protein Subunits; Proteins; Public Health; Recycling; Relative (related person); Research; Resistance; Role; Simulate; Site; Sodium; Space Perception; Structure; Sulfhydryl Compounds; System; Tertiary Protein Structure; Therapeutic; Therapeutic Agents; Toxic effect; Transmembrane Domain; Transport Process; Vitamins; Work; absorption; analog; bile acid transporter; bile salts; clinically significant; crosslink; design; dimer; ear helix; hypercholesterolemia; ileum; lactose permease; molecular dynamics; monomer; mutant; novel strategies; novel therapeutics; research study; restraint; solute; structural biology; three dimensional structure; three-dimensional modeling; uptake

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.

描述（由申请人提供）：顶端钠依赖性胆汁酸转运蛋白（ASBT）在胆汁盐的肠肝循环、胆固醇稳态中发挥关键作用，并作为高胆固醇血症药物和药物前药策略的分子靶点。尽管 ASBT 具有临床意义，但其在分子水平上的特征却很差。拟议的研究将集中于 ASBT 的结构生物学。使用结合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、配体结合域和细胞运输机制。将解决以下具体目标：1）确定参与底物和钠结合和易位的关键蛋白质结构域中的氨基酸；我们将结合使用定点硫醇修饰、第二位点抑制诱变和关键突变体的动力学分析来解决 ASBT 亲水裂隙内的氨基酸参与底物易位的假设。 2) 确定ASBT跨膜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试剂来研究双半胱氨酸突变体构建体中的螺旋接近度和方向；此外，我们的目标是确定 ASBT 在功能性多聚体状态中的组织。 3）利用分子动力学模拟来完善ASBT的同源模型，并探测与运输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 蛋白同源模型，该模型将在完全溶剂化的脂质双层系统中进行模拟。这些研究获得的信息将显着增加我们对驱动胆汁酸转运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结构了解。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。公众健康相关性：胆汁酸在肠道吸收食物来源的脂质以及脂溶性维生素和药物方面发挥着不可估量的作用。人类胆汁酸库通过远端回肠和肝脏中表达的胆汁酸转运蛋白的再循环得到有效保存。粪便中胆汁酸的损失是通过肝脏中胆汁酸的前体胆固醇从头合成来补偿的。因此，胆汁酸转运蛋白在胆固醇分解代谢中发挥着复杂的作用，它们可以用作抗高胆固醇血症药物的靶标。此外，肠道胆汁酸转运蛋白 ASBT 可用作渗透性差的治疗药物的药物递送靶点。目前的提案建立在我们之前的工作基础上，该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 关键残基；这反过来又促进了人类 ASBT 功能性三维模型的开发，该模型可用于合理设计旨在降低血浆胆固醇水平的新型疗法或旨在增强肠道通透性的前药。