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（由申请方提供）：顶端钠依赖性胆汁酸转运蛋白（ASBT）在胆盐的肝肠循环、胆固醇稳态中起关键作用，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很差。拟议的研究将重点关注ASBT的结构生物学。使用一种新的方法，结合分子和计算生物学，我们的长期目标是描绘的三维结构，配体结合域，和细胞运输机制的ASBT。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;我们将使用定点巯基修饰，第二位点抑制突变和关键突变体的动力学分析的组合来解决ASBT的亲水性裂缝内衬的氨基酸参与底物易位的假设。2)为了确定ASBT跨膜结构域的结构组织和螺旋包装;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和方向;此外，我们的目标是确定ASBT在功能多聚体状态下的组织。3)采用分子动力学模拟来完善ASBT的同源性模型，并探测与转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蛋白的同源性模型，该模型将在完全溶剂化的脂质双层系统中进行模拟。通过这些研究获得的信息将显着增加我们的理解，驱动胆汁酸转运的结构相互作用，并进一步我们的溶质载体蛋白的结构知识一般。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：胆汁酸在食物来源的脂质和脂溶性维生素和药物的肠道吸收中起着非常重要的作用。人胆汁酸库通过回肠末端和肝脏中表达的胆汁酸转运蛋白的再循环而有效保存。胆汁酸的粪便损失通过肝脏中其前体胆固醇的从头合成来补偿;因此，胆汁酸转运蛋白在胆固醇代谢中起着复杂的作用，并且它们可用作抗高胆固醇血症药物的靶点。此外，肠道胆汁酸转运蛋白，ASBT，可开发作为药物递送靶点渗透性差的治疗。本提案建立在我们以前的工作基础上，这些工作帮助我们鉴定了在药物-蛋白质相互作用中起作用的ASBT的关键残基;这反过来又允许开发人ASBT的功能性三维模型，其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。