Canonical & non-canonical regulation of the HDL receptor by PDZK1's PDZ domains

典范

• 批准号: 9198970
• 负责人: MONTY KRIEGER
• 金额: $ 49.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9198970
• 关键词: Adaptor Signaling Protein; Adrenal Glands; Adult; Affinity Chromatography; Alpha Cell; Arteries; Binding; Binding Sites; Biochemistry; Biological Assay; Biological Process; Biology; Biophysics; Blood Cells; Blood Platelets; Blood coagulation; C-terminal; Calorimetry; Cell Culture System; Cell Surface Receptors; Cell membrane; Cells; Cellular Structures; Cellular biology; Cholesterol; Cholesterol Homeostasis; Cognition; Computer Simulation; Coronary heart disease; Crystallography; Cystic Fibrosis Transmembrane Conductance Regulator; Deep Vein Thrombosis; Deuterium; Development; Disease; Endothelial Cells; Erythrocytes; Female; Fertility; HDL receptor; Heart Diseases; Hepatic; Hepatitis C; Hepatocyte; High Density Lipoproteins; Histologic; Hydrogen; In Vitro; Individual; Infection; Inflammation; Ion Channel; Knock-in Mouse; Knockout Mice; Length; Link; Lipid Binding; Lipids; Lipoproteins; Liver; Mass Spectrum Analysis; Mediating; Medicine; Membrane; Membrane Lipids; Membrane Proteins; Metabolism; Methods; Modeling; Mutation; Myocardial Infarction; Names; Neutrons; Ovary; Oxygen; Peptides; Pharmaceutical Chemistry; Pharmacology; Physiological; Physiology; Plasma; Play; Post-Translational Regulation; Process; Property; Prostaglandins I; Proteins; Reagent; Regulation; Role; SR-BI receptor; Signal Transduction; Site; Specificity; Stroke; Structure; Structure-Activity Relationship; Synaptic plasticity; System; Techniques; Tertiary Protein Structure; Testing; Testis; Tissues; Titrations; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Work; X-Ray Crystallography; analog; base; biological systems; egg; experimental study; hypercholesterolemia; in vivo; inhibitor/antagonist; knockin animal; mutant; protein E; protein expression; protein function; public health relevance; receptor; scaffold; screening; small molecule; small molecule inhibitor; tool

 DESCRIPTION (provided by applicant): PDZ domains are ~90 residue-long protein domains that usually bind the C-termini of their protein targets. PDZ-containing proteins regulate diverse biological systems and frequently have multiple, distinct PDZ domains, thus acting as scaffolds/adaptors simultaneously binding multiple targets. We will study PDZK1 (four PDZ domains, PDZ1-PDZ4) to understand 1) how distinct PDZ domains in multi-PDZ domain proteins collaborate to mediate function, and 2) how a single target (SR-BI) requiring a PDZ protein in one tissue (liver) requires a different PDZ protein in other tissues (e.g., steroidogeni tissue (ST)). PDZK1 is expressed in diverse tissues, and has many targets, e.g., CFTR, prostacyclin receptor, ion channels, and the HDL receptor SR-BI. We will focus on the tissue-specific PDZ domain-mediated regulation of SR-BI, which controls plasma HDL metabolism. SR-BI influences many processes, including platelet & red blood cell structure/function, female fertility, development, synaptic plasticity & cognition, inflammation, deep vein thrombosis, endothelial physiology, infection (e.g., hepatic Hepatitis C Virus entry), and lipoprotein metabolism and associated diseases (e.g., coronary heart disease). In vivo in hepatocytes SR-BI's normal localization, abundance and function, and thus normal HDL metabolism, depend on its productive interaction with PDZK1. Normal PDZK1/SR-BI interaction requires 1) binding of SR-BI's C-terminus to either PDZ1 or PDZ3, 2) the presence of either PDZ2 or PDZ3 and 3) the presence of PDZ4, which in vitro can non-canonically bind directly to inner plasma membrane-like bilayers. PDZK1's localization to the hepatocyte plasma membrane in vivo requires PDZ4 and co-expression of SR-BI. Knock-in mice with a C-terminal deletion in SR-BI suggest a PDZK1 analog is required for SR-BI expression in ST. We will test two hypotheses (I &II): I. PDZK1's regulation of SR-BI requires two- or three-pronged binding in which 1) PDZ4 tethers PDZK1 to the membrane when SR-BI binds to PDZ1 (or PDZ3) and 2) PDZ2 or PDZ3 either binds to other target membrane proteins or plays a structural role. We will perturb PDZK1's PDZ domains individually or within the full-length protein. Perturbations will include targeted mutations or inhibition by small molecules identified using in silico screening and optimized by medicinal chemistry. The mutants & inhibitors will be used in vitro (biochemistry, biophysics) and/or in vivo (cell biology, transgenic and knock-in mice & physiology). Techniques will include analysis of purified proteins (e.g., isothermal titration calorimetry, hydrogen/deuterium exchange mass spectrometry, X-ray crystallography, neutron reflectometry) and histologic and physiologic studies with transgenic and knock-in animals (including analysis of plasma lipids and lipoproteins). Analysis of the PDZK1/SR-BI system will serve as a model for understanding in detail how other multi-PDZ proteins regulate the important biological functions of cell surface receptors. II. A PDZK1 analog(s) in ST regulates SR-BI protein expression and function (as does PDZK1 in the liver). We will isolate the analog(s) and determine the mechanism of its regulation of SR-BI.

 描述（由申请人提供）：PDZ 结构域是约 90 个残基长的蛋白质结构域，通常结合其蛋白质靶标的 C 末端。含有 PDZ 的蛋白质调节不同的生物系统，并且通常具有多个不同的 PDZ 结构域，从而充当同时结合多个靶标的支架/适配器。我们将研究 PDZK1（四个 PDZ 结构域，PDZ1-PDZ4），以了解 1) 多 PDZ 结构域蛋白中的不同 PDZ 结构域如何协作介导功能，以及 2) 一个组织（肝脏）中需要 PDZ 蛋白的单一靶标 (SR-BI) 如何需要其他组织（例如类固醇组织 (ST)）中的不同 PDZ 蛋白。 PDZK1在多种组织中表达，并具有许多靶标，例如CFTR、前列环素受体、离子通道和HDL受体SR-BI。我们将重点关注 SR-BI 的组织特异性 PDZ 结构域介导的调节，它控制血浆 HDL 代谢。 SR-BI影响许多过程，包括血小板和红细胞结构/功能、女性生育力、发育、突触可塑性和认知、炎症、深静脉血栓形成、内皮生理学、感染（例如丙型肝炎病毒进入肝脏）以及脂蛋白代谢和相关疾病（例如冠心病）。体内肝细胞中 SR-BI 的正常定位、丰度和功能以及正常的 HDL 代谢取决于其与 PDZK1 的有效相互作用。正常的 PDZK1/SR-BI 相互作用需要 1) SR-BI 的 C 末端与 PDZ1 或 PDZ3 结合，2) PDZ2 或 PDZ3 的存在，以及 3) PDZ4 的存在，PDZ4 在体外可以非规范地直接与内部质膜样双层结合。 PDZK1 在体内定位于肝细胞质膜需要 PDZ4 和 SR-BI 的共表达。 SR-BI C 端缺失的敲入小鼠表明，ST 中的 SR-BI 表达需要 PDZK1 类似物。我们将测试两个假设 (I 和 II)：I. PDZK1 对 SR-BI 的调节需要双管齐下或三管齐下的结合，其中 1) 当 SR-BI 与 PDZ1（或 PDZ3）结合时，PDZ4 将 PDZK1 束缚在膜上；2）PDZ2 或 PDZ3 与其他靶膜蛋白结合或发挥结构作用。我们将单独或在全长蛋白质内扰动 PDZK1 的 PDZ 结构域。扰动将包括通过计算机筛选识别并通过药物化学优化的小分子的靶向突变或抑制。突变体和抑制剂将用于体外（生物化学、生物物理学）和/或体内（细胞生物学、转基因和敲入小鼠和生理学）。技术包括纯化蛋白质的分析（例如等温滴定量热法、氢/氘交换质谱法、X射线晶体学、中子反射法）以及转基因和基因敲入动物的组织学和生理学研究（包括血浆脂质和脂蛋白分析）。 PDZK1/SR-BI 系统的分析将作为详细了解其他多 PDZ 蛋白如何调节细胞表面受体的重要生物学功能的模型。二. ST 中的 PDZK1 类似物调节 SR-BI 蛋白的表达和功能（与肝脏中的 PDZK1 一样）。我们将分离类似物并确定其调节 SR-BI 的机制。