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，因此充当同时结合多个靶标的支架/衔接子。我们将研究PDZK 1（四个PDZ结构域，PDZ 1-PDZ 4）以了解1）多PDZ结构域蛋白中不同的PDZ结构域如何协作介导功能，以及2）在一个组织（肝脏）中需要PDZ蛋白的单个靶标（SR-BI）如何在其他组织（例如，类固醇生成组织（ST））。PDZK 1在多种组织中表达，并且具有许多靶点，例如，CFTR、前列环素受体、离子通道和HDL受体SR-BI。我们将重点关注组织特异性PDZ结构域介导的SR-BI调节，其控制血浆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，肝丙型肝炎病毒进入），以及脂蛋白代谢和相关疾病（例如，冠心病）。在肝细胞中，SR-BI的体内正常定位、丰度和功能以及因此的正常HDL代谢取决于其与PDZK 1的有效相互作用。正常的PDZK 1/SR-BI相互作用需要1）SR-BI的C-末端与PDZ 1或PDZ 3结合，2）PDZ 2或PDZ 3的存在和3）PDZ 4的存在，其在体外可以非典型地直接结合到内质膜样双层。PDZK 1在体内定位于肝细胞质膜需要PDZ 4和SR-BI的共表达。在SR-BI中具有C-末端缺失的敲入小鼠表明在ST中SR-BI表达需要PDZK 1类似物。PDZK 1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结合PDZ 1（或PDZ 3）时，PDZ 4将PDZK 1拴在膜上，2）PDZ 2或PDZ 3结合其他靶膜蛋白或起结构作用。我们将干扰PDZK 1的PDZ结构域单独或在全长蛋白质。干扰将包括靶向突变或使用计算机模拟筛选鉴定并通过药物化学优化的小分子抑制。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）。技术将包括分析纯化的蛋白质（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）。PDZK 1/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PDZK 1类似物调节SR-BI蛋白的表达和功能（如肝脏中的PDZK 1）。我们将分离类似物并确定其调节SR-BI的机制。