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（由适用提供）：PDZ结构域是〜90个居住的蛋白质结构域，通常结合其蛋白质靶标的C末端。含PDZ的蛋白质调节潜水生物系统，并经常具有多个不同的PDZ结构域，因此充当支架/适配器，仅结合多个靶标。 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 tissue (e.g., steroidogeni tissue (ST)). PDZK1在不同的组织中表达，并且具有许多靶标，例如CFTR，前列环素受体，离子通道和HDL受体SR-BI。我们将专注于组织特异性PDZ结构域介导的SR-BI调节，该调节控制了血浆HDL代谢。 SR-BI影响了许多过程，包括血小板和红细胞结构/功能，女性生育，发育，突触可塑性和认知，感染，深静脉血栓形成，内皮生理学，感染（例如，乙型肝炎病毒的进入）和脂蛋白的代谢性疾病和相关疾病（例如，心脏病）。体内肝细胞中SR-BI的正常定位，抽象和功能，因此HDL代谢正常，取决于其与PDZK1的产物相互作用。正常的PDZK1/SR-BI相互作用需要1）SR-BI的C-末端与PDZ1或PDZ3的结合，2）存在PDZ2或PDZ3和3）PDZ4的存在，PDZ4的存在，PDZ4的存在，这些PDZ4在体外可以非典型地直接与内部质膜膜的纤维膜直接结合。 PDZK1在体内的肝细胞质膜定位需要PDZ4和SR-BI的共表达。 SR-BI中具有C末端缺失的敲入小鼠表明SR-BI表达在ST中需要PDZK1类似物。我们将检验两个假设（I＆II）：I。PDZK1对SR-BI的调节需要两种或三个良好的结合，其中1）当SR-BI与PDZ1（或PDZ3）和2）PDZ2或PDZ2或PDZ2或PDZ2或PDZ3 bite to to to n tote Membrane protem prote蛋白蛋白蛋白蛋白蛋白pdzk1时PDZ4 TETHERS PDZK1与膜上。我们将单独或全长蛋白质内扰动PDZK1的PDZ域。扰动将包括靶向突变或使用用于计算机筛选并通过医学化学优化的小分子抑制。突变体和抑制剂将在体外（生物化学，生物物理学）和/或体内使用（细胞生物学，转基因和敲门小鼠和生理学）。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，X射线晶体学，中子反射测定法）以及对转基因和敲门动物的组织学和生理研究（包括血浆脂质脂质和脂蛋白的分析）。 PDZK1/SR-BI系统的分析将作为一个模型，以详细了解其他多PDZ蛋白如何调节细胞表面受体的重要生物学功能。 ii。 ST中的PDZK1类似物（S）调节SR-BI蛋白的表达和功能（肝脏中的PDZK1也是如此）。我们将分离类似物，并确定其调节SR-BI的机理。