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

 描述(申请人提供)：PDZ结构域是~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的蛋白质调节多种生物系统，经常具有多个不同的PDZ结构域，从而充当同时结合多个靶点的支架/接头。我们将对PDZK1(四个PDZ结构域，PDZ1-PDZ4)进行研究，以了解1)多个PDZ结构域蛋白中不同的PDZ结构域如何协作调节功能，以及2)一个组织(肝脏)中需要PDZ蛋白的单个靶点(SR-BI)如何需要其他组织(如类固醇激素组织(ST))中不同的PDZ蛋白。PDZK1在多种组织中表达，具有多种靶点，如CFTR、前列环素受体、离子通道和高密度脂蛋白受体SR-BI。我们将专注于组织特异性PDZ结构域介导的SR-BI的调节，SR-BI控制血浆高密度脂蛋白的代谢。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、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(如丙型肝炎病毒进入)、脂蛋白代谢和相关疾病(如冠心病)。在体内，SR-BI在肝细胞中的正常定位、丰度和功能，以及正常的高密度脂蛋白代谢，依赖于它与PDZK1的生产性相互作用。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，它在体外可以非规范地直接与内质膜样双层结合。PDZK1‘S在体内定位于肝细胞质膜需要PDZ4和SR-BI的共表达。SR-BI中C端缺失的敲入小鼠提示，ST中SR-BI的表达需要PDZK1类似物。我们将检验两个假设(I和II)：1.PDZK1‘S对SR-BI的调控需要两个或三个方面的结合：1)当SR-BI与PDZ1(或PDZ3)结合时，PDZ4将PDZK1拴在膜上；2)PDZ2或PDZ3要么与其他靶蛋白结合，要么发挥结构作用。我们将单独或在全长蛋白内干扰PDZK1‘S的PDZ结构域。干扰将包括用于电子筛查并通过药物化学优化的小分子的靶向突变或抑制。这些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、转基因和转基因小鼠和生理学)。技术将包括对纯化蛋白质的分析(例如，等温滴定量热法、氢/重离子交换质谱仪、X射线结晶学、中子反射法)以及转基因和转基因动物的组织学和生理学研究(包括血浆脂质和脂蛋白的分析)。对PDZK1/SR-BI系统的分析将成为详细了解其他多PDZ蛋白如何调控细胞表面受体的重要生物学功能的模型。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(S)，并确定其调节SR-BI的机制。

 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.