Structures and Mechanisms of Polycystic Kidney Disease Proteins

多囊肾病蛋白的结构和机制

• 批准号: 9982295
• 负责人: Erhu Cao
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-16 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982295
• 关键词: Address; Adopted; Antibodies; Antigens; Architecture; Autosomal Dominant Polycystic Kidney; Biochemical; Biological; Biophysics; Calcium; Cardiovascular system; Cells; Characteristics; Chemicals; Cilia; Complex; Cryoelectron Microscopy; Data; Detection; Detergents; Development; Disease; EF Hand Motifs; EF-Hand Domain; Electrophysiology (science); Environment; Family; Foundations; Future; Genetic Diseases; Goals; In Vitro; Individual; Ion Channel; Ion Channel Gating; Ions; Life; Ligands; Lipid Bilayers; Lipids; Liposomes; Liquid substance; Mechanics; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Mutagenesis; Mutation; Outcome; PKD1 gene; PKD2 gene; Patients; Pharmaceutical Preparations; Pharmacology Study; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C; Physiological; Physiology; Polycystic Kidney Diseases; Preparation; Property; Protein Biochemistry; Proteins; Regulation; Resolution; Role; Second Messenger Systems; Signal Pathway; Site; Stimulus; Structure; System; TRP channel; Therapeutic; Therapeutic Agents; United States; base; biophysical properties; biophysical techniques; cell growth regulation; desensitization; design; extracellular; insight; mechanical force; member; nanodisk; novel therapeutics; particle; patch clamp; polycystic kidney disease 1 protein; receptor; reconstitution; voltage

Project Summary Autosomal dominant polycystic kidney disease (ADPKD) is a systemic disorder with various extrarenal manifestations, including life-threatening complications involving the cardiovascular system. ADPKD represents one of the most common genetic diseases, with over 600,000 cases in the United States and about 12.5 million cases worldwide. ADPKD is caused by mutations in PKD1 or PKD2 genes that encode two founding members of the functionally enigmatic polycystin family of membrane proteins. Structurally, PKD1 is an 11-transmembrane spanning receptor-like protein with a remarkably large extracellular N-terminus that is thought to participate in the detection of unknown extracellular stimuli or ligands. PKD2 is a transient receptor potential (TRP) channel that shares a similar architecture with well-characterized tetrameric voltage-gated ion channels. PKD1 and PKD2 co-assemble into a heteromultimeric receptor/ion channel complex at primary cilia, likely responding to mechanical force of fluid shear and/or chemical stimuli. Currently, structural information of polycystin proteins is only available for several individual soluble domains despite the urgent need for structures of a complete receptor or channel to inform physiological and pharmacological studies and to guide rational design of effective strategies to treat ADPKD patients. Moreover, the basic biophysical properties of polycystin proteins and how these properties are altered in disease-associated mutations remain largely unknown. Here, we propose two specific aims that will use a range of biophysical and structural approaches to address these fundamental questions, which are of both basic and translational significance. Our preliminary data include a structure of PKD2 in the closed conformation in lipid nanodiscs at 2.9Å resolution. We have also prepared a construct that includes the EF hand domain and obtained preliminary EM data that are expected to reveal similar resolution structures of the open and/or desensitized states in the presence of regulatory cellular factors such as calcium and PIP2. These structures form the basis for precisely designed mutagenesis structure-function studies. Successful outcome will provide a detailed view of PKD2 in multiple functionally important states and establish how unique structural features of PKD2 correlate with its physiological functions. The primary goal of Aim 2 is to characterize the biophysical properties of PKD1, PKD2, and the PKD1/PKD2 complex, with the longer-term goal of providing relevant structural information. Toward this goal, our preliminary data include preparation of relevant proteins in biochemically tractable states. Successful outcome will clarify the currently obscure mechanistic roles of these disease-associated channel proteins, provide a basis to understand the impact of disease-associated mutations, and inform future efforts to develop novel therapeutics.

项目摘要 常染色体显性多囊性肾脏疾病（ADPKD）是一种全身性疾病 表现形式，包括威胁生命的并发症涉及心血管系统。 ADPKD 代表最常见的遗传疾病之一，在美国及其大约有60万例 全球1250万例案件。 ADPKD是由编码两个的PKD1或PKD2基因中的突变引起的 膜蛋白的功能神秘多囊这家族的创始成员。从结构上讲，pkd1是 一个11跨膜跨越接收器样蛋白，其细胞外N末端非常大 被认为参与了未知的细胞外刺激或配体的检测。 PKD2是瞬态接收器 电势（TRP）通道，该通道与特征良好的四聚电压离子共享类似的架构 频道。 PKD1和PKD2共组装成原发性纤毛的异源接收器/离子通道复合物， 可能响应流体剪切和/或化学刺激的机械力。目前，结构信息 多囊蛋白蛋白仅适用于几个单个固体域目的地的迫切需求 完整接收器或渠道的结构，以告知生理和药物研究并指导 有效治疗ADPKD患者的有效策略的合理设计。此外，基本的生物物理特性 多囊蛋白蛋白以及如何改变与疾病相关的突变中这些特性的变化很大程度上仍然存在 未知。在这里，我们提出了两个具体目标，这些目标将使用一系列生物物理和结构方法 解决这些基本问题，这些问题既具有基本意义又具有翻译意义。我们的初步 数据包括在2.9Å分辨率的脂质纳米散发中的闭合构象中的PKD2结构。我们也有 准备了包括EF手部域并获得的初步EM数据的结构 在有调节性细胞的情况下，揭示开放和/或脱敏状态的类似分辨率结构 钙和PIP2等因素。这些结构构成了精确设计诱变的基础 结构功能研究。成功的结果将在多个功能上提供PKD2的详细视图 重要状态并确定PKD2的独特结构特征与其物理功能如何相关。 AIM 2的主要目标是表征PKD1，PKD2和PKD1/PKD2的生物物理特性 复杂的，其长期目标是提供相关的结构信息。朝着这个目标，我们 初步数据包括制备生化典型状态中相关蛋白质。成功的结果 将阐明这些疾病相关的通道蛋白的当前晦涩的机理作用，提供 了解与疾病相关的突变的影响，并为未来开发新颖的努力提供信息 疗法。