Structures and Mechanisms of Polycystic Kidney Disease Proteins

项目摘要 常染色体显性遗传性多囊肾病（ADPKD）是一种具有多种肾外病变的系统性疾病， 表现，包括危及生命的并发症，涉及心血管系统。ADPKD 是最常见的遗传疾病之一，在美国有超过60万例， 12.5全球百万病例。ADPKD是由PKD 1或PKD 2基因突变引起的， 功能上神秘的膜蛋白多囊蛋白家族的创始成员。在结构上，PKD 1是 一种11跨膜受体样蛋白，具有非常大的细胞外N-末端， 被认为参与检测未知的细胞外刺激物或配体。PKD 2是一种瞬时受体， 一种与四聚体电压门控离子具有相似结构的TRP通道 渠道PKD 1和PKD 2在初级纤毛处共组装成异源多聚体受体/离子通道复合物， 可能响应于流体剪切的机械力和/或化学刺激。目前， 多囊蛋白仅可用于几个单独的可溶性结构域， 完整受体或通道的结构，以告知生理学和药理学研究，并指导 合理设计有效的策略治疗ADPKD患者。此外， 多囊蛋白以及这些特性如何在疾病相关突变中改变， 未知在这里，我们提出了两个具体的目标，将使用一系列生物物理和结构的方法， 解决这些既具有基础性又具有翻译意义的基本问题。我们的初步 数据包括在2.9 μ m分辨率下在脂质纳米盘中处于闭合构象的PKD 2的结构。我们还 准备了一个包括EF手域的结构，并获得了预期 揭示了在调节性细胞因子存在下开放和/或脱敏状态的类似分辨率结构， 如钙和PIP 2。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提供PKD 2在多个功能中的详细视图 重要的状态和建立PKD 2的独特结构特征如何与其生理功能相关。 目的2的主要目标是表征PKD 1、PKD 2和PKD 1/PKD 2的生物物理性质。 复杂，长期目标是提供相关的结构信息。为了实现这一目标，我们 初步数据包括制备生物化学易处理状态的相关蛋白质。成功结果 将澄清目前模糊的机制作用，这些疾病相关的通道蛋白，提供了一个 了解疾病相关突变影响的基础，并为未来开发新的 治疗学

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.