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器官的全身性疾病 临床表现，包括危及生命的心血管系统并发症。ADPKD 代表着最常见的遗传病之一，在美国有超过60万例病例，大约 全球有1250万例。ADPKD是由编码两种蛋白的PKD1或PKD2基因突变引起的 功能神秘的膜蛋白多囊蛋白家族的创始成员。从结构上讲，PKD1是 一种11-跨膜跨膜受体样蛋白，具有非常大的细胞外N-末端，即 被认为参与检测未知的细胞外刺激或配体的。PKD2是一种短暂性受体 与特性良好的四聚体电压门控离子共享相似结构的电位(Trp)通道 频道。PKD1和PKD2在初级纤毛上共同组装成异多聚体受体/离子通道复合体， 可能对流体剪切和/或化学刺激的机械力做出反应。目前，的结构信息 多囊藻毒素蛋白只对几个单独的可溶区可用，尽管迫切需要 一个完整的受体或通道的结构，以告知生理和药理学研究并指导 合理设计治疗ADPKD患者的有效策略。此外，它的基本生物物理性质 多囊蛋白和这些特性在疾病相关突变中如何改变在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，这两个目标将使用一系列生物物理和结构方法来 解决这些基本问题，它们既具有基本意义，也具有翻译意义。我们的预赛 数据包括脂类纳米盘中闭合构象中的PKD2的结构，分辨率为2.9？我们还有 准备了一个包括EF手域的结构，并获得了初步的EM数据，预计 揭示在调节细胞存在的情况下开放和/或脱敏状态的相似的分辨结构 钙和PIP2等因素。这些结构构成了精确设计的突变的基础。 结构-功能研究。成功的结果将提供PKD2的多个功能的详细视图 并确定PKD2独特的结构特征与其生理功能之间的关系。 目标2的主要目标是表征PKD1、PKD2和PKD1/PKD2的生物物理性质 复杂，较长期的目标是提供相关的结构信息。为了这个目标，我们的 初步数据包括在生物化学易处理状态下制备相关蛋白质。成功的结果 将阐明这些与疾病相关的通道蛋白目前鲜为人知的机制作用，提供 为了解疾病相关突变的影响并为未来开发新的 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.