Develop enabling biochemical and structural tools for dissecting the roles of PKD2L2 in metabolism

项目摘要 多囊蛋白家族分为两类完整的膜蛋白：1)PKD1型分支 包括5个11-跨膜(TM)跨受体样蛋白(PKD1、PKD1L1、PKD1L2、PKD1L3和 PKD1REJ)，其特征是具有可能识别未知配体的大的N末端胞外结构域(S)；以及 2)PKD2型分支由三个跨越瞬时受体电位(Trp)的6-TM离子通道(PKD2， PKD2L1和PKD2L2)。PKD1和PKD2是目前研究最广泛的两种多囊蛋白 因为PKD1或PKD2中的失活突变会导致一种常见的、危及生命的多系统 难治性常染色体显性遗传多囊肾病(ADPKD)。我们和其他人证明了PKD2本身 充当非选择性的、结构域交换的阳离子通道。PKD2与PKD1额外结合形成 肾上皮感觉纤毛上的异构体受体/离子通道复合体可能对其产生反应 未知的化学配体(S)和/或机械力，有助于建立和维持 肾脏中肾单位的精致管状结构。血管紧张素转换酶的生理功能与疾病相关性 其他多囊蛋白家族成员虽然也组装成不同的复合体，但人们对它们知之甚少 它们可能作为细胞传感器来检测和响应一系列不同的生理和 环境刺激。PKD2L2可能参与男性生殖，因为它在睾丸和 参与精子中的钙信号转导。照亮毒品产生的PKD2L2-/-小鼠 基因组(IDG)联盟表现出糖耐量改变，这是一个令人兴奋的发现，表明 PKD2L2可能作为一条钙通道，调节胰腺内的钙信号和胰岛素分泌。已建成 我们对PKD_1和PKD_2的生化、结构和功能研究的成功和最新突破 在确定3.0äPKD2L2冷冻-EM结构的过程中，我们将在这里继续开发使能生化 试剂和结构模型，降低其他共享的研究人员的进入门槛 热衷于PKD2L2通道的候补。具体地说，我们将利用我们在膜方面的专业知识 蛋白质生物化学和结构生物学：1)确定PKD2L2的额外低温EM结构， 沿其选通周期捕获处于新功能状态的通道；以及2)开发特定和敏感的通道 PKD2L2的抗体和纳米抗体，可用于定位天然组织和细胞中的通道， 将通过照亮通道运行的细胞类型和组织来深入了解PKD2L2的功能。

Project Summary The polycystin family falls into two classes of integral membrane proteins: 1) the PKD1-type clade comprises five 11-transmembrane (TM) spanning receptor-like proteins (PKD1, PKD1L1, PKD1L2, PKD1L3, and PKD1REJ) characterized by a large N-terminal ectodomain that likely recognizes as-yet unknown ligand(s); and 2) the PKD2-type clade consists of three 6-TM spanning transient receptor potential (TRP) ion channels (PKD2, PKD2L1, and PKD2L2). PKD1 and PKD2 are the two most extensively studied polycystin proteins largely because inactivating mutations in either PKD1 or PKD2 cause a common, life-threatening, multisystem, and incurable autosomal dominant polycystic kidney disease (ADPKD). We and others showed that PKD2 itself functions as a non-selective, domain swapped cation channel. PKD2 additionally associates with PKD1 to form a heteromeric receptor/ion channel complex at sensory cilia of renal epithelia where they may respond to as-yet unknown chemical ligand(s) and/or mechanical force, contributing to the establishment and maintenance of the exquisite tubular architecture of nephrons in the kidneys. The physiological functions and disease relevance of other polycystin family members are poorly understood, although they also assemble into various complexes that possibly serve as cellular sensors for detecting and responding to a diverse range of physiological and environmental stimuli. PKD2L2 may participate in male reproduction as it is expressed in the testis and contributes to Ca2+ signaling in sperms. The PKD2L2-/- mice generated by the Illuminating the Druggable Genome (IDG) consortium exhibit altered glucose tolerance, which is an exciting discovery that suggests that PKD2L2 may function as a Ca2+ channel that regulates Ca2+ signaling and insulin secretion in the pancreas. Built on our successful biochemical, structural, and functional studies on PKD1 and PKD2 and a recent breakthrough in determining a 3.0 Å PKD2L2 cryo-EM structure, here we will continue to develop enabling biochemical reagents and structural models to lower the barriers to entry for other researchers who share the same enthusiasm for the understudies PKD2L2 channel. Specifically, we will leverage our expertise in membrane protein biochemistry and structural biology to: 1) determine additional cryo-EM structures for PKD2L2 that capture the channel in new functional states along its gating cycle; and 2) develop specific and sensitive antibodies and nanobodies for PKD2L2 that can be used to localize the channel in native tissues and cells, which will provide insights into PKD2L2 functions by illuminating the cell types and tissues where the channel operates.