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

项目摘要 多囊蛋白家族福尔斯分为两类膜蛋白：1）PKD 1型进化枝 包括五种11跨膜（TM）跨受体样蛋白（PKD 1、PKD 1 L1、PKD 1 L2、PKD 1 L3和PKD 1 L4）， PKD 1 REJ），其特征在于大的N-末端胞外域，其可能识别一种或多种未知配体;和 2)PKD 2型进化枝由三个跨越6-TM的瞬时受体电位（TRP）离子通道（PKD 2， PKD 2L 1和PKD 2L 2）。PKD 1和PKD 2是研究最广泛的两种多囊蛋白， 因为PKD 1或PKD 2中的失活突变导致共同的、危及生命的、多系统的， 常染色体显性遗传性多囊肾病（ADPKD）我们和其他人发现PKD 2本身 作为非选择性的域交换阳离子通道。PKD 2还与PKD 1结合形成 在肾上皮感觉纤毛上的异聚受体/离子通道复合物，在那里它们可能对迄今为止的 未知的化学配体和/或机械力，有助于建立和维持 肾脏中肾单位精致的管状结构。的生理功能和疾病相关性 其他多囊蛋白家族成员的了解很少，尽管它们也组装成各种复合物 其可能用作细胞传感器，用于检测和响应各种生理和 环境刺激PKD 2L 2可能参与雄性生殖，因为它在睾丸中表达， 有助于精子中的Ca 2+信号传导。通过照亮可药用药物产生的PKD 2L 2-/-小鼠 基因组（IDG）财团表现出改变葡萄糖耐量，这是一个令人兴奋的发现，表明， PKD 2L 2可能作为Ca 2+通道发挥作用，调节胰腺中的Ca 2+信号传导和胰岛素分泌。建造 我们对PKD 1和PKD 2成功的生物化学、结构和功能研究以及最近的一项突破 在确定3.0 μ PKD 2L 2 cryo-EM结构的过程中，我们将继续开发使生物化学 试剂和结构模型，以降低其他研究人员的进入门槛， 对PKD 2L 2通道的热情。具体来说，我们将利用我们在膜方面的专业知识， 1）确定PKD 2L 2的另外的冷冻-EM结构， 沿着其门控周期捕获处于新功能状态的通道;以及2）开发特异性和敏感性 PKD 2L 2的抗体和纳米抗体，其可用于在天然组织和细胞中定位通道， 将通过阐明通道运作的细胞类型和组织来深入了解PKD 2L 2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.