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

开发有利的生化和结构工具来剖析 PKD2L2 在代谢中的作用

• 批准号: 10452211
• 负责人: Erhu Cao
• 金额: $ 15.35万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452211
• 关键词: Animals; Antibodies; Architecture; Autosomal Dominant Polycystic Kidney; Binding; Biochemical; Biological Process; Biological Products; Biology; Biophysics; Biotechnology; Bypass; Cations; Cells; Chemicals; Cilia; Complex; Congenital Heart Defects; Cryoelectron Microscopy; Detection; Development; Disease; Drug or chemical Tissue Distribution; Exhibits; Family; Family member; Genome; Human; Hybridomas; Integral Membrane Protein; Ion Channel; Ion Channel Gating; Kidney; Left; Life; Ligands; Lipids; Liposomes; Maintenance; Mechanics; Membrane Proteins; Mendelian disorder; Metabolism; Modeling; Molecular Conformation; Mus; Mutagenesis; Mutate; Mutation; N-terminal; Nephrons; Organ; Pancreas; Pattern; Physiological; Play; Property; Protein Biochemistry; Proteins; Proteomics; Reagent; Reproduction; Reproductive Biology; Research; Research Personnel; Resolution; Role; Sensory; Sensory Receptors; Signal Pathway; Signal Transduction; Site; Source; Stimulus; Structural Models; Structure; TRP channel; Technology; Testis; Time; Tissues; Tubular formation; Variant; causal variant; cell type; diadenosine pyrophosphate; falls; frontier; gain of function; glucose tolerance; insight; insulin secretion; male; mechanical force; member; nanobodies; receptor; reconstitution; renal epithelium; response; sensor; sperm cell; structural biology; tool; voltage

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.

项目摘要 polycystin家族分为两类的整体膜蛋白：1）PKD1型进化枝 包括五个跨越接收器样蛋白（PKD1，PKD1L1，PKD1L2，PKD1L3和PKD1L3）的五个11跨膜（TM）和 PKD1REJ）的特征是大型N末端细胞域，该域可能识别尚未知道的配体；和 2）PKD2型进化枝由三个6-TM跨越瞬态接收器电势（TRP）离子通道组成（PKD2，PKD2， PKD2L1和PKD2L2）。 PKD1和PKD2是两个最广泛研究的多囊蛋白蛋白 因为PKD1或PKD2中的灭活突变会导致常见，威胁生命的多系统和 无法治愈的常染色体显性多囊性肾脏疾病（ADPKD）。我们和其他人表明PKD2本身 充当非选择性域交换阳离子通道。 PKD2另外与PKD1相关联 肾上皮的感觉纤毛的杂体接收器/离子通道复合 未知的化学配体和/或机械力，有助于建立和维护 孩子们的精美肾上腺素结核。身体功能和疾病相关性 其他多囊菌家族成员也很少理解，尽管他们也聚集了各种复合物 这可能是用于检测和响应潜水员生理和生理和响应的细胞传感器 环境刺激。 PKD2L2可以参与男性繁殖，因为它在睾丸中表达 有助于精子中的Ca2+信号传导。由照明可吸毒产生的PKD2L2 - / - 小鼠 基因组（IDG）财团暴露了改变的葡萄糖耐受性，这是一个令人兴奋的发现，表明 PKD2L2可能充当Ca2+通道，可调节胰腺中的Ca2+信号传导和胰岛素分泌。建造 在我们成功的PKD1和PKD2的成功生化，结构和功能研究以及最近的突破 在确定3.0ÅPKD2L2冷冻EM结构时，我们将继续开发启用生化 试剂和结构模型，以降低共享相同的研究人员的入境障碍 对理解PKD2L2频道的热情。具体来说，我们将利用我们在膜上的专业知识 蛋白质生物化学和结构生物学：1）确定PKD2L2的其他冷冻EM结构 沿其门控循环中捕获新功能状态的通道； 2）发展特定和敏感 PKD2L2的抗体和纳米生物剂，可用于将通道定位在天然组织和细胞中，这些通道 通过照亮通道运行的细胞类型和组织，将提供对PKD2L2功能的见解。