PKD2的S4-S5连接域与C末端间相互作用介导的调控其离子通道功能及相关疾病的机制

Autosomal dominant polycystic kidney disease (ADPKD) is due to mutations in PKD1 or PKD2 and affects affects over 10 millions people worldwide. ADPKD patients still rely on hemodialysis and transplantation. Clinical trials were recently conducted based on known PKD1 or PKD2 regulatory pathways. The outcomes were rather negative except that Tolvaptan was approved as the first ADPKD drug in Canada and Japan. However, its effectiveness is marginal and it is not approved in USA due to an unknown risk/benefit ratio. Of note, the tested drugs all act on regulatory pathways but not directly on PKD1 or PKD2; eg, Tolvaptan is a vasopressin receptor 2 antagonist. Because PKD1 and PKD2 are regulated by multiple pathways which also regulate other targets, the efficacy and specificity of a single drug are questionable. Our goal is to identify molecules directly acting on PKD2 to compensate for pathogenic PKD2 function. .PKD2 has 6 membrane spans (S1-S6) and an intracellular N- and C-terminus. Our preliminary experiments found that the S4-S5 linker interacts with the C-terminus (termed L-C interaction) of PKD2 and its homologue PKD2L1, and that the interaction is required for channel function. We also found that phospholipid PIP2 inhibits the PKD2L1 L-C interaction. We hypothesize that PIP2 binds to a cationic residue in the PKD2 S4-S5 linker, which undermines the functionally critical L-C interaction thereby inhibiting PKD2 function. .RESEARCH PLAN and METHODOLOGY.Aim 1. Characterization of the PKD2 L-C interaction and its requirement for channel function and regulation by PIP2. We will use epithelial mouse collecting duct cells and Xenopus oocytes to first characterize the PKD2 intramolecular L-C interaction using mutagenesis, co-immunoprecipitation, GST pull-down and biotinylation. We will then determine that the L-C interaction is functionally essential by use of electrophysiology, immunofluorescence and surface biotinylation. Finally we will examine how PIP2 modulates the PKD2 L-C interaction through which it inhibits PKD2 channel function. .Aim 2. Compensation for pathogenic PKD2 function by PKD2 blocking peptides acting on the PIP2/L-C regulation axis. We will use cell models to first express 4 selected PKD2 pathogenic mutants to determine how their channel function or surface membrane targeting is altered, using similar techniques as in Aim 1. We will then identify and optimize PKD2 blocking peptides that compensate for altered function of these mutant channels through inhibiting the L-C or PIP2-PKD2 interaction, and will be further characterized in animal models (Aims 3 and 4). .Aim 3. Regulation of PKD2-dependent diseases in PKD2 knockdown and pathogenic mutant knockin zebrafish via modulation of PKD2 intramolecular interactions. We will use zebrafish models to examine how tail curling and pronephric cyst due to PKD2 morpholino knockdown or pathogenic PKD2 knockin are alleviated by fish PKD2 blocking peptides that inhibit the PIP2-PKD2 or L-C interaction. PKD2 blocking peptides will be expressed in fish through injection of mRNAs or plasmids. .Aim 4. Regulation of PKD2-dependent diseases in PKD2+/- knockout mice and PKD2 pathogenic mutant knockin mice via modulation of the PIP2/L-C regulation axis. We will determine the effects of blocking peptides on renal cyst progression using our existing PKD2+/- mice through peritoneal injection. We will also generate PKD2 pathogenic mutant knockin mice using CRISPRs and perform similar experiments to test the effects of blocking peptides on alleviating renal cyst progression. .SIGNIFICANCE. In contrast to recent clinical trials that all targeted to PKD1 or PKD2 regulatory pathways our project will directly target to PKD2 intra-protein interactions and identify PKD2 blocking peptides that compensate for pathogenic PKD2 function and alleviate the associated diseases in zebrafish and mice, which will provide novel insights into design of effective peptide drugs for treating ADPKD.

PKD1受体或PKD2离子通道的致病性突变导致全世界有数千万遗传性多囊肾疾病（ADPKD）患者，目前缺乏有效治疗药物。近年来多个临床试验药物大都以失败告终，仅在少量国家（不含美国）批准的药物Tolvaptan疗效也不明显。究其原因是药物都靶向PKD1或PKD2的调控因子/通路而不是PKD蛋白本身。我们的新思路是通过揭示PKD2通道作用机制而找到直接作用于PKD2的特异性短肽而补偿PKD2功能反常及减缓肾囊肿。本课题首先用细胞模型研究PKD2中S4-S5连结域和C端TRP域间的结合并揭示此结合和PIP2磷脂是如何调控通道功能的，并依此优化补偿PKD2致病突变体功能的阻断肽。其次构建模拟多囊肾患者的PKD2敲低和致病突变体并敲入斑马鱼和小鼠模型中，从而验证细胞模型中揭示的调控机制及阻断肽减缓肾囊肿的作用。本课题有望得到更具特异性的临床药物前体，为多囊肾疾病创新药物研制提供理论基础。

常染色体显性多囊肾病(ADPKD)是由分别编码膜受体PKD1和阳离子通道PKD2的PKD1或PKD2基因突变引起的。PKD2，也称为瞬时受体电位多囊蛋白-2(TRPP2)，是位于细胞表面、初级纤毛和内质网(ER)膜上的Ca2+可渗透通道。本研究阐明TACAN作为 PKD2 抑制剂并介导PKD2-TACAN通道复合体的机械敏感性。利用膜片钳和双电极电压钳技术分别对非洲爪蟾卵母细胞和哺乳动物细胞进行电生理学研究，发现TACAN抑制PKD2和突变体F604P的单通道电导和开放概率。首次发现TACAN N端S片段T160X与PKD2 C端片段N580-L700相互作用，C端S6的片段L296-D343与PKD2 N端A594X相互作用，通过物理相互作用抑制PKD2通道的功能。在斑马鱼幼虫实验中，TACAN会加重斑马鱼幼虫中PKD2依赖性尾部弯曲和前肾囊肿。此外，TRPV6对钙稳态至关重要。通过在非洲爪蟾卵母细胞和癌细胞中的电生理学和Ca2+成像、分子生物学和数值模拟研究，揭示了TRPV6中的分子内S4-S5、S5/S6螺旋相互作用，与C端TRP螺旋（L/C）以及N端前S1螺旋与TRP螺旋（N/C）之间存在相互作用，从而维持TRPV6在基础状态的功能。PIP2通过结合在S5或C末端上的三个阳离子残基，能够抑制两者相互作用，从而激活TRPV6型。. 总而言之，本研究揭示了PKD2通道作用机制，并阐明了TACAN作为PKD2抑制剂并介导PKD2-TACAN通道复合体的机械敏感性，此外，“PIP2-分子内相互作用” 涉及TRPV6激活的自抑制作用的调控机制。本课题揭示的调控机制有助于设计PKD2或TACAN的阻断肽，可成为治疗多囊肾疾病的具有特异性的前期药物，以及乳腺癌治疗的药物开发提供了新的分子靶标。

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