Novel signaling pathways in the ciliary inversin compartment

睫状体反相室中的新信号通路

• 批准号: 10711865
• 负责人: Peter G Czarnecki
• 金额: $ 16.45万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10711865
• 关键词: Address; Adenylate Cyclase; Affect; Affinity; Alleles; Applications Grants; Autosomal Dominant Polycystic Kidney; Biochemical; Biology; Catalysis; Catalytic Domain; Cells; Central Nervous System; Cilia; Communication; Complex; Congenital Heart Defects; Coupling; Cyclic AMP; Cystic Kidney Diseases; Data; Defect; Development; Disease; Enzymes; Fluorescent Probes; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; Generations; Genes; Genetic; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Heart; Heterotrimeric GTP-Binding Proteins; Hydrolysis; Individual; Kidney; Kidney Diseases; Kinetics; Knock-out; Left; Length; Link; Maps; Measures; Missense Mutation; Molecular; Mus; Mutation; Nucleotides; Organ; Organelles; Other Genetics; Outcome; Pathogenesis; Pathway interactions; Phenocopy; Polycystic Kidney Diseases; Positioning Attribute; Process; Property; Proteins; Recombinants; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Structure; Syndrome; Tertiary Protein Structure; Testing; Vertebrates; cardiogenesis; ciliopathy; design; disease-causing mutation; gene function; gene product; nephrogenesis; novel; novel therapeutic intervention; novel therapeutics; protein complex; protein protein interaction; receptor; recruit; response

Project summary Primary cilia function is essential for the development of normal left-right asymmetry, kidneys, heart, central nervous system and many other organs. Current data indicate that the cilium functions as a signal transduction organelle, compartmentalizing receptor-, adaptor- and downstream transduction mechanisms for multiple signaling pathways. However, the identity of essential cilia signals and how signals are transduced by the cilium remains unclear. A complex of four ciliary proteins, inversin, NEK8, ANKS6 and nephrocystin-3 constitutes the so-called ciliary inversin compartment (IC), localized in the proximal portion of the ciliary shaft. We and others found that the IC is required for cilium function in vertebrates, with mutations in INVS, NEK8, ANKS6 and NPHP3 all leading to ciliopathy syndromes that include L-/R-asymmetry perturbation, congenital heart defects and polycystic kidneys. These defects phenocopy the full knockout of PKD2, one of the two major autosomal-dominant polycystic kidney disease genes, suggesting that signaling downstream of ADPKD- and IC-gene products occurs along the same pathway. We found that IC proteins also control ciliary length and intraflagellar transport (IFT) velocity, processes that require cAMP signaling. Unexpectedly, we find that nephrocystin-3 acts as a novel GTPase, suggesting a causal link between IC- and cAMP-signaling. In this grant proposal, we aim to investigate the functional significance of the nephrocystin-3 GTPase mechanism, defining the catalytic parameters of the enzyme, and interrogating molecular connections among the IC constituents and to upstream- and downstream signaling factors. We hypothesize that nephrocystin-3 acts analogous to a stimulatory heterotrimeric G-protein α-subunit, switching its activity state off and on, along a GTP-hydrolysis cycle. In Aim #1, we will investigate nucleotide affinitiy and -turnover of the purified recombinant nephrocystin-3, along with the pcy allele that affects the catalytic site, and that gives rise to polycystic kidney disease in mice; compared to GαS. We will test, whether ciliary G-protein coupled receptors may utilize nephrocystin-3 as a signal adaptor, and whether adenylyl cyclases may be stimulated by nephrocystin-3, to generate cAMP. In Aim #2, we investigate the molecular details of the IC assembly hierarchy. We seek to identify what protein domains are necessary to recruit nephrocystin-3 to its proper localization, to maintain the integrity of the whole complex, and if disease-causing mutations destabilize the structure. We will ultimately measure ciliary cAMP levels, as a function of IC manipulations, and test whether deletion or mutation of individual IC proteins change ciliary cAMP levels. These studies connect ciliopathy gene function to cAMP signaling, thereby addressing outstanding issues in ciliary biology with important implications for cystic kidney disease pathogenesis. The results will help identifying druggable enzymatic targets within the IC that may lead to the design of novel therapies.

项目摘要 原发性纤毛功能对于发展正常左右不对称，肾脏，心脏，中央至关重要 神经系统和许多其他器官。当前数据表明纤毛作为信号转导功能 细胞器，隔室化受体，适应器和下游转移机制 信号通路。但是，必需纤毛信号的身份以及信号如何翻译为 纤毛仍不清楚。四个睫状蛋白的复合物，inversin，nek8，anks6和肾囊蛋白3 构成所谓的睫状室（IC），位于睫状轴的代理部分中。 我们和其他人发现，IC是脊椎动物中纤毛功能所必需的，其中突变为Invs，Nek8， ANKS6和NPHP3全部导致纤毛病综合征，包括L-/R-空气对称扰动，先天性 心脏缺陷和多囊肾脏。这些缺陷的表观拷贝是PKD2的完整敲除，这是两者之一 主要常染色体肾脏肾脏疾病基因，表明ADPKD-下游信号传导 和IC-Gene产品沿着相同的途径出现。我们发现IC蛋白还控制睫状长度和 Flagellar内传输（IFT）速度，需要cAMP信号的过程。出乎意料的是，我们发现 肾环胞菌3充当一种新颖的GTPase，表明IC和营地信号之间存在因果关系。在这个 授予建议，我们旨在研究肾囊蛋白-3 GTPase机制的功能意义， 定义酶的催化参数，并询问IC之间的分子连接 组成以及上游和下游信号传导因子。 我们假设肾环胞菌3的作用类似于刺激性异三聚体G蛋白α-Subunit， 沿GTP - 水溶液周期关闭其活性状态。在AIM＃1中，我们将调查核苷酸 纯化的重组肾上腺囊蛋白-3的伴侣和 - 弯曲，以及影响纯的PCY等位基因 催化部位，这导致小鼠多囊性肾脏疾病；与Gα相比。我们将测试，是否 睫状G蛋白偶联受体可以利用肾环胞蛋白3作为信号适配器，以及是否腺苷酸化。 肾环胞菌3可能会刺激周期，以产生cAMP。在AIM＃2中，我们研究了分子 IC组装层次结构的详细信息。我们试图确定招募需要哪些蛋白质域 肾环胞菌3至适当的定位，以维持整个复合物的完整性，如果是引起疾病的情况 突变破坏了结构。我们最终将根据IC的函数来衡量睫状营的水平 操纵并测试单个IC蛋白的缺失或突变会改变睫状营的水平。 这些研究将纤毛病基因功能与营地信号传导联系起来，从而解决了 睫状生物学对囊性肾脏疾病发病机理具有重要意义。结果将有所帮助 在IC内识别可吸毒的酶促靶标，这可能会导致新型疗法的设计。