Novel signaling pathways in the ciliary inversin compartment

睫状体反转蛋白室中的新信号通路

基本信息

批准号：
10321218
负责人：
Peter G Czarnecki
金额：
$ 16.96万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10321218
关键词：
Address Adenylate Cyclase Affect Affinity Alleles Applications Grants Autosomal Dominant Polycystic Kidney Biochemical Biology Catalysis Catalytic Domain Cells Cilia Communication Complex Complex Analysis 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 Lead Left Length Link Maps Measures Missense Mutation Molecular Mus Mutation Neuraxis 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

项目摘要

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.

项目概要初级纤毛功能对于正常左右不对称、肾脏、心脏、中枢神经系统的发育至关重要目前的数据表明纤毛具有信号转导的功能。细胞器，区分受体、接头和下游转导机制然而，重要纤毛信号的身份以及信号如何转导。纤毛是由四种纤毛蛋白（反转蛋白、NEK8、ANKS6 和 nephrocystin-3）组成的复合物。构成所谓的睫状体反相室（IC），位于睫状干的近端部分。我们和其他人发现 IC 是脊椎动物纤毛功能所必需的，INVS、NEK8、 ANKS6 和 NPHP3 都会导致纤毛病综合征，包括 L-/R-不对称扰动、先天性心脏缺陷和多囊肾这些缺陷的表型是 PKD2（两者之一）的完全敲除。主要常染色体显性多囊肾病基因，表明 ADPKD 下游信号传导我们发现 IC 蛋白也控制纤毛长度和 IC 基因产物。出乎意料的是，我们发现鞭毛内运输（IFT）速度是需要 cAMP 信号传导的过程。 nephrocystin-3 充当一种新型 GTP 酶，表明 IC 信号传导和 cAMP 信号传导之间存在因果关系。拨款提案，我们旨在研究 nephrocystin-3 GTPase 机制的功能意义，定义酶的催化参数，并询问 IC 之间的分子连接成分以及上游和下游信号因子。我们发现 nephrocystin-3 的作用类似于刺激性异源三聚体 G 蛋白 α 亚基，沿着 GTP 水解循环关闭和打开其活性状态在目标 #1 中，我们将研究核苷酸。纯化的重组 nephrocystin-3 的亲和力和周转率，以及影响 pcy 等位基因催化位点，与 GαS 相比，这是否会引起小鼠多囊肾病；纤毛 G 蛋白偶联受体可能利用肾囊肿蛋白 3 作为信号适配器，并且腺苷酸是否环化酶可能会受到肾囊肿蛋白 3 的刺激，产生 cAMP 在目标#2 中，我们研究了该分子。我们试图确定招募哪些蛋白质结构域是必需的。 nephrocystin-3 的正确定位，以维持整个复合物的完整性，如果引起疾病我们最终将测量纤毛 cAMP 水平，作为 IC 的函数。操作，并测试单个 IC 蛋白的缺失或突变是否会改变纤毛 cAMP 水平。这些研究将纤毛病基因功能与 cAMP 信号传导联系起来，从而解决了纤毛病基因功能与 cAMP 信号传导之间的突出问题。纤毛生物学对囊性肾病的发病机制具有重要意义，这些结果将有所帮助。识别 IC 内可药物化的酶靶标，这可能会导致新疗法的设计。