Novel signaling pathways in the ciliary inversin compartment

睫状体反相室中的新信号通路

• 批准号: 10550024
• 负责人: Peter G Czarnecki
• 金额: $ 5.4万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10550024
• 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

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 初级纤毛功能对于正常的左右不对称、肾脏、心脏、中枢神经系统的发育至关重要。 神经系统和许多其他器官。目前的资料表明，纤毛的功能作为一个信号转导 细胞器、区室化受体、接头和下游转导机制， 信号通路然而，必需纤毛信号的身份以及信号如何被纤毛传递， 纤毛仍不清楚。一种由四种睫状蛋白，inversin，NEK 8，ANKS 6和肾囊蛋白-3组成的复合物 构成了所谓的睫状体反转区室（IC），位于睫状体干的近端部分。 我们和其他人发现，IC是脊椎动物纤毛功能所必需的，INVS，NEK 8， ANKS 6和NPHP 3均导致纤毛病变综合征，包括L-/R-不对称扰动、先天性 心脏缺陷和多囊肾这些缺陷表型复制了PKD 2的完全敲除，PKD 2是两个缺陷之一。 主要常染色体显性多囊肾病基因，这表明ADPKD- 和IC基因产物沿着相同的途径发生。我们发现IC蛋白也控制纤毛长度， 鞭毛内转运（IFT）速度，需要cAMP信号传导的过程。出乎意料的是，我们发现， 肾囊蛋白-3作为一种新的GTP酶，提示IC-和cAMP-信号传导之间的因果关系。在这 我们的目的是研究肾囊蛋白-3 GT3受体机制的功能意义， 定义酶的催化参数，并询问IC之间的分子连接 成分和上游和下游信号传导因子。 我们假设肾囊蛋白-3的作用类似于刺激性异源三聚体G蛋白α亚基， 沿着GTP水解循环切换其活性状态关闭和打开。在目标#1中，我们将研究核苷酸 纯化的重组肾囊蛋白-3的亲和力和-周转，沿着影响肾囊蛋白-3的pcy等位基因， 催化位点，并导致小鼠多囊肾病;与GαS相比。我们将测试， 睫状体G蛋白偶联受体可能利用肾囊蛋白-3作为信号接头， 环化酶可被肾囊蛋白-3刺激以产生cAMP。在目标#2中，我们研究了 IC组装层次结构的细节。我们试图确定哪些蛋白质结构域是必要的招募 肾囊蛋白-3的适当定位，以维持整个复合体的完整性，如果致病 突变使结构不稳定。我们将最终测量纤毛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改变纤毛cAMP水平。 这些研究将纤毛病变基因功能与cAMP信号传导联系起来，从而解决了纤毛病变中的突出问题。 睫状体生物学对囊性肾病发病机制具有重要意义。这些结果将帮助 识别IC内的可药用酶靶点，这可能导致新疗法的设计。