Molecular dissection of signal transduction at primary cilia

描述(申请人提供)：初级纤毛是天线状的细胞器，从我们身体大多数细胞的表面伸出，在脊椎动物的发育中起着重要的信号中心的作用。纤毛缺陷会导致许多人类遗传性疾病，即“纤毛疾病”，表现为器官系统的表型。信号从纤毛传递到细胞核的分子机制仍然知之甚少。刺猬(Hedgehog，HH)途径与发育、癌症和再生有关，在脊椎动物的纤毛上受到调控。我们使用蛋白质组学方法鉴定了一种纤毛蛋白复合体(“EVC复合体”)，它积极地调节初级纤毛上的HH信号。其中的两种蛋白质 EfCab7和Iqce是HH信号的新的正调节因子，另外两个Evc和Evc2在Ellis van Creveld综合征和Weyers肢端牙齿发育不良症中发生突变，这是一种以骨骼、心脏和口腔面部组织HH信号缺陷为特征的人类纤毛疾病。在这个提议的三个目标中，我们将测试这个复合体在纤毛底部的一个独特的信号微域(EVC区)介导膜蛋白平滑(Smo)和Gli转录因子之间的信号传递的模型。Smo调节Gli蛋白的机制仍然是脊椎动物HH信号的长期谜团之一，尽管这一步骤是HH药物在肿瘤学中的靶点。在目标1中，我们将通过测试与已知HH组分的相互作用以及通过CRISPR/Cas9介导的单基因和双基因敲除在培养细胞中实现的详细体外上位性分析来确定EVC复合体调控信号的特定步骤。根据初步数据，将特别强调对蛋白激酶A和Sufu的调控，这是两个在Smo和Gli蛋白之间发挥作用的普遍负调控因子。在深入的系统发育分析的指导下，我们已经确定了EVC复合蛋白的保守结构域和序列元件，并使用了一系列结合分析来定位这四种蛋白之间的接触点。在目标2中，我们使用基因替换策略来测试这些结构域在介导复杂组装、EVC区定位和HH信号转导中的细胞功能。最后，在目标3中，我们将扩展我们成功的蛋白质组流水线，通过串联亲和纯化和邻近生物素化来鉴定与EfCab7、Iqce和EVC结合的蛋白质 这项工作有望揭示HH信号中一个神秘和治疗相关步骤的分子机制，并阐明纤毛相关先天性畸形综合征的病理生理学。

DESCRIPTION (provided by applicant): Primary cilia are antenna-like organelles that project from the surfaces of most cells in our bodies and serve as important signaling centers in vertebrate development. Ciliary defects cause a number of inherited human diseases, the "ciliopathies," manifested by phenotypes across organ systems. The molecular mechanisms by which signals are transmitted from the cilium to the nucleus remain poorly understood. The Hedgehog (Hh) pathway, implicated in development, cancer and regeneration, is orchestrated at cilia in vertebrates. We have used proteomic methods to identify a ciliary protein complex (the "EvC complex") that positively regulates Hh signaling at primary cilia. Two of the proteins in this complex, Efcab7 and Iqce, are novel positive regulators of Hh signaling, and the other two, Evc and Evc2, are mutated in Ellis van Creveld syndrome and Weyers acrodental dysostosis, human ciliopathies characterized by defective Hh signaling in skeletal, cardiac and orofacial tissues. In the three aims of this proposal, we will test the model that this complex mediates signaling between the membrane protein Smoothened (Smo) and the Gli transcription factors at a unique signaling microdomain (the "EvC Zone") at the base of cilia. The mechanism by which Smo regulates the Gli proteins remains one of the long-standing mysteries in vertebrate Hh signaling, despite the fact that this step is the target for Hh drugs in oncology. In Aim 1, we wil identify the specific step in signaling regulated by the EvC complex by testing for interactions with known Hh components and by detailed in vitro epistasis analysis enabled by CRISPR/Cas9-mediated single and double gene knockouts in cultured cells. Based on preliminary data, particular emphasis will be placed on the regulation of Protein Kinase A and Sufu, two universal negative regulators that function between Smo and the Gli proteins. Guided by deep phylogenetic analysis, we have identified conserved domains and sequence elements in EvC complex proteins and used a battery of binding assays to map the contact points between the four proteins. In Aim 2, we use a gene replacement strategy to test the cellular functions of these domains in mediating complex assembly, EvC zone localization, and Hh signaling. Finally, in Aim 3 we will expand our successful proteomic pipeline to identify proteins that bind to Efcab7, Iqce and Evc using tandem affinity purification and proximity biotinylation to complement the more directed investigations outlined in Aims 1 and 2. This work promises to reveal the molecular mechanism of a mysterious and therapeutically relevant step in Hh signaling and to illuminate the pathophysiology of a cilia-related congenital malformation syndrome.