Molecular dissection of signal transduction at primary cilia

描述（由申请人提供）：初级纤毛是一种天线状的细胞器，从我们身体中大多数细胞的表面伸出，是脊椎动物发育过程中重要的信号中心。纤毛缺陷引起许多遗传性人类疾病，即“纤毛病”，表现为跨器官系统的表型。信号从纤毛传递到细胞核的分子机制尚不清楚。Hedgehog （Hh）通路与发育、癌症和再生有关，在脊椎动物的纤毛上进行调控。我们使用蛋白质组学方法鉴定了一种纤毛蛋白复合物（“EvC复合物”），该复合物正调控初级纤毛Hh信号。这里面有两个蛋白质

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.