Molecular dissection of signal transduction at primary cilia

初级纤毛信号转导的分子解剖

• 批准号: 8990973
• 负责人: RAJAT ROHATGI
• 金额: $ 30.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990973
• 关键词: Affinity Chromatography; Architecture; Binding; Binding Proteins; Biochemical; Biological Assay; Biotin; Biotinylation; CRISPR/Cas technology; Cardiac; Cell Communication; Cell Nucleus; Cell physiology; Cells; Chimeric Proteins; Cilia; Complement; Complex; Congenital Abnormality; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Development; Disease; Dissection; Drug Targeting; Dysostoses; Elements; Ellis-Van Creveld Syndrome; Erinaceidae; Family; Functional disorder; Genes; Genetic Epistasis; Gli2 protein; Goals; Health; Hereditary Disease; Human; Human Development; Human Genetics; In Vitro; Indium; Inherited; Integral Membrane Protein; Investigation; Life; Ligands; Ligation; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Methods; Microtubules; Modeling; Molecular; Mutate; Mutation; Natural regeneration; Organelles; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Proteins; Proteomics; Regenerative Medicine; Regulation; Role; Signal Transduction; Structure; Surface; Syndrome; System; Testing; Tissues; Transducers; Vertebrates; Work; base; body system; ciliopathy; gene replacement; hedgehog signal transduction; human disease; human tissue; knockout gene; novel; oncology; orofacial; protein complex; protein function; reconstitution; signal processing; skeletal; smoothened signaling pathway; tissue regeneration; trafficking; transcription factor

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.

描述（由申请人提供）：初级纤毛是触角样细胞器，从我们体内大多数细胞的表面伸出，并在脊椎动物发育中作为重要的信号中心。纤毛缺陷导致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仍然知之甚少。Hedgehog（Hh）通路涉及发育、癌症和再生，在脊椎动物中在纤毛处被编排。我们已经使用蛋白质组学方法来鉴定在初级纤毛处正调节Hh信号传导的纤毛蛋白复合物（“EvC复合物”）。其中两种蛋白质 Efcab 7和Iqce是新的Hh信号传导正调控因子，另外两个Evc和Evc 2在Ellis货车Creveld综合征和Weyers肢端牙骨发育不全中发生突变，这两种人类纤毛疾病的特征是骨骼、心脏和口面组织中Hh信号传导缺陷。在这一建议的三个目标，我们将测试模型，该复合物介导的膜蛋白Smoothened（Smo）和Gli转录因子之间的信号在一个独特的信号微域（“EvC区”）在纤毛的基础。Smo调节Gli蛋白的机制仍然是脊椎动物Hh信号传导中长期存在的谜团之一，尽管这一步骤是肿瘤学中Hh药物的目标。在目的1中，我们将通过测试与已知Hh组分的相互作用以及通过在培养细胞中通过CRISPR/Cas9介导的单基因和双基因敲除实现的详细体外上位性分析来鉴定由EvC复合物调节的信号传导中的特定步骤。基于初步数据，将特别强调蛋白激酶A和Sufu的调节，这两个通用的负调节器在Smo和Gli蛋白之间发挥作用。在深入系统发育分析的指导下，我们已经确定了EvC复合物蛋白中的保守结构域和序列元件，并使用了一系列结合试验来绘制四种蛋白质之间的接触点。在目标2中，我们使用基因置换策略来测试这些结构域在介导复合物组装、EvC区定位和Hh信号传导中的细胞功能。最后，在目标3中，我们将扩展我们成功的蛋白质组学管道，以使用串联亲和纯化和邻近生物素化来鉴定与Efcab 7、Iqce和Evc结合的蛋白质， 补充目标1和2所述的更有针对性的调查。这项工作有望揭示一个神秘的和治疗相关的步骤Hh信号的分子机制，并阐明纤毛相关的先天性畸形综合征的病理生理。