Proteomics of Primary Cilia through Proximity Labeling

 描述(申请人提供)：初级纤毛在专门的环境中组织信号通路，如视觉、嗅觉和刺猬发育通路。纤毛的可溶物和膜被扩散屏障与细胞的其余部分隔开，从而赋予纤毛一个独特的物理化学环境。根据Hedgehog信号的状态，特定的信号中间产物进入或离开纤毛，据预测，Hedgehog信号改变了纤毛组成。然而，由于纤毛不容易从哺乳动物细胞中分离出来，目前还不可能评估Hedgehog途径激活时纤毛蛋白质组变化的程度。初级纤毛功能障碍是一系列遗传性疾病的根本原因，称为纤毛病，其特征是肥胖、肾囊肿、骨骼畸形和感觉缺陷。获得主要纤毛蛋白的目录，并将纤毛疾病的纤毛蛋白质组与健康细胞进行比较，将提供一种快速而有效的方法来表征纤毛疾病。因此，初级纤毛的蛋白质组学目前是发现新的纤毛机制的主要技术障碍。我们没有分离纤毛，而是试图通过将生物素化酶定位于初级纤毛来将小分子生物素附着到所有纤毛蛋白上。这种被称为邻近标记的方法在获得线粒体蛋白质组方面显示了巨大的前景。我们提供了有希望的初步数据，证明了这种方法对于纤毛蛋白质组学的有效性，并希望将其应用于比较不同信号状态之间的纤毛蛋白质组，或者在睫状肌病变突变体和健康细胞之间进行比较。通过比较纤毛蛋白质组学获得的结果将为几个重要问题提供独特的见解，并产生可测试的分子模型。该提案旨在开发一种有前景的技术，并作为快速有效的比较纤毛蛋白质组学平台的垫脚石。

 DESCRIPTION (provided by applicant): Primary cilia organize signaling pathways such as vision, olfaction and the Hedgehog developmental pathway inside a specialized environment. The soluble contents and the membrane of the cilium are separated from the rest of the cell by diffusion barriers, thus endowing cilia with a unique physico-chemical environment. Depending on the status of Hedgehog signaling, specific signaling intermediates move into or out of cilia and it is predicted that the ciliary composition is modified by Hedgehog signaling. However, it has not been possible to assess the extent of the changes in the ciliary proteome upon Hedgehog pathway activation because cilia cannot be readily isolated from mammalian cells. Dysfunction of primary cilia is the underlying cause of a collection of hereditary disorders named ciliopathies characterized by obesity, kidney cysts, skeletal malformations and sensory defects. Obtaining a catalogue of primary cilium proteins and comparing the ciliary proteome of ciliopathy to healthy cells would provide a rapid and effective way to characterize ciliopathies. Thus, proteomics of primary cilia is currently a major technical blockage in the discovery of novel ciliary mechanisms. Rather than isolating cilia, we sought to attach the small molecule biotin to all ciliary protein by targeting a biotinylation enzyme to the primary cilium. This approach named proximity labeling has shown great promise in obtaining the proteome of mitochondria. We present promising preliminary data showing the validity of this approach for ciliary proteomics and wish to apply it to compare the ciliary proteome between different signaling states or between ciliopathy mutants and healthy cells. The results obtained by comparative ciliary proteomics will provide unique insights into several important questions and generate testable molecular models. The proposal is intended to develop a promising technology and to serve as a stepping-stone towards a rapid and effective comparative ciliary proteomics platform.