Molecular Mechanisms of SCUBE2-Mediated Sonic Hedgehog Release and Delivery

项目摘要/摘要 Hedgehog(HH)细胞-细胞信号通路在胚胎发育中起着关键作用。 成人干细胞的维持，如果调控不当，会导致各种先天缺陷和癌症。在……里面 脊椎动物，HH途径的典型激活物是分泌蛋白Sonic hedgehog(SHH)，它 必须从产生它的细胞(“发送细胞”)到它向其发出信号的细胞(“接收细胞”)远行。 然而，矛盾的是，SHH与胆固醇是共价连接的，这种修饰将胆固醇与血浆捆绑在一起 发送细胞的膜。最近的研究发现，膜蛋白Dispatched1(DISP1)和 SCUBE家族的分泌蛋白是SHH从发送细胞释放所必需的。然而，准确的 SCUBE2驱动SHH释放的机制尚不清楚。此外，目前还不清楚 SCUBE2积极促进SHH向受体细胞的传递，其中SHH必须与其受体Patched1结合 (Ptch1)，以及至少三个功能冗余的辅助受体中的一个，CDO，BOC和GAS1。 我的初步数据表明：(1)SCUBE2可以释放一种异源胆固醇修饰的 蛋白质，但释放SHH的效率较低；(2)SCUBE2直接增强SHH的传递到接收 (3)SCUBE2与CDO结合；(4)SCUBE2的不同部分参与SHH的释放和SHH 送货。这些数据表明：(1)SCUBE2通过在发送时首先结合SHH蛋白来驱动SHH的释放 细胞和随后结合其胆固醇锚，和(2)SCUBE2促进SHH递送到接收 细胞通过直接结合SHH辅助受体。 目前的建议旨在：(1)确定SCUBE2如何识别SHH的胆固醇锚， 并利用新颖的光交联法，测试胆固醇结合在细胞释放SHH中的作用 光反应胆固醇类似物和质谱学；(2)阐明SCUBE2如何选择性释放 利用活细胞荧光显微镜和释放试验；以及(3)阐明其机制 以及SCUBE2和SHH共受体之间的相互作用在SHH传递中的作用 荧光显微镜和基于细胞的信号分析中的基因敲除和过表达方法。 这些实验将阐明膜锚定的SHH是如何释放并传递到遥远的细胞的， 为我们对HH信号的理解提供关键的缺失信息。这些信息反过来又将定义 新的分子靶点，在配体释放和传递的水平上，用于阻断癌症中异常的HH信号。

Project Summary/Abstract The Hedgehog (Hh) cell–cell signaling pathway plays key roles in embryonic development, in maintenance of adult stem cells, and, when improperly regulated, in various birth defects and cancers. In vertebrates, the prototypical activator of the Hh pathway is the secreted protein Sonic hedgehog (SHH), which must travel far from the cells that produce it (“sending cells”) to the cells to which it signals (“receiving cells”). Paradoxically, though, SHH is covalently attached to cholesterol, a modification that tethers it to the plasma membrane of sending cells. Recent studies discovered that the membrane protein Dispatched1 (DISP1) and the SCUBE family of secreted proteins are required for SHH release from sending cells. However, the precise mechanism through which SCUBE2 drives SHH release is unknown. Furthermore, it is unknown whether SCUBE2 actively contributes to SHH delivery to receiving cells, where SHH must bind to its receptor, Patched1 (PTCH1), and at least one of three functionally redundant co-receptors, CDO, BOC, and GAS1. My preliminary data demonstrates that: (1) SCUBE2 can release a heterologous cholesterol-modified protein, but less efficiently than it releases SHH; (2) SCUBE2 directly enhances SHH delivery to receiving cells; (3) SCUBE2 binds to CDO; and (4) different parts of SCUBE2 are involved in SHH release and SHH delivery. These data suggest that (1) SCUBE2 drives SHH release by first binding SHH protein on sending cells and subsequently binding its cholesterol anchor, and (2) SCUBE2 facilitates SHH delivery to receiving cells by directly binding SHH co-receptors. The present proposal aims to: (1) determine how SCUBE2 recognizes the cholesterol anchor of SHH, and to test the function of cholesterol-binding in SHH release from cells, using photocrosslinking with novel photoreactive cholesterol analogs and mass spectrometry; (2) elucidate how SCUBE2 selectively releases SHH from cells, using live-cell fluorescence microscopy and release assays; and (3) elucidate the mechanism and function of interaction between SCUBE2 and SHH co-receptors in SHH delivery, using live-cell fluorescence microscopy and knockout and overexpression approaches in cell-based signaling assays. These experiments will clarify how membrane-anchored SHH is released and delivered to distant cells, providing critical missing information in our understanding of Hh signaling. This information, in turn, will define novel molecular targets, at the level of ligand release and delivery, for blocking aberrant Hh signaling in cancer.