The role of cholesterol in patched/hedgehog signalling during mammalian development.

哺乳动物胚胎发育期间胆固醇水平的波动已被证明具有灾难性的影响，导致严重畸形，特别是在大脑和面部发育方面。发育中的胚胎对胆固醇的需求与补丁刺猬信号通路有关，我们之前已经证明，该信号通路是哺乳动物发育和肿瘤形成的中心。特别是，负责通过这种复杂的蛋白质相互作用级联来调节信号的补丁蛋白具有与其他蛋白质中被证明检测和响应细胞内胆固醇水平的结构域相似的结构域。补丁蛋白与细胞表面的刺猬结合，并介导刺猬信号的转导进入细胞。通过类似于类固醇敏感结构域在其他蛋白质中的作用，我们假设Patted中的这个结构域检测细胞内胆固醇水平的波动，这反过来又改变了Patted到细胞表面的运输，在那里它可以参与刺猬受体复合体。这一假说得到了我们的初步数据的支持，该数据表明Patted通常定位于细胞表面和细胞内。我们提出了一系列实验来验证我们的假设，其中大部分实验涉及确定Patted在细胞培养系统中的定位，这些细胞培养系统暴露于旨在改变细胞内胆固醇水平的试剂。免疫荧光、电子显微镜和免疫印迹分析将分析Patched的亚细胞定位。我们还将测试Patted与其他在接收和发送刺猬信号方面至关重要的分子在细胞表面聚集的能力。这项提议中的实验很可能给出第一个线索，即在Patted中类固醇敏感结构域的功能，以及它在调节胆固醇和胚胎发育之间的重要联系中的作用。

Fluctuations in levels of cholesterol during development of the mammalian embryo have been shown to have catastrophic affects leading to gross deformities particularly in terms of brain and facial development. The requirement of the developing embryo for cholesterol has been linked to the patched-hedgehog signalling pathway which we have previously shown to be central to mammalian development as well as tumour formation. In particular, the patched protein which is responsible for regulating signalling through this complex cascade of protein interactions has a domain similar to that which in other proteins has been shown to detect and respond to intracellular levels of cholesterol. The patched protein binds to hedgehog at the surface of the cell and mediates the transduction of the the hedgehog signal into the cell. By analogy to the role of sterol sensing domains in other proteins, we hypothesise that this domain in patched detects fluctuations in intracellular cholesterol levels which in turn alter trafficking of patched to the cell surface where it can participate in the hedgehog receptor complex. This hypothesis is supported by our preliminary data which suggests that patched is normally localised both at the cell surface and intracellularly. We are proposing a series of experiments to test our hypothesis, most of which deal with determing the localisation of patched in a cell culure system exposed to agents aimed at varying the intracellular levels of cholesterol. Subcellular localisation of patched will be analysed by immunofluorescence, electron microscopy and immunoblotting analysis. We will also test the ability of patched to aggregate at the cell surface with other molecules important in receiving and sending the hedgehog signal. The experiments in this proposal are likely to give the first clues as to the function of the sterol sensing domain in patched and its role in mediating the vital link between cholesterol and embryonic development.