Biochemical Mechanisms of Hedgehog Signaling

描述（由申请人提供）：Hedgehog信号通路的异常激活与多种器官系统中人类癌症的发生和维持有关，包括皮肤、脑、肺、前列腺和胰腺。据推测，该通路通过引起组织干细胞的激活和扩增来驱动肿瘤发生。我们对哺乳动物Hedgehog信号是如何被接收和转导的理解有很大的差距。破译刺猬信号传导的详细生化机制将有助于开发针对这些致命癌症的新疗法和预防策略。在Marc Kirschner博士的博士培训期间，我使用蛋白质生物化学和显微镜相结合的方法来剖析连接细胞表面信号和肌动蛋白细胞骨架的途径。为了将类似的生化和细胞生物学方法应用于刺猬信号传导，我开发了新的抗体和基于共聚焦显微镜的检测方法来研究该途径中蛋白质的动力学和相互作用。基于这项工作，我构建了一个新的刺猬信号模型，强调了初级纤毛的重要性，初级纤毛是大多数细胞表面上发现的一个微小的突起，最近与刺猬通路和人类疾病有关。在本提案的独立阶段，我计划使用上述工具来了解受体Patched 1在初级纤毛的定位如何影响其感知Sonic Hedgehog信号的能力，并激活小鼠胚胎成纤维细胞和人类肿瘤细胞的下游信号。我还将使用无偏倚的免疫亲和纯化方法来发现新的相互作用蛋白和翻译后修饰，将Hedgehog途径成分与初级纤毛联系起来。在该提案的K99阶段，人类和小鼠癌症中Hedgehog信号分析的先驱Matthew Scott博士将担任导师。我在他的实验室的时间将提供一个重要的机会，以获得在组织培养成纤维细胞、肿瘤细胞和小鼠中分析刺猬信号转导的技术经验，并为拟议的成像和蛋白质相互作用分析开发光学和生化探针。最重要的是，这将使我成为肿瘤科终身教授的理想人选，在那里我计划花80-90%的时间在研究和教学上，10-20%的时间在照顾癌症患者上。

DESCRIPTION (provided by applicant): Aberrant activation of the Hedgehog signaling pathway has been implicated in the genesis and maintenance of human cancers that develop in multiple organ systems, including the skin, brain, lung, prostate, and pancreas. It is hypothesized that the pathway drives tumorigenesis by causing the activation and expansion of tissue stem cells. There are significant gaps in our understanding of how the mammalian Hedgehog signal is received and transduced. Deciphering the detailed biochemical mechanism of Hedgehog signaling will allow the development of novel therapeutics and preventative strategies for these lethal cancers. During my PhD training with Dr. Marc Kirschner, I used a combination of protein biochemistry and microscopy to dissect a pathway that links cell surface signals to the actin cytoskeleton. To apply a similar biochemical and cell-biological approach to Hedgehog signaling, I have developed novel antibodies and confocal microscopy- based assays to study the dynamics and interactions of proteins in the pathway. Based on this work, I have constructed a new model for Hedgehog signaling that highlights the importance of the primary cilium, a tiny projection found on the surface of most cells that has been recently implicated in the Hedgehog pathway and in human disease. In the independent phase of this proposal, I plan to use the above tools to understand how localization of the receptor, Patched 1, to the primary cilium affects its ability to sense the Sonic Hedgehog signal and to activate downstream signaling in mouse embryonic fibroblasts and human tumor cells. I will also use an unbiased immunoaffinity purification approach to discover novel interacting proteins and post-translational modifications that link Hedgehog pathway components to the primary cilium. In the K99 phase of this proposal, Dr. Matthew Scott, a pioneer in the analysis of Hedgehog signaling in human and mouse cancer, will serve as mentor. My time in his laboratory will provide a critical opportunity to gain experience in techniques for the analysis of Hedgehog signal transduction in tissue culture fibroblasts, tumor cells and mice and to develop optical and biochemical probes for the proposed imaging and protein- interaction analysis. Most importantly, it will put me in an ideal position for a tenure-track position in an oncology department, where I plan to spend ~80-90% of my time in research and teaching and 10-20% of my time in the care of cancer patients.