Biochemical Mechanisms of Hedgehog Signaling

Hedgehog信号通路的异常激活涉及在多个器官系统（包括皮肤、脑、肺、前列腺和胰腺）中发展的人类癌症的发生和维持。假设该途径通过引起组织干细胞的活化和扩增来驱动肿瘤发生。我们对哺乳动物Hedgehog信号如何被接收和转导的理解存在重大差距。破译Hedgehog信号传导的详细生化机制将允许开发针对这些致命癌症的新疗法和预防策略。在我与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的组合来剖析将细胞表面信号与肌动蛋白细胞骨架联系起来的途径。为了将类似的生物化学和细胞生物学方法应用于Hedgehog信号传导，我开发了新的抗体和基于共聚焦显微镜的方法。 分析以研究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，突出了初级纤毛的重要性，初级纤毛是一种在大多数细胞表面发现的微小突起，最近与刺猬信号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在初级纤毛上的定位如何影响其感知Sonic Hedgehog信号并激活小鼠胚胎成纤维细胞和人类肿瘤细胞中下游信号传导的能力。我还将使用一个公正的免疫亲和纯化方法，发现新的相互作用的蛋白质和翻译后修饰，连接刺猬途径组件的主要纤毛。在该提案的K99阶段，Matthew Scott博士，人类和小鼠癌症中Hedgehog信号传导分析的先驱，将担任导师。我在他的实验室的时间将提供一个关键的机会，以获得经验的技术分析刺猬信号转导在组织培养成纤维细胞，肿瘤细胞和小鼠，并开发光学和生化探针的建议成像和蛋白质相互作用分析。最重要的是，这将使我在肿瘤科获得终身职位的理想位置，我计划将80-90%的时间用于研究和教学，10-20%的时间用于护理癌症患者。

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 microscopybased 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 proteininteraction 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.