Biochemical and cell biological mechanisms of signal transduction through the Hedgehog pathway

 描述(申请人提供)：Hedgehog(HH)途径是一种细胞间通讯系统，在发育、再生和癌症中发挥重要作用。HH信号是在脊椎动物的初级纤毛上协调的，初级纤毛是一种天线状的细胞器，从我们身体大多数细胞的表面伸出，在发育过程中充当信号中心。纤毛基因的突变导致了许多被称为“纤毛疾病”的遗传性人类疾病，其中许多疾病的特征是出生缺陷和其他可归因于HH信号异常的表型。尽管它在癌症和再生医学中是一个重要的靶点，但HH信号的许多步骤在生化和细胞生物学水平上仍然知之甚少。我的研究计划集中在脊椎动物HH途径中尚未解决的主要机制问题上，特别强调介导信号传播和转录激活的纤毛机制。HH信号通过7次跨膜蛋白Smo传递，Smo是所有临床使用的抗HH药物的靶点。然而，我们不了解Smo被HH配体激活的机制，也不知道激活的Smo如何反过来向胶质母细胞瘤(Gli)转录因子家族发出信号。初级纤毛在这两个步骤中都扮演着重要的角色--HH配体促进Smo在纤毛膜上的积累，这是信号传递的关键步骤，最终导致G1蛋白在通过纤毛室时被激活。阐明这一机制将为TM受体如何将信号从纤毛膜传递到细胞核提供一个有价值的范例。正在研究的三个主要问题是：(1)主要的HH受体1如何通过内源性小分子配体激活Smo；(2)纤毛膜上激活的Smo如何向Gli蛋白传递信号；(3)Gli蛋白如何转化为转录激活剂。我们在这些领域的每一个方面都取得了进展。利用新的化学工具来探索Smo与氧固醇类内源性脂类之间的相互作用，我们鉴定并鉴定了Smo中一个先前未知的具有调节潜力的配体结合部位。利用比较蛋白质组学，我们鉴定了一个纤毛膜蛋白复合体，它与纤毛上的Smo结合以响应HH信号，并且是Smo信号所必需的。最后，我们对在Gli激活中起关键作用的动态磷酸化和蛋白质结合事件进行了表征。我们的工作得到了与在合成化学、结构生物学、质谱学和胚胎学方面拥有专业知识的研究人员的富有成效的合作的支持。这个项目的成功完成将提供(1)Smo调节问题的答案，这可能是HH途径中存在时间最长的谜团，(2)了解纤毛蛋白运输如何驱动信号传递，以及这些过程在纤毛疾病中是如何被破坏的，以及(3)在HH相关疾病中监测和调节该途径的新策略。

 DESCRIPTION (provided by applicant): The Hedgehog (Hh) pathway is a cell-cell communication system that plays important roles in development, regeneration and cancer. Hh signaling is orchestrated in vertebrates at primary cilia, antenna-like organelles that project fro the surfaces of most cells in our bodies and serve as signaling centers in development. Mutations in cilia genes cause a number of inherited human diseases called "ciliopathies," many of which are characterized by birth defects and other phenotypes attributable to aberrant Hh signaling. Despite its importance as a target in cancer and regenerative medicine, many of the steps in Hh signaling remain poorly understood at the biochemical and cell biological level. My research program is focused on the major unsolved mechanistic questions in the vertebrate Hh pathway, with a particular emphasis on ciliary mechanisms that mediate signal propagation and transcriptional activation. The Hh signal is transmitted across the membrane by the 7-pass transmembrane protein Smo, the target for all anti-Hh drugs in clinical use. However, we do not understand the mechanism by which Smo is activated in response to Hh ligands, nor do we know how activated Smo in turn signals to the Glioblastoma (Gli) family of transcription factors. Primary cilia play an important role in both steps- Hh ligands promote the accumulation of Smo in the ciliary membrane, a critical step in signaling that eventually leads to the activation of Gl proteins as they traffic through the ciliary compartment. Delineating this mechanism will provide a valuable paradigm for how TM receptors relay signals from the ciliary membrane to the nucleus. Three major questions under investigation are (1) how Smo is activated by the main Hh receptor Patched 1 through endogenous small molecule ligands, (2) how activated Smo in the ciliary membrane transmits signals to the Gli proteins and (3) how Gli proteins are converted into transcriptional activators. We have made progress in each of these areas. Using new chemical tools to probe the interaction between Smo and oxysterols, endogenous lipids that can activate Hh signaling, we identified and structurally characterized a previously unknown ligand-binding site with regulatory potential in Smo. Using comparative proteomics, we identified a ciliary membrane protein complex that engages Smo at cilia in response to Hh signals and is required for Smo signaling. Finally, we have characterized both dynamic phosphorylation and protein association events that play critical role in Gli activation. Our work is supported by productive collaborations with investigators who have expertise in synthetic chemistry, structural biology, mass spectrometry and embryology. The successful completion of this project will provide (1) an answer to the question of Smo regulation, perhaps the longest-standing mystery in the Hh pathway, (2) an understanding of how ciliary protein trafficking drives signaling and how these processes are corrupted in ciliopathies, and (3) new strategies to monitor and modulate the pathway in Hh-related diseases.