Structural and Biophysical Characterization of Hedgehog Signaling

描述（由申请人提供）：刺猬（Hh）是一种分泌的信号蛋白，在动物发育过程中许多组织的模式中起关键作用。Hh通过在特化组织中表达并以浓度依赖的方式诱导邻近组织中的细胞生长和分化，符合形态因子的经典定义。不适当的Hh信号导致胚胎严重的发育异常，并与成人的许多致命癌症有关。由于其强大的模式活动，Hh和Hh反应性的分布受到严格调节。例如，Hh被双重脂化并包装成脂蛋白颗粒以供分泌，并且需要硫酸肝素蛋白聚糖来运输Hh跨越多个细胞层。至少有5种完整的膜蛋白（Patched、Smoothened、Ihog、dally样蛋白和刺猬相互作用蛋白）参与Hh应答细胞中Hh信号的转导或调节。我们研究的总体目标是了解Hh信号中涉及的分子相互作用以及这些相互作用在正常发育过程中如何被调节。通过了解正常情况下这些相互作用的性质，我们也希望了解该途径在疾病中是如何被激活的，以及在这种情况下如何最好地设计针对Hh途径的抑制剂。Ihog是一种I型整体膜蛋白，在其细胞外区域具有多个免疫球蛋白和III型纤维连接蛋白（FNIII）重复序列。Hh已被证明在肝素存在的情况下与Ihog FNIII重复序列之一直接相互作用，我们最近确定了果蝇Hh和Ihog FNIII结构域复合物的晶体结构。不幸的是，肝素没有在这种结构中可视化，我们的第一个目标是使用生化，生物物理和结构方法来表征肝素在介导Hh和Ihog之间相互作用中所涉及的性质和作用。奇怪的是，脊椎动物Ihog同源物上的Hh结合位点发生在与果蝇Ihog不同的Ihog FNIII结构域上，我们的第二个目标是表征脊椎动物Hh和Ihog同源物之间的相互作用，并确定这种相互作用对肝素或其他辅助因子的依赖性。我们的最终目标是表达和纯化参与Hh反应的每个细胞表面成分的细胞外区域，并通过生化，生物物理和结构手段研究这些成分与Hh之间任何相互作用的性质。

DESCRIPTION (provided by applicant): Hedgehog (Hh) is a secreted signaling protein that plays a key role in the patterning of many tissues during animal development. Hh fits the classical definition of a morphogen by being expressed in specialized tissues and inducing cell growth and differentiation in neighboring tissues in a concentration dependent manner. Inappropriate Hh signaling leads to severe developmental abnormalities in embryos and is associated with many lethal cancers in adults. Because of its potent patterning activity, the distribution of both Hh and Hh responsiveness is tightly regulated. For example, Hh is dually lipidated and packaged into lipoprotein particles for secretion, and heparan sulfate proteoglycans are required for transport of Hh across multiple cell layers. At least 5 integral membrane proteins - Patched, Smoothened, Ihog, Dally-like protein, and Hedgehog-interacting protein - have been implicated in transducing or modulating Hh signals in Hh responsive cells. The overall goal of our studies is to understand the molecular interactions involved in Hh signaling and how these interactions are regulated during normal development. By understanding the nature of these interactions in normal circumstances, we also hope to understand how the pathway becomes activated in disease and how best to design inhibitors targeting the Hh pathway in such cases. Ihog is a type I integral membrane protein with multiple immunoglobulin and fibronectin type III (FNIII) repeats in its extracellular region. Hh has been shown to interact directly with one of the Ihog FNIII repeats in the presence of heparin, and we recently determined the crystal structure of a complex of Drosophila Hh and the Ihog FNIII domains. Heparin was unfortunately not visualized in this structure, and our first aim is to use biochemical, biophysical, and structural approaches to characterize the nature and role of the heparin involved in mediating interactions between Hh and Ihog. Curiously, the Hh binding site on vertebrate Ihog homologs occurs on a different Ihog FNIII domain than is observed for Drosophila Ihog, and our second aim is to characterize the interactions between vertebrate Hh and Ihog homologs and identify any dependence of this interaction on heparin or other co-factors. Our final aim is to express and purify the extracellular regions of each of the cell-surface components involved in Hh responsiveness and investigate the nature of any interactions among these components and Hh by biochemical, biophysical, and structural means.