Structural and Biophysical Characterization of Hedgehog Signaling

描述（由申请人提供）：Hedgehog（Hh）是一种分泌型信号蛋白，在动物发育过程中许多组织的形成中起关键作用。Hh通过在特化组织中表达并以浓度依赖性方式在邻近组织中诱导细胞生长和分化而符合形态发生素的经典定义。不适当的Hh信号传导导致胚胎严重发育异常，并与成人的许多致命癌症有关。由于其有效的图案化活性，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用于分泌，并且硫酸乙酰肝素蛋白聚糖是Hh跨多个细胞层运输所需的。至少有5种膜蛋白- Patched、Smoothened、Ihog、Dally样蛋白和Hedgehog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参与Hh信号传导的分子相互作用，以及这些相互作用在正常发育过程中如何调节。通过了解正常情况下这些相互作用的性质，我们还希望了解该途径如何在疾病中被激活，以及如何在这种情况下最好地设计针对Hh途径的抑制剂。Ihog是一种I型整合膜蛋白，在其胞外区具有多个免疫球蛋白和III型纤连蛋白（FNIII）重复序列。Hh已被证明在肝素存在下直接与Igog FNIII重复序列之一相互作用，并且我们最近确定了果蝇Hh和Igog FNIII结构域的复合物的晶体结构。不幸的是，肝素在这种结构中没有可视化，我们的第一个目标是使用生物化学，生物物理学和结构方法来表征参与介导Hh和Ihog之间相互作用的肝素的性质和作用。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所观察到的不同的Ihog FNIII结构域上，并且我们的第二个目的是表征脊椎动物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因子的任何依赖性。我们的最终目的是表达和纯化的细胞表面的每个组件参与Hh的反应和研究的性质，这些组件和Hh之间的任何相互作用的细胞外区域的生化，生物物理和结构手段。

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.