Structural and Biophysical Characterization of Hedgehog Signaling

Hedgehog 信号传导的结构和生物物理表征

• 批准号: 7247440
• 负责人: DANIEL J LEAHY
• 金额: $ 27.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7247440
• 关键词: Adult; Affinity; Amino Acid Sequence Homology; Animals; Area; Binding; Binding Sites; Biochemical; Buffers; Cell Surface Proteins; Cell surface; Cells; Chemicals; Collaborations; Complex; Condition; Crystallization; Dependence; Development; Dimerization; Drosophila genus; Effectiveness; Embryo; Erinaceidae; Fibronectins; Goals; Heparan Sulfate Proteoglycan; Heparin; Heparin Binding; Heterogeneity; Homologous Gene; Immunoglobulins; Inorganic Sulfates; Institutes; Integral Membrane Protein; Laboratories; Length; Ligands; Lipoproteins; Malignant Neoplasms; Mediating; Molecular; Mutate; Nature; Oligosaccharides; Pathway interactions; Pattern; Play; Proteins; Relative (related person); Research Personnel; Roentgen Rays; Role; Screening procedure; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Sodium Chloride; Structure; Tissues; Unspecified or Sulfate Ion Sulfates; Vitelliform macular dystrophy; Work; base; cell growth; design; extracellular; human SHH protein; human SMO protein; inhibitor/antagonist; inorganic phosphate; morphogens; particle; programs; receptor; response; smoothened signaling pathway

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.

描述(申请人提供)：Hhedgehog(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发挥关键作用。HH在特定的组织中表达，并以浓度依赖的方式在邻近组织中诱导细胞生长和分化，符合形态发生的经典定义。不适当的HH信号会导致胚胎严重的发育异常，并与许多成人致命性癌症有关。由于其强大的构图活性，HH和HH响应性的分布受到严格调控。例如，HH被双重脂化并包装成脂蛋白颗粒进行分泌，而HH在多个细胞层的运输需要硫酸乙酰肝素蛋白多糖。至少有5种完整的膜蛋白--Patted、Smoothens、Ihog、Dally-like和Hedgehog相互作用蛋白--参与了HH反应细胞中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是如何调节的。通过了解这些相互作用在正常情况下的性质，我们也希望了解在疾病中该途径是如何被激活的，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型完整膜蛋白，胞外区有多种免疫球蛋白和纤维连接蛋白III(FNIII)重复序列。HH已被证明在肝素存在下与Ihog FNIII重复序列之一直接相互作用，我们最近确定了果蝇HH与Ihog FNIII结构域的复合体的晶体结构。不幸的是，肝素没有在这个结构中可视化，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参与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结合位点与果蝇Ihog上观察到的Ihog FNIII结构域不同，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是否依赖于肝素或其他辅助因子。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相互作用的性质。