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: 7840374
• 负责人: DANIEL J LEAHY
• 金额: $ 26.89万
• 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/7840374
• 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; 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（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着关键作用。 HH通过在专业组织中表达并以浓度依赖性方式诱导相邻组织的细胞生长和分化，从而适合形态学的经典定义。不适当的HH信号导致胚胎的严重发育异常，并且与成人的许多致命癌有关。由于其有效的图案活动，HH和HH响应性的分布都受到严格的调节。例如，将HH进行双重脂化并包装到脂蛋白颗粒中进行分泌，硫酸乙酰肝素蛋白聚糖是在多个细胞层上运输HH所必需的。至少有5种整合性膜蛋白（修补，平滑，IHOG，DALLY蛋白和刺猬相互作用蛋白）与HH响应细胞中的HH信号有关。我们研究的总体目标是了解HH信号传导所涉及的分子相互作用以及在正常发育过程中如何调节这些相互作用。通过了解在正常情况下这些相互作用的性质，我们也希望了解途径如何在疾病中激活，以及如何最好地设计针对HH途径的抑制剂。 IHOG是一种I型积分膜蛋白，在其细胞外区域具有多种免疫球蛋白和纤连蛋白III（FNIII）重复。 HH已被证明在存在肝素的情况下直接与IHOG FNIII重复进行了直接相互作用，并且我们最近确定了果蝇HH和IHOG FNIII域的复合物的晶体结构。不幸的是，在这种结构中没有看到肝素，我们的第一个目的是使用生化，生物物理和结构方法来表征与HH与IHOG之间介导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结合位点发生在不同的IHOG FNIII结构域上，与果蝇IHOG所观察到的不同，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HEPARIN或其他共同因素的任何依赖性。我们的最终目的是表达和净化与HH响应相关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和HH之间的任何相互作用的性质。