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Structual & Biophysical Characterization of Hedgehog Signaling

结构性

基本信息

批准号：
8692227
负责人：
DANIEL J LEAHY
金额：
$ 4.2万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8692227
关键词：
Adhesions Affinity Animals Binding Biochemical Biological Assay Breast Calcium Cell Differentiation process Cell Proliferation Cell Surface Proteins Cell surface Cells Clinical Trials Co-Immunoprecipitations Colon Complex Conflict (Psychology)Congenital Abnormality Core Protein Defect Development Differentiation and Growth Drosophila genus Erinaceidae Event Fibronectins Genetic Glycosylphosphatidylinositols Glypican Goals Growth Factor Receptors Heparan Sulfate Proteoglycan Heparin Homologous Gene Homologous Protein Human Immunoglobulins In Vitro Integral Membrane Protein Invertebrates Label Laboratories Lead Lung Malignant Neoplasms Malignant neoplasm of brain Maps Measures Mediating Molecular Nature Organism Pancreas Pathway interactions Pattern Polysaccharides Positioning Attribute Process Protein Binding Proteins Reporting Roentgen Rays Role Signaling Molecule Site Site-Directed Mutagenesis Skin Structure Surface Therapeutic Tissues Vertebrates Work X-Ray Crystallography base cancer therapy cell type fly glypican 3 inhibitor/antagonist insight meetings milligram morphogens patched protein receptor reconstitution research study smoothened signaling pathway therapeutic target transmission process vertebrate hedgehog protein

项目摘要

Hedgehog proteins are secreted signaling molecules that regulate the growth and differentiation of cells during animal development. Hedgehogs meet the classical definition of a morphogen by forming gradients of decreasing concentration that emanate from sites of secretion and induce differentiation of distinct cell types at different positions along the gradient. In this manner Hedgehog proteins mediate the formation of complex tissue patterns. Abnormal Hedgehog signaling can result in severe developmental defects and cancer, and clinical trials assessing the efficacy of Hedgehog signaling inhibitors are underway in cancers of the brain, breast, lung, colon, pancreas, and skin. Perhaps owing to its importance for patterning multiple tissues, the activity of the Hedgehog signaling pathway is tightly regulated. Hedgehog proteins appear to interact with multiple protein and glycan partners that modulate its activity, distribution, or both. The 12-pass integral membrane protein Patched is required for normal reception of Hedgehog signals and has been widely identified as the "Hedgehog receptor", but evidence for direct binding of Hedgehog to Patched is absent in flies and indirect in vertebrates. Several additional cell-surface proteins have been shown to bind directly to Hedgehog proteins with high affinity including Ihog in flies and Cdo, Boc, and Hedgehog-interacting protein in vertebrates. Others, including the protein cores of glypicans and a vertebrate-specific protein Gas1, associate with Hedgehog proteins with weaker affinity or as part of larger complexes and have been suggested as co- receptors for Hedgehog proteins. We have expressed and purified active fragments of Hedgehog, Ihog, Cdo, Boc, glypican, and Gas1 proteins, determined crystal structures of Hh:Ihog, Hh:Cdo, and Hh:Boc complexes, and demonstrated that these interactions depend on additional co-factors including heparin and Ca2+. We have also recently expressed and purified small amounts (~40 ug) of Patched. To investigate the molecular mechanisms governing Hedgehog signaling and understand the nature of the Hedgehog receptor at the molecular level we propose X-ray crystallographic, biophysical, and biochemical studies of these Hh pathway components and their complexes. These studies will elucidate the molecular mechanisms governing activity of the Hedgehog signaling pathway and thus the molecular strategies employed to generate complex tissue pattern. Such studies are also likely to uncover molecular interactions or processes that may be targeted for therapeutic inhibition of Hedgehog signaling. We will achieve these goals by pursuing the following specific aims: (1) determine the molecular mechanisms by which glypicans modulate vertebrate and invertebrate Hedgehog signaling, (2) determine the molecular mechanisms by which Gas1 modulates vertebrate Hedgehog signaling and the interplay between Gas1 and other modulators of Hedgehog signaling, and (3) determine the nature of vertebrate and invertebrate Hedgehog receptor complexes by reconstituting the minimal Hedgehog binding complex in vitro with purified components.

Hedgehog蛋白是分泌的信号分子，在动物发育过程中调节细胞的生长和分化。刺猬符合形态发生素的经典定义，通过形成从分泌位点发出的浓度递减的梯度，并诱导沿梯度不同位置的不同细胞类型的分化。以这种方式，刺猬蛋白介导复杂组织模式的形成。异常的 Hedgehog 信号传导可能导致严重的发育缺陷和癌症，评估 Hedgehog 信号传导抑制剂在脑癌、乳腺癌、肺癌、结肠癌、胰腺癌和皮肤癌中的功效的临床试验正在进行中。也许由于其对多种组织模式的重要性，Hedgehog 信号通路的活性受到严格调控。 Hedgehog 蛋白似乎与多种蛋白质和聚糖伴侣相互作用，调节其活性、分布或两者。正常接收 Hedgehog 信号需要 12 遍整合膜蛋白 Patched，并且已被广泛鉴定为“Hedgehog 受体”，但在果蝇中不存在 Hedgehog 与 Patched 直接结合的证据，而在脊椎动物中则没有间接结合的证据。几种其他细胞表面蛋白已被证明能够以高亲和力直接与 Hedgehog 蛋白结合，包括果蝇中的 Ihog 以及脊椎动物中的 Cdo、Boc 和 Hedgehog 相互作用蛋白。其他蛋白，包括磷脂酰肌醇蛋白核心和脊椎动物特异性蛋白 Gas1，以较弱的亲和力与 Hedgehog 蛋白结合或作为较大复合物的一部分，并被建议作为 Hedgehog 蛋白的共受体。我们表达并纯化了 Hedgehog、Ihog、Cdo、Boc、磷脂酰肌醇蛋白聚糖和 Gas1 蛋白的活性片段，确定了 Hh:Ihog、Hh:Cdo 和 Hh:Boc 复合物的晶体结构，并证明这些相互作用依赖于其他辅助因子，包括肝素和 Ca2+。我们最近还表达并纯化了少量（约 40 ug）的 Patched。为了研究控制 Hedgehog 信号传导的分子机制并在分子水平上了解 Hedgehog 受体的性质，我们建议对这些 Hh 通路成分及其复合物进行 X 射线晶体学、生物物理和生化研究。这些研究将阐明控制 Hedgehog 信号通路活性的分子机制，从而阐明用于生成复杂组织模式的分子策略。此类研究还可能揭示分子相互作用或过程，这些相互作用或过程可能是刺猬信号传导治疗抑制的目标。我们将通过追求以下具体目标来实现这些目标：（1）确定磷脂酰肌醇蛋白聚糖调节脊椎动物和无脊椎动物 Hedgehog 信号传导的分子机制，（2）确定 Gas1 调节脊椎动物 Hedgehog 信号传导的分子机制以及 Gas1 与 Hedgehog 信号传导的其他调节剂之间的相互作用，以及（3）确定脊椎动物和无脊椎动物的性质 Hedgehog 受体复合物是通过用纯化的成分在体外重建最小的 Hedgehog 结合复合物来实现的。