Structural and Biophysical Characterization of Hedgehog Signaling

描述(申请人提供)：Hedgehog蛋白质是分泌的信号分子，在动物发育过程中调节细胞的生长和分化。刺猬符合形态素的经典定义，它从分泌部位产生浓度递减的梯度，并在沿梯度的不同位置诱导不同类型的细胞分化。通过这种方式，Hedgehog蛋白介导了复杂组织模式的形成。Hedgehog信号异常会导致严重的发育缺陷和癌症，评估Hedgehog信号抑制剂对脑癌、乳腺癌、肺癌、结肠癌、胰腺癌和皮肤癌疗效的临床试验正在进行中。也许由于其对多种组织构图的重要性，Hedgehog信号通路的活性受到严格调控。刺猬蛋白似乎与调节其活性、分布或两者兼而有之的多种蛋白质和葡聚糖伙伴相互作用。Hedgehog信号的正常接收所需的12遍完整膜蛋白Patted被广泛认为是“Hedgehog受体”，但在苍蝇中没有直接结合Hedgehog的证据，在脊椎动物中没有间接结合的证据。一些额外的细胞表面蛋白已被证明直接与Hedgehog蛋白高亲和力结合，包括苍蝇中的Ihog和脊椎动物中的CDO、Boc和Hedgehog相互作用蛋白。其他的，包括龟头鱼的蛋白质核心和脊椎动物特有的蛋白质Gas1，与Hedgehog蛋白质结合，亲和力较弱或作为较大复合体的一部分，已被认为是Hedgehog蛋白质的辅助受体。我们已经表达和纯化了Hedgehog、Ihog、CDO、Boc、Glypcan和Gas1蛋白的活性片段，测定了HH：IHOG、HH：CDO和HH：BOC复合体的晶体结构，并证明了这些相互作用依赖于额外的辅助因素，包括肝素和钙。我们最近还表达和纯化了少量(~40微克)的补丁。为了研究Hedgehog信号转导的分子机制，并在分子水平上了解Hedgehog受体的性质，我们对这些HH途径成分及其复合体进行了X射线结晶学、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，从而阐明用于产生复杂组织模式的分子策略。这类研究还可能揭示可能针对Hedgehog信号的治疗抑制的分子相互作用或过程。我们将通过以下具体目标来实现这些目标：(1)确定Glypicans调节脊椎动物和无脊椎动物Hedgehog信号的分子机制，(2)确定Gas1调节脊椎动物Hedgehog信号的分子机制以及Gas1与其他Hedgehog信号调节器之间的相互作用，以及(3)通过在体外用纯化的成分重组最小的Hedgehog结合复合体来确定脊椎动物和无脊椎动物Hedgehog受体复合体的性质。

DESCRIPTION (provided by applicant): 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.