Structural and Biophysical Characterization of Hedgehog Signaling

描述（由申请人提供）：刺猬蛋白是动物发育过程中分泌的调节细胞生长和分化的信号分子。刺猬符合形态原的经典定义，它们在分泌部位形成浓度递减的梯度，并在梯度的不同位置诱导不同类型的细胞分化。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疗效的临床试验正在进行中。可能由于其对多种组织的重要意义，Hedgehog信号通路的活性受到严格调控。刺猬蛋白似乎与多种蛋白和聚糖伙伴相互作用，调节其活性、分布或两者兼而有之。12过的完整膜蛋白Patched是正常接收刺猬信号所必需的，被广泛认为是“刺猬受体”，但在苍蝇和脊椎动物中没有证据表明Hedgehog与Patched直接结合，而在脊椎动物中则间接结合。一些其他的细胞表面蛋白已被证明可以直接与高亲和力的刺猬蛋白结合，包括苍蝇中的Ihog和脊椎动物中的Cdo、Boc和刺猬相互作用蛋白。其他的，包括glypicans的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，被认为是Hedgehog蛋白的共受体。我们表达并纯化了Hedgehog、Ihog、Cdo、Boc、glypican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，并证明了这些相互作用依赖于其他辅助因子，包括肝素和Ca2+。我们最近也表达和纯化了少量的patch （~40 ug）。为了研究调控Hedgehog信号传导的分子机制，并在分子水平上理解Hedgehog受体的性质，我们提出对这些Hh通路组分及其复合物进行x射线晶体学、生物物理和生化研究。这些研究将阐明调控Hedgehog信号通路活性的分子机制，从而阐明产生复杂组织模式的分子策略。这样的研究也可能揭示分子相互作用或过程，可能是靶向治疗抑制刺猬信号传导。为达致这些目标，我们将致力达致以下具体目标：(1)确定glypicans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与其他Hedgehog信号传导调节剂之间的相互作用；(3)通过纯化组分体外重组最小的Hedgehog结合复合物，确定脊椎动物和无脊椎动物Hedgehog受体复合物的性质。

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.