RECONSTITUTION OF HEDGEHOG SIGNALING COMPLEX

Hedgehog(HH)信号转导通路组件的突变是许多人类发育障碍的基础，并有助于多种肿瘤的发生。戈林氏综合征是这一途径在发育和癌症中所起作用的一个例子。除了许多发育异常外，患有这种疾病的人还具有基底细胞癌和各种原始神经外胚层肿瘤(如髓母细胞瘤)的遗传易感性。类似的成分也发现在这些相同的恶性肿瘤的零星形式中发生突变，或者在胶质母细胞瘤中高度放大。我们的长期目标是阐明这一信号转导途径是如何在这些不同的人类病理中被篡夺的。Hedgehog的信号转导通过一个大型的多组分微管结合蛋白复合体进行，当该复合体被破坏时，就会阻止信号传递。Hedgehog信号复合体(HSC)是一个约1000 kDa的多蛋白复合体，由3个已知成员组成，即蛋白激酶融合蛋白(FU)、转录因子Cubitus interuptus(Ci)和激动素样蛋白Costa2(Cos2)。除了Fu、Ci和Cos2，我们假设HSC包含一些未知的但功能重要的蛋白质成分。本项目的目标是确定这些组成部分，并评估它们在HSC职能和组织中的作用。我们研究的具体目的是：1)利用杆状病毒表达系统为HSC成员建立体外重建系统，这将使我们能够大量分离各种HSC组分的修饰形式，用于各种体外研究；2)鉴定HH依赖的信号复合体的更多成员。这将通过结合亲和层析和传统层析以及使用候选基因方法来完成。识别这些其他成分将有助于深入了解这台大型蛋白质机器在HH信号转导中所起的作用。它还将提供必要的框架，以确定人类HSC的同源基因，并阐明它们在肿瘤发生中的作用(在未来的工作中)。

Mutations in components of the Hedgehog (Hh) signal transduction pathway underlie a number of human developmental disorders, and contribute to a diverse array of tumors. Gorlin's syndrome is one example of the role this pathway plays in both development and cancer. Besides numerous developmental abnormalities, individuals afflicted with this disorder have an inherited predisposition to basal cell carcinoma as well as to a variety of primitive neuroectodermal tumors, such as medulloblastoma. Similar components are also found mutated in sporadic forms of these same malignancies, or highly amplified in glioblastoma. Our long-term goals are to elucidate how this signal transduction pathway is usurped in these different human pathologies. Hedgehog signal transduction occurs through a large multi-component microtubule-bound protein complex, that when disrupted blocks signaling. This Hedgehog signaling complex (HSC) is an approximately 1000 kDa multi-protein complex with three known members, the protein kinase Fused (Fu), the transcription factor Cubitus Interuptus (Ci), and the kinesin-like protein Costal2 (Cos2). In addition to Fu, Ci, and Cos2, we hypothesize that the HSC contains a number of as yet unknown, but functionally important protein components. The goals of this project are to identify these components, and evaluate their role in HSC function and organization. The specific aims of our studies are: 1) Develop an in vitro reconstitution system for members of the HSC using a baculovirus expression system, this will allow us to isolate modified forms of the various HSC components in large amounts for various in vitro studies; 2) Identify additional members of a Hh dependent signaling complex. This will be done through a combination of both affinity and conventional chromatography, as well as using a candidate gene approach. Identifying these other components will provide insight into the role this large protein machine plays in Hh signal transduction. It will also provide the framework necessary to identify the human orthologs of the HSC, and elucidate their role in oncogenesis (in future work).