RECONSTITUTION OF HEDGEHOG SIGNALING COMPLEX

Hedgehog（Hh）信号转导通路的组分中的突变是许多人类发育障碍的基础，并导致多种肿瘤。 Gorlin综合征是这种途径在发育和癌症中发挥作用的一个例子。 除了许多发育异常外，患有这种疾病的个体还具有遗传性的基底细胞癌易感性以及各种原始神经外胚层肿瘤，如髓母细胞瘤。在这些相同恶性肿瘤的散发形式中也发现类似的组分突变，或在胶质母细胞瘤中高度扩增。我们的长期目标是阐明这种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信号转导通过一个大的多组分微管结合蛋白复合物发生，当被破坏时会阻断信号传导。这种Hedgehog信号复合物（HSC）是一种约1000 kDa的多蛋白复合物，具有三个已知成员，蛋白激酶融合（Fu）、转录因子Cubitus Interuptus（Ci）和驱动蛋白样蛋白Costal 2（Cos 2）。 除了Fu，Ci和Cos 2，我们假设HSC含有一些未知的，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，并评估其在HSC功能和组织中的作用。 我们研究的具体目的是：1）使用杆状病毒表达系统开发HSC成员的体外重建系统，这将允许我们大量分离各种HSC组分的修饰形式用于各种体外研究; 2）鉴定Hh依赖性信号传导复合物的其他成员。这将通过亲和层析和常规层析的组合以及使用候选基因方法来完成。 识别这些其他组件将提供洞察这个大蛋白机器在Hh信号转导中发挥的作用。 它还将提供必要的框架，以确定人类的HSC直系同源物，并阐明其在肿瘤发生中的作用（在未来的工作）。

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).