Characterization of a soluble form of Sonic Hedgehog

描述(由申请人提供)：Sonic Hedgehog(Shh)信号通路的人类成分在癌症的发病机制中发挥着重要作用。这一发现也许并不令人惊讶，因为这一途径在果蝇的细胞增殖和细胞命运决定中发挥着重要作用。人类的SHH、FTC、SMO和GLI-1基因都与肿瘤的发生有关，这四个基因都是人类Shh信号通路的成员。基于这些数据，我们推测Shh信号通路的扰动是肿瘤发生过程中的一个关键事件。这些基因产物促进肿瘤进展的机制尚不清楚。我们研究的目的是通过首先阐明Shh通路的正常信号通路，来阐明Shh通路在人类肿瘤发生中是如何被篡夺的。Shh发挥生物学效应的能力受一系列翻译后过程的调控。这些过程包括分子内裂解、胆固醇和/或棕榈酸酯的共价加成，以及转化为多膜自由扩散形式。Shh的加工影响其运输、效力和在许多细胞直径上的信号传递能力。因此，这些过程所需的基因产物的丢失会使HH蛋白丧失发挥其生物学效应的能力。虽然来自一些实验系统的令人信服的证据现在支持HH家族成员直接发挥其许多长期影响的想法，但关于HH的天然形式可以自由传播的证据一直缺失。S-ShhNp的发现提供了这样一个缺失的环节，因为我们假设S-ShhNp是负责远程信号传递的生理相关形式的Shh。这种可自由扩散的Shh是胆固醇修饰的、多聚体和生物有效的。我们建议，在这里，描述S-ShhNp，并阐明脂类修饰和蛋白质-蛋白质相互作用对多聚体的重要作用。此外，我们还将对S-ShhNp进行纯化，以确定其蛋白质组成，因为S-ShhNp中可能存在除Shh以外的额外蛋白质，以及翻译后修饰。完成后，这项工作的结果将阐明Shh不寻常的生物化学如何转化为Shh在长距离、短距离或两者兼而有之地发挥其生物效应的能力。

DESCRIPTION (provided by applicant): Human components of the Sonic Hedgehog (Shh) signaling pathway play an important role in the pathogenesis of cancer. This finding was perhaps not surprising given the significant role this pathway plays in cellular proliferation and cell fate determination in Drosophila. The human genes SHH, FTC, SMO and GLI- 1 have all been implicated in oncogenesis, all four are members of the human Shh signaling pathway. Based on these data, we speculate that perturbation of the Shh signaling pathway is a critical event during tumorigenesis. The mechanisms by which these gene products contribute to tumor progression remain unknown. The goal of our research is to elucidate how the Shh pathway is usurped in human oncogenesis, by first elucidating its normal signaling pathway. The ability of Shh to exert its biological effects is regulated by a series of post-translational processes. These processes include an intramolecular cleavage, covalent addition of cholesterol and/or palmitate, and conversion into a multimenc freely diffusible form. The processing of Shh affects its trafficking, potency, and ability to signal over many cell diameters. Accordingly, the loss of gene products required for these processes abrogates the ability of the Hh proteins to exert their biological effects. While convincing evidence from a number of experimental systems now support the idea that Hh family members exert many of their long-range effects directly, the evidence for a native form of Hh that is freely diffusible had been missing. The discovery of s-ShhNp provided such a missing link, as we hypothesize that s-ShhNp is the physiologically relevant form of Shh responsible for long-range signaling. This freely diffusible form of Shh is cholesterol modified, multimeric and biologically potent. We propose here, to characterize s-ShhNp, and to elucidate the lipid modifications and protein-protein interactions important for multimerization. Additionally, we will purify s-ShhNp to homogeneity to identify its protein composition, as there may be additional proteins in s-ShhNp besides Shh, and post-translational modifications. Upon its completion, results from this work will have elucidated how the unusual biochemistry of Shh is translated into the ability of Shh to exert its biological effects at a long range, short range, or both.