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.
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