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Title: The role of hedgehog acyltransferase & heparan sulphate proteoglycans in human sonic hedgehog signalling
Author: Chang, Shu-Chun
ISNI:       0000 0004 2703 6863
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Hedgehog proteins (Hh) are morphogens and major mediators in many developmental processes. Hh signalling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancers. Hh proteins require dual lipidation and Heparan Sulfate Proteoglycans (HSPGs) for their proper distribution and signalling activity. My first aim was to study the role of HSPGs in human (h) Sonic Hedgehog (Shh) signalling and clarify the biological function of hShh/HSPGs complexes in hShh signalling, by investigating the interaction between human hShh and HSPGs. I used DNA mutagenesis and heparin affinity chromatography to determine key residues in hShh involved in heparin binding (K37/38 and K178). The activity of these mutants was tested by detecting induced Alkaline Phosphatase activity in C3H10T1/2 cells and hShh-inducible gene expression in PANC1 human pancreatic carcinoma cells. I examined the biological function of mutated hShhs (K37/38S, K178S and K37/38/178S) that cannot interact with heparin efficiently and showed that they had reduced signalling activity compared to wild type hShh and a control mutation (K74S). Also, I showed that mutant hShh proteins mediate reduced proliferation and invasion of PANC1 cells following hShh RNAi knockdown (KD), and this correlated with reduced Shh multimeric complex formation. Structurally, Shh proteins are unusual in being dually lipid-modified to be fully active. During the post-translational modifications of Shh, N-terminal palmitoylation is facilitated by the product (Hhat) of the hedgehog acyltransferase gene. I have carried out a thorough analysis of Hhat in PANC1 cells. First, I characterised an antibody prepared in the lab to hHhat. I confirmed the specificity of the antibody by immunoblotting using a self-constructed hHhat-EGFP clone, and a control mGup1-EGFP clone. By subcellular fractionation and Western blotting I found Hhat to be a membrane protein. In addition, I used the hHhat antibody to determine the intracellular localisation of hHhat in PANC1 cells by confocal microscopy and showed that hHhat localised in ER mainly but not in Golgi apparatus. I confirmed this using the hHhat-EGFP clone for fluorescence microscopy in transfected cells. To illuminate the biological function of palmitoylation of hShh in production of active hShh and in the formation of hShh multimeric complex I optimised hHhat RNAi knockdown (KD) in PANC1 cells and confirmed this by a cell-based palmitoylation assay. Using semi-quantitative RT-PCR and immunoblot analyses, I showed that hHhat KD caused decreased signalling through the Shh pathway due to reduced production of active hShh. In addition, I investigated the effect of the addition of palmitate to hShh on its association with cells by comparing hHhat KD cells with control cells. Immunoblotting suggests that palmitoylation of Shh improves its ability to associate to cell membranes. Using hHhat KD, gel filtration of high molecular weight complexes of hShh and immunoblotting of hShh I characterised the role of palmitoylation of hShh in multimeric complex formation. Lastly, I investigated the effect of hHhat KD on PANC1 proliferation and invasion, showing that it represses PANC1 proliferation and invasion. These studies provide a firm basis for understanding the functional roles of hShh palmitoylation and its interactions with HSPGs, and provide proof-of-principle for targeting these aspects of hShh biology in tumour cell therapeutics, specifically in the pancreatic carcinoma context.
Supervisor: Magee, Tony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral