The Role of Hedgehog Acyltransferase & Heparan Sulphate Proteoglycans in Human Sonic Hedgehog Signalling
Author(s)
Chang, Shu-Chun
Type
Thesis
Abstract
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.
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.
Date Issued
2011
Date Awarded
2011-04
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Magee, Tony
Creator
Chang, Shu-Chun
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)