Summary:
Hepcidin (HAMP) is a 25-aa peptide hormone that regulates iron homeostasis
through degradation of the only known cellular iron exporter ferroportin. The
present thesis presents our efforts to produce recombinant hepcidin in its
monomeric form and to investigate the putative regulation of hepcidin gene
expression by the Hepatitis C virus (HCV), during HCV infection of the liver.
Thus, the results are divided in two parts.
Firstly, we purified the soluble form of the recombinant peptide Hep25-His,
which carries a 6Ηis-tag, from the yeast P. pastoris. Hep25-His was used for
the quantitation of secreted hepcidin in hepatic cell cultures. We investigated
the contribution of lyophilization to aggregate formation during hepcidin
purification. We also tried to purify the recombinant peptide Hep25 using
specific monoclonal antibodies, but the isolation of its monomer through size
exclusive chromatography was not successful. In order to solve this problem, we
measured the elution volume of other compounds that were present in the eluate,
tested the separating efficiency of the column and changed the elution buffer
with a higher concentration salt solution.
In the second part, we investigated the HCV core protein-mediated regulation of
hepcidin gene expression in HepG2 hepatic cells. HCV core is a multifunctional
structural protein that plays important roles in HCV life cycle. We measured
the hamp gene expression and protein levels in core-expressing HepG2 cells with
qRT-PCR and competitive ELISA, respectively. Results demonstrate that HCV core
up-regulates hepcidin expression in a transcriptional manner. Western blot
analysis revealed differential modulation of other important components of iron
homeostasis, such as up-regulation of the iron storage protein ferritin and
down-regulation of TfR1, matriptase and ferroportin. Using specific inhibitors,
we demonstrated that HCV core increased hepcidin gene expression via a complex
signaling network that requires the involvement of BMP/SMAD and STAT3 signaling
pathways, as well as HCV core-mediated activation of CK2 kinase.
Keywords:
recombinant hepcidin, hamp gene, core protein, Hepatitis C, CK2 kinase
