Adams, AL;
(2014)
Developing Novel Peptide Ligation Methodology for the Synthesis and Labelling of Bioactive Hepcidin (Not applicable, , Trans.).
Doctoral thesis , UCL (University College London).
Abstract
Hepcidin is a 25 amino-acid peptide hormone with an essential role in the systemic regulation of iron. The dysregulation of hepcidin is linked to a number of iron loading disorders such as hereditary haemochromatosis and anaemia of inflammation. Procuring synthetic, recombinant or endogenous hepcidin is challenging and expensive; therefore strategies to improve the synthesis of hepcidin and add modifications to extend its short half-life in vivo are required for potential therapeutic applications. Industrial scale production of a peptide such as hepcidin for therapeutic use would likely be performed using a convergent approach, involving the ligation of peptide fragments. Native chemical ligation (NCL) is a powerful tool in peptide and protein synthesis, allowing peptide fragments to be joined together through a chemoselective ligation between a peptide-thioester and a cysteinyl peptide. Preparation of peptide¬thioesters is challenging, however thioester formation via 1 U 6 acyl transfer at cysteine residues allows generation of thioesters in both synthetic and recombinant peptides and proteins. Here, investigations into optimising the preparation of peptide-thioesters via 1 U 6 acyl transfer have been carried out to extend its application to the synthesis of complete hepcidi n, analogues and labelled variants. This led to the development of a novel, faster route to peptide thioesters via 1 U 6e acyl transfer, which was applied to the cyclisation of peptides via in situ NCL following thioester formation. Additionally a novel methodology via 1 U 6 acyl transfer has been established for the preparation of peptide hydrazides, which are stable alternatives to peptide-thioesters in NCL. These novel strategies have been applied to full-length hepcidin and fragments of hepcidin, which in conjunction with NCL, have been used to generate a variety of hepcidins. A measurable reduction in the iron efflux membrane protein, ferroportin, in cells treated with these analogues, confirmed their biological activity, indicating hepcidin-induced internalisation of ferroportin, including for hepcidin biotinylated using the hydrazide-NCL strategy.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Developing Novel Peptide Ligation Methodology for the Synthesis and Labelling of Bioactive Hepcidin |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1427640 |
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