Peptide hormone regulation and synthesis / Karine Caron.

Abstract

The work presented in this thesis focuses on the study of peptide hormones, their post-translational biosynthesis and regulation, and the synthesis of unnatural peptide hormone analogues using cell-fee protein synthesis. In the first instance, the enzymatic activity of peptidylglycine a-amidating monooxygenase (PAM) was studied, an enzyme that catalyses the final post-translational step for many hormones. A novel whole-cell assay is described in Chapter 2, utilising HPLC, which allows simultaneous detection of multiple species of calcitonin (CT). Regulation of PAM activity was examined through measurement of variation in CT and pro-CT levels, in a small cell lung carcinoma cell line (DMS53), to evaluate the effect of PAM inactivators on CT amidation, and in extension, on cancer cell survival. It was found that CT levels relative to CT precursor levels could not be decreased below a certain level in a dose-dependent manner, which signifies the presence of a mechanism for maintenance of homeostasis. Furthermore, through the identification of CT degradation products generated in DMS53 cells medium, it was possible to further probe the degradative pathways for CT in this cell line. To better understand the interactions of PAM with natural substrates, the binding affinities of a series of peptide prohormones were determined, in Chapter 3, using PAM from DMS53 cell medium. While the role of the penultimate amino acid in substrate recognition has been previously reported for short synthetic substrates, the work presented herein highlights the role of the entire peptidic sequence in substrate recognition. It was found that though trends are similar, the extent of the effect of the penultimate amino acid on substrate binding is exaggerated in synthetic substrates, when compared to natural substrates. Furthermore, this variation in peptide hormone binding does not appear to govern the relative levels of those hormones in vivo. Due to our interest in the study of peptide hormones, a new methodology was developed as an alternative to laborious solid phase synthesis for the production of peptide hormones and peptide analogues. As presented in Chapter 4, cell-free protein synthesis, which is commonly used for the production of natural proteins and proteins containing non-canonical amino acids, was applied as a simple and cheap means for generating peptides and unnatural peptide analogues. To do this a new strategy was employed, making use of fusion partner methodology. An expression plasmid was designed and produced, which expresses the peptide of choice as a fusion with a soluble protein, through an enterokinase cleavable linker. This allowed production of CTG and CTG analogues containing the non-canonical amino acids chloro-Val, chloro-Ile, chloro-Tyr, fluoro-Leu and fluoro-Phe. Finally, with the aim of making this new technique more efficient and cost-effective for the production of peptides, in Chapter 5, the use of acetyl phosphate to drive nucleoside triphosphate-dependent protein synthesis together with the generation of the expensive and unstable nucleoside triphosphates was undertaken. This process required characterisation of the endogenous phosphorylating enzymes of the commonly used E. coli BL21(DE3) star strain used for cell-free protein synthesis. Show more