Regulation of Peptide Hormones.

Abstract

C-Terminal amidated peptide hormones regulate numerous physiological process and are associated with many pathological conditions. Their C-terminal amidation is exclusively catalysed by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM), which has two subunits: peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). In the present study, the substrate specificity and inhibition of the PAM sourced from DMS53 human small cell lung cancer cells were investigated. A peptide consensus sequence N-Ac-Gly-(S)-Pro-Gln-(S)-Arg-(S)-Phe-Gly-COOH was constructed from the human amidated peptide hormone database, and a library of peptide substrates was designed by varying the amino acids located at the penultimate and antepenultimate positions from the C-terminus. These peptides were synthesised by solid phase peptide synthesis and studied in a competitive PHM binding assay. The results indicated that human glycine-extended hormone precursors having -Arg-Phe-Gly-COOH are likely to bind to PHM effectively in living bodies with IC50 values around twenty micro molar and those having -Asp-Phe-Gly-COOH and -Ala-Phe-Gly-COOH are likely to bind with IC50 values around seventy micro molar. The results also show that antepenultimate amino acids affect binding to PHM but to a lesser degree than the effect of penultimate amino acids.

In a prodrug study, the anti-cancer drug Bexarotene was synthetically extended with a glycine to obtain a derivative that was expected to be cleaved by PAM and then deliver a cytotoxic drug. However, the derivative compound showed poor binding to PHM at the millimolar level, but had a similar cytotoxic activity against DMS53 cells to its parent agent. Unlike Bexarotene, a previous study showed that the glycine derivative of Temozolomide (another anti-cancer drug) has strong binding to PHM at micromolar concentration, but somehow does not convert to Temozolomide by PAM. One possible reason is that the glycine derivative possessing an imidazole moiety could inhibit PHM by metal chelation. To investigate this, a structurally-similar molecule N-Ac-(S)-His-Gly-COOH was tested and found to be processed by PAM and not undergo copper coordination. This implies that the glycine derivative of Temozolomide is unlikely to chelate the copper of the PHM active site.

To investigate PHM and PAL activity simultaneously, a new PAM assay with a direct, rapid and sensitive HPLC-MS system was established. With this new assay the PAL inhibitor N-Ac-Phe-pyruvate and analogues were evaluated in terms of PAM inhibition through isolated enzyme competitive assays. This demonstrated that N-Ac-Phe-pyruvate is a PAL inhibitor at the nanomolar level against the medium PAM from DMS53 cells and deprotonation of its enol form results in a decrease of PAL inhibition.

By using the established HPLC-MS detection system, the intermediate of the C-terminal amidation of calcitonin-Gly (CTG), alpha-hydroxyglycine-extended calcitonin (HO-CTG), was detected in the culture medium of DMS53 cells. This is the first time HO-CTG produced by cells has been detected. With and without a PAL inhibitor, the concentration of the HO-CTG in the medium sample was found to be more than two orders of magnitude lower than those of the corresponding substrate and amidated product, suggesting the PAL catalysis is much faster than PHM catalysis, and PHM catalysis is the rate-limiting step of amidation of calcitonin in human cells. Show more