Investigating the role of C-terminal amidation in neurokinin B amyloid fibril formation

Abstract

The brain functions through the signalling of neuropeptides that elicit specialised biological activities. This study observed the neuropeptide neurokinin B (NKB) which typically participates in signalling for the release of reproductive hormones. Some neuropeptides need posttranslational processing to fully establish their bioactivity, and an important modification is Cterminal amidation. Some modifications have been previously observed to influence the aggregation and storage of neuropeptides in dense core vesicles. NKB is well known to form amyloid fibrils, and the aim of this study was to determine if amidation of NKB is important for fibril formation and correct cellular packaging. To begin investigating the role of NKB amidation, an in vitro study was conducted on nonamidated, glycine-extended and amidated wild type peptides. Using a range of techniques, nonamidated peptide was found to aggregate into amorphous aggregates rather than fibrils, in contrast to wild-type amidated NKB which rapidly aggregated into parallel b-sheet amyloid fibrils.This highlights the importance of C-terminal amidation for fibril formation by NKB. The amyloid fibril formation of NKB following amidation was further explored in within the intracellular environment of astrocytes. Amidation was inhibited by 4-phenyl-3-butenoic acid to prevent glycine-extended NKB precursors from being amidated. The resulting NKB peptide aggregates were observed using polyacrylamide gel electrophoresis to changes in the peptide aggregation by monitoring variations in molecular weight. Unexpectedly, the absence of amidation did not result in the cellular accumulation of glycine-extended precursors as hypothesised. This was further explored at a genetic level to determine if peptide production was genetically regulated. When amidation was inhibited, NKB gene expression was downregulated, explaining the observed lack of accumulation of non-amidated, glycine extended NKB. This work extends the role of amidation beyond that of receptor activation to show that it is important for the correct structure of NKB. An absence of amidation would lead to incorrectly folded NKB in the dense core vesicles and the cell downregulates transcription in response. This has profound implications for the biosynthesis NKB and the storage of this neuropeptides within neuronal cells.

Date of Thesis submission	2025
Original language	English
Awarding Institution	Western Sydney University
Sponsors	Australian Government Research Training Program Scholarship
Supervisor	Christopher Jones (Supervisor), Nathan Absalom (Supervisor) & Roland Gamsjaeger (Supervisor)