nvestigating the role of the N-terminal disulphide loop structure for receptor activation

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Analysing spontaneous peptide oxidation

To achieve solubility at 1 mM stock concentrations for biological assays, human amylin is dissolved in DMSO and stored at -30°C. Since DMSO is a natural oxidising agent, we needed to ensure that solvation and storage in DMSO did not cause oxidation of the linear peptide.
The retention time for crude linear human amylin was 13.66 minutes (Figure 3.12), and following a 1 hour oxidation with DTNP, this shifts rightwards to 13.91 minutes, eluting on a linear gradient of 5 – 65% B at 3% B per minute at 210 nm (Figure 3.13). If linear human amylin oxidises in DMSO at room temperature, a similar shift in retention time would be expected with a gradual splitting to two peaks over time, indicating a mixed species of unoxidised and oxidised amylin. Therefore, a time course comparing linear human amylin in DMSO was conducted to observe how quickly it oxidised under these conditions (Figure 3.14).
The retention time for linear human amylin in water was 13.66 minutes (Figure 3.12), and at time 0 in DMSO, the retention time was similar at 13.63 minutes (Figure 3.14 A), indicating that linear human amylin does not oxidise instantly in DMSO. In DMSO at room temperature, the oxidised peptide begins to appear after 2 hours and the retention time shifts to 14.05 minutes (Figure 3.14 C). Oxidation proceeds over the next 4 hours up to 6hrs (Figure 3.14 D – E). Therefore, it appears that linear human amylin can be made up in DMSO for assays, provided it remains frozen prior to assaying as per our normal procedure. Since the drug-stimulation step in the cAMP functionality assay used to test these peptides is only 15 minutes, presumably the peptide should retain linearity in this time-frame.

Confirming linearity of human amylin under cAMP assay conditions

WT pharmacology of the linear variant was unexpected. Given the supposed importance of the activation loop, potency reductions were anticipated from the linear peptide. The results prompted further investigation into the linearity of the peptide as it could not be ruled out that the peptide may oxidise under assay conditions. The linear human amylin had been made up in DMSO and stored at -30°C two weeks prior to analysis. Analysis of this peptide confirmed that under the above conditions, the linear human amylin had not oxidised (Figure 3.16 A, C).
However, after being made up in DMSO and added to assay media (serum-free media, IBMX and BSA) at a final concentration of 100 μM and incubated for 15 minutes at 37°C, there was a shift in retention time from 15.57 minutes (Figure 3.16 A) to 15.96 minutes (Figure 3.16 B), which eluted on a linear gradient of 5 – 65% B at 3% B per minute. The additional peaks observed in the LCMS trace correspond to other components that eluted from the cAMP media (Figure 3.16 B). The m/z ratios changes revealed that under these conditions, linear human amylin oxidises and cannot remain linear (Figures 3.16 C, D). Unfortunately, this outcome indicates that we are unable to test a linear variant of human amylin towards receptor activation under these assay conditions at this
time.

READ DIETARY FIBRE IN GROWING PIG NUTRITION

Chapter 1 Introduction
1.1 Amylin and the calcitonin family of peptides
1.2 Control of food Intake
1.3 Amylin physiology and expression
1.3.1 Amylin discovery
1.3.2 Pancreatic amylin
1.3.3 Amylin in the circulation
1.3.4 Amylin physiology
1.3.5 Amylin dysregulation
1.3.6 Extrapancreatic amylin
1.3.7 Amylin actions are centrally-mediated
1.3.8 Pramlintide
1.4 Amylin receptors and pharmacology
1.4.1 Family B GPCRs and RAMPs
1.4.2 CGRP and AM receptors
1.4.3 Amylin receptors
1.7 Introduction summary
Chapter 2 Materials and Methods .
2.1 Materials
2.2 Methods
Chapter 3 Investigating the role of the N-terminal disulphide loop structure for receptor activation
3.1 Introduction
3.2 Peptide Synthesis Methodology – Overview
3.3 Pharmacological Characterisation Methodology – Overview
3.4 Results: WT human amylin
3.5 Results: Linear human amylin
3.6 Results: Linear human amylin variant C2S-C7S
3.7 Results: Truncated human amylin8-37
3.8 Results: Chapter 3 summary
Chapter 4 Identifying residues within the N-terminal disulphide loop of human amylin important for receptor activation
Chapter 5 Identifying residues within the proposed 8-17 amphipathic alpha helical region of human amylin that are important for receptor activation 1
Chapter 6 Investigating further sequence or structurally modified analogues and their impact on receptor activation
Chapter 7 General discussion and conclusion