The influence of copper (II) ions on the structure and stability of the prion protein and its interaction with the amyloid-beta peptides.

PhDThe prion protein (PrPC) is a cell surface glycoprotein that binds Cu2+ ions. The misfolding and oligomerisation of PrPC is responsible for a range of transmissible spongiform encephalopathies (TSEs) in mammals. As changes in PrPC conformation are intimately linked with disease pathogenesis, the effect of Cu2+ ions on the structure and stability of PrPC has been investigated. In chapter 3, urea unfolding studies indicate that Cu2+ ions destabilise the native fold of PrPC. The mid-point of the unfolding transition is reduced by 0.73 ± 0.05 M urea in the presence of Cu2+ ions equating to an appreciable difference in free energy of unfolding (∆∆GU[D]50%), 2.02 ± 0.05 kJmol-1. This suggests that in Prion diseases, Cu2+ ions could destabilise the native fold of PrPC and make the transition to a misfolded state more favourable. Furthermore, Cu2+ induced changes in secondary structure observed for small fragments of the protein are related to the full-length prion protein. An increase in -sheet like character is observed when Cu2+ ions are present, this is due to local Cu2+ ion coordination to the individual binding sites of the amyloidgenic region. Cu2+ induced changes in the secondary structure of the N-terminal domain, PrP(23-126) and full-length PrP(23-231), are attributed to Cu2+ ions binding within the octarepeat region of PrPC. Oxidative stress is also a well-recognised feature of Prion diseases. In chapter 4, the effects of Cu2+ catalysed oxidation of PrP on the structure and stability are discussed. 2D 1H-15N HSQC NMR studies of PrPC indicate that specific key residues are perturbed upon methionine (Met) oxidation by H2O2. These residues are involved in the hydrophobic packing of the structured core of the protein, stabilising its ternary structure. Urea unfolding studies indicate that the oxidation of PrPC by H2O2 and to a greater extent Cu2+ ions with peroxide significantly reduce the thermodynamic stability of PrPC. Cu2+ catalysed oxidation of PrP causes much more significant alteration of the structure. 2D 1H-15N HSQC NMR spectroscopy indicates that the structured C-terminal portion of PrP becomes a large molten-globule, made of monomeric species. FT-IR and far-UV-CD spectroscopy indicate that this molten-globule is rich in β-sheet. These observations supports a hypothesis that oxidation of PrP destabilises the native fold of PrPC, making the transition to PrPSc more energetically favourable. This study gives a structural and thermodynamic explanation for the high levels of oxidised Met residues in scrapie isolates. Finally, in chapter 5 the interaction of PrPC with Aβ peptide, responsible for Alzheimer’s disease (AD), is investigated. In particular, the influence of full length PrP and fragments on the kinetics of Aβ fibril growth is investigated. The complete inhibition Aβ fibril formation is observed when as little as one-twentieth of the molar ratio of PrPC is used. The unstructured N-terminus of PrPC, residues 23 to115, is thought to be crucial for this inhibition, while, residues 116 to 231 have no influence on the fibril formation. Gel filtration chromatography indicates that the complex formed by PrPC with an Aβ oligomer is 12 to 24 monomers in size.Biotechnology and Biological Sciences Research Counci

Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2

The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation

Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: The N-terminal region of S2 facilitates self-association

The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo

The cellular prion protein traps Alzheimer's A beta in an oligomeric form and disassembles amyloid fibers

This work was supported by a Wellcome Trust project grant (093241/Z/10/Z) and UK Biotechnology and Biological Sciences Research Council Quota studentships. The authors thank Harold Toms (Queen Mary, University of London) and the UK National Institute for Medical Research for NMR support, and Graham McPhail for assistance with TEM

The Rapid Exchange of Zinc (2+) Enables Trace Levels to Profoundly Influence Amyloid-beta Misfolding and Dominates Assembly Outcomes in Cu (2+)/Zn (2+) Mixtures

This work was supported by the Biotechnology and Biological Sciences Research Council Project GrantBB/M023877/1 and Quota Studentship