| dc.description.abstract | Neurodegeneration, the progressive and irrevocable loss of neuronal structure, is quickly becoming an
imposing health concern in a globally ageing society. While specific neurodegenerative conditions
exhibit specific clinical symptoms and progressions, a common neuropathological feature is the
misfolding, oligomerisation and fibrillation of certain proteins causing neuronal stress and death.
Parkinson’s disease, PD, has long been characterised by the death of nerve cells focused in the
substantia nigra pars compacta region of the midbrain and deposition of large protein aggregates, called
Lewy Bodies, throughout the central nervous system. More recently, the protein which forms these
inclusion bodies was identified as alpha synuclein, αSyn, a ubiquitous neuroprotein with no known
function. Furthermore, persons with mutations in the SNCA gene, which codes for αSyn, exhibit PD
progression at a far younger age with a more severe phenotype, positively linking αSyn with PD.
αSyn is an intrinsically disordered protein, IDP, and generally persists as such in solution and inside
bacterial and mammalian cells. However, when in contact with a lipid bilayer the protein will embed
upon the surface in an amphipathic alpha helical conformation and can also aggregate, forming toxic
oligomeric and fibrillar species containing significant β-sheet identity. Its function as a helical
apolipoprotein and subcellular localisation to both the nucleus and synapse has led researchers to
suggest that αSyn has a role synaptic transmission and release. However, knocking out the protein does
not reduce viability or produce pathological abnormalities in neuronal structure. The helical form of the
protein may also persist as transient, metastable helical bundles which are non-toxic and resist
aggregation. While a number of studies and tools have been reported and developed to investigate the
toxic oligomeric/fibrillar forms of αSyn, very little attention has been accorded to the helical
conformation. This thesis will redress this balance by producing tools which will allow us to mimic the
helical form of αSyn, promote the active refolding of the full-length protein using a stable, helical
peptide template and produce antibodies which recognise helical αSyn specifically for use in discovery
and chaperone-like refolding.
In Chapter 2 a region of αSyn (14 amino acids) was identified with a unique primary sequence located
within a mutation prone section of the protein. Peptide ‘stapling’ technologies were then employed
using a panel of monosubstituted ‘staple’ diastereomers, to produce a highly helical portion of αSyn.
Using several other protein targets particular diastereomeric ‘staple’ combinations were analysed for
obvious trends in helical content. Using solution NMR, backbone refined three dimensional structures
of these helical peptides were produced which showed that they were faithful structural homologues of
their parent helical proteins.
In Chapter 3 the drug-like properties and therapeutic potential of stable, helical αSyn peptides were
investigated. Using fluorescently labelled peptide substrates, ‘stapled’ peptides were shown to be far
more cell penetrant than their wild type equivalents and demonstrated that the mechanism for cellular
uptake appears to be specific. Furthermore, under harsh proteolytic conditions the ‘stapled’, helical
peptides were far more resistant to hydrolysis than wild type or ‘stapled’, poorly helical peptides. The
‘stapled’ peptides were also highly soluble and did not appear to aggregate in a time-dependent manner.
Using ion mobility mass spectrometry, it was shown that incubation of full-length protein with the
‘stapled’, helical peptides caused a contraction in the hydrodynamic radius of the protein. However,
using solution NMR no active refolding of αSyn was observed when under the same conditions. Rather
small perturbations in chemical shift were apparent which did not suggest that the αSyn protein folded
into a discrete structural conformation, such as an alpha helix.
In Chapter 4 the stable, helical αSyn peptide was employed as a conformational model and unique
antigen in antibody discovery. Immunisation with the ‘stapled’, helical αSyn peptide initially produced
a pool of polyclonal antibodies with a half log specificity for the helical peptide. After bespoke affinity
chromatography this was increased to three log orders of specificity. Initial immunocytochemistry did
not detect any helical αSyn protein in SH-SY5Y cells. To validate the helical epitope on the full-length
protein in vitro an assay based around flow cytometry of synthetic vesicle structures was developed,
with their synthesis, characterisation and binding of the αSyn protein described. | en |
