Bio21 - Theses
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ItemMolecular characterisation of the amyloid precursor protein: a key biomolecule in Alzheimer's disease( 2017)The amyloid precursor protein (APP) is strongly implicated in Alzheimer’s disease pathogenesis due to the observed aggregation of its Aβ sequence in the brain of AD patients. Despite being a molecule of intense interest, a sound molecular understanding of the normal functions of APP remains elusive. This work provides molecular insights into two ostensibly significant interactions involving the extracellular domain of APP, namely: (i) copper binding; and (ii) heparin-induced homo-dimerization. Biological copper is available in two oxidation states: Cu(I) and Cu(II). Well-characterised spectroscopic probes for Cu(I) binding studies are available, but reliable probes for Cu(II) binding studies are lacking. This work has developed a new method for in vitro characterisation of Cu(II) binding affinities. This involved the design and characterisation of four fluorescent peptide-based probes which respond to the binding of paramagnetic Cu(II) ions. Each displays a different affinity for the metal and together they are capable of detecting Cu(II) binding from micromolar to femtomolar range. This allows quantification of the Cu(II) binding affinities of individual protein targets on a unified scale. These probes facilitated studies of copper binding in the APP ectodomain. Biological studies performed over the last two decades have suggested that APP has a role in maintaining cellular copper homeostasis and it is commonly cited as a copper-binding protein. Several putative copper binding sites in the extracellular domain have previously been identified using structural techniques (NMR, X-ray crystallography). However, the chemistry of these has never been systematically studied under uniform conditions and the preferred metal coordination site is unknown. This work has characterised the thermodynamics of Cu(I) and Cu(II) binding in the APP ectodomain. The preferred binding site for both metal oxidation states is a (His)4 centre of picomolar affinity in the α-helical E2 domain. Interestingly, copper coordination leads to conformational changes in this flexible subdomain which could be significant in a cellular context. Homo- and hetero-dimeric interactions between APP and its ‘amyloid precursor-like protein’ homologues (APLP1 and APLP2) have been observed in many previous in vivo studies. These interactions are promoted by the binding of heparins (glycosaminoglycan chains abundant in the extracellular space). The dimers are thought to play a critical role in the normal functions of this protein family including trans-cellular adhesion and synaptogenesis. Detailed structural studies of these dimeric assemblies have not previously been performed. This is largely due to the size and heterogeneity of these complexes which present challenges for high-resolution techniques such as NMR and X-ray crystallography. However, homo-dimeric structures have been solved for two isolated extracellular domains (E1 and E2) which each contain heparin-binding regions. In this work, a chemical cross-linking mass spectrometry approach is used to provide structural information about the dimeric assembly of the complete secreted ectodomain sAPPα. The results offer direct proof of a parallel dimeric interaction between two sAPPα monomers. Additionally, this provides a means to evaluate E1 and E2 homo-dimeric models within the context of the overall ectodomain structure. This preliminary study provides a basis for further investigation of homo- and hetero-dimeric interactions involving APP homologues.