Structural and biophysical characterisation of Alzheimer’s disease amyloid precursor protein

© 2015 Dr. Chen GaoAlzheimer’s disease (AD) is an irreversible, neurodegenerative disorder most commonly affecting the aged population. Despite the growing incidence world wide, the disease remains untreatable due to a lack of understanding of the disease mechanism. Central to AD etiology is the amyloid cascade hypothesis, which states that the accumulation of a toxic peptide amyloid-beta (Abeta) sets off a cascade of cytotoxic events leading to the death of neurons. Abeta is derived from the amyloid precursor protein (APP). APP is a type-I ransmembrane protein with a large ectodomain (sAPP), a single transmembrane domain and a cytoplasmic tail. Its dimerisation has been closely linked to Aβ overproduction, and is also implicated in signalling as APP is proposed to be a membrane receptor. There are four putative dimerisation sites in APP, three of which are located in the sAPP region. However, the mechanisms of APP dimerisation remains unclear. Understanding APP dimerisation mechanisms at the molecular level will not only provide insights into how APP signals but also have therapeutic implications. Several factors are found to regulate APP proteolysis in vivo. To test their impact on sAPP dimerisation, different fragments of sAPP containing distinct dimerisation domains were expressed as recombinant proteins and subjected to extensive dimerisation studies in vitro. pH, zinc and long-chain heparin were found to be the three major modulators of sAPP stability and dimerisation. Interestingly, sAPP fragments containing different dimerisation domains respond differently to the three modulators: the C-terminal fragment of sAPP containing three of the four dimerisation sites can be stabilised by low pH and zinc, but only forms dimers in the presence of long-chain heparin. In contrast, the N-terminal fragment of sAPP containing two of the dimerisation domains was found to dimerise at higher pH and in the presence of zinc. Such differences in the dimerisation behaviour between different regions of sAPP suggest the dimerisation mechanism of the full-length APP is much more complex than previously thought. Results from the dimerisation studies led to the successful crystallisation of the sAPP C-terminal region in the presence of zinc and long-chain heparin in unique dimeric forms. The structure derived from the crystals reveals the dimer is stabilised by six zinc ions. The observation of a heparin disaccharide at the two-fold symmetry axis also led to the construction of two long-chain heparin mediated APP dimer models, one of which is consistent with the requirement of long-chain heparin to stabilise the dimer. A dodecapeptide from the juxtamembrane region of APP was observed to interact with the metal-binding E2 domain of APP via two zinc ions; this interaction is accompanied by the formation of a partial helix in the otherwise unstructured peptide. Interestingly, a familial AD mutation P545L (P620L in the longest APP isoform) is located near one of the zinc binding sites in the peptide, suggesting the zinc-mediated peptide/E2 interaction may have significant implications in APP processing and AD pathogenesis. Together with results from the dimerisation studies, the crystal structure has provided a leap forward in understanding the mechanism of APP dimerisation and its regulation by physiologically relevant modulators. The pathogenic and functional consequences of the proposed dimers remains to be investigated in vivo, while structural information can be used to assist the development of APP dimerisation modulators as potential therapeutics to treat Alzheimer’s disease