Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect beta-secretase cleavage of APP
ABSTRACT We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.
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ABSTRACT: Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2014; 34(33):11159-11172. DOI:10.1523/JNEUROSCI.0180-14.2014 · 6.75 Impact Factor
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ABSTRACT: The amyloid precursor protein (APP) and its proteolytic cleavage product Aβ are widely believed to be central to the etiology of Alzheimer’s disease (AD). APP and its family members are also essential for proper neuronal development and homeostasis. APP is located at the cell surface and within intracellular compartments, cellular regions that exhibit different pH-values. The AD-associated amyloidogenic processing of APP is initiated predominantly in intracellular acidic compartments, whereas its non-amyloidogenic cleavage is initiated at the cell surface at slightly basic pH. We analyzed the influence of pH on the APP-E1-domain and found that its two constituting subdomains, GFLD and CuBD, interact with each other in a pH-dependent manner. Dynamic light scattering (DLS) showed that APP-E1 represents a more open conformation at neutral and a more closed conformation at acidic pH. Analyzing a 1.4 Å high resolution X-ray structure of E1 derived from merohedrally twinned crystals resulted in the identification of individual residues that are responsible for these pH-dependent interactions. Mutational studies and DLS-measurements further proved that specific hydrogen bonds between the two carboxylates of D177 and E87 as well as between N89 and H147 are major determinants of this pH-driven conformational switch in APP-E1. These findings show how APP can adopt different conformations depending on pH and suggest that the protein fulfils different functions at distinct localizations within the cell. Additionally, our data suggest a novel strategy for treating AD based on regulating the amyloidogenic processing of APP by the specific interruption of the interaction between the APP-E1-subdomains.Journal of Molecular Biology 12/2014; 368(2). DOI:10.1016/j.jmb.2014.12.005 · 3.96 Impact Factor