Homophilic Interactions of the Amyloid Precursor Protein (APP) Ectodomain Are Regulated by the Loop Region and Affect β-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.
- SourceAvailable from: Corinna van den Heuvel
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- "It is yet to be determined the functional effects mediated by homo-and heterodimerisation of the APP family members, although it has been hypothesized that cis-dimerisation of membrane bound APP with sAPPa may allow activation of intracellular signalling pathways (Gralle et al. 2009; Reinhard et al. 2005). Alternatively cis dimerisation may simply influence the processing of APP, possibly by changing its conformational state, thereby decreasing its amyloidogenic processing by preventing access of b-secretase to its cleavage site (Kaden et al. 2008). It is possible that sAPPa may act in a similar manner to known growth factors, whereby heparin is required to facilitate binding of APP to a co-receptor, through the APP96-110 region. "
ABSTRACT: We have previously shown that following traumatic brain injury (TBI) the presence of the amyloid precursor protein (APP) may be neuroprotective. APP knockout mice have increased neuronal death and worse cognitive and motor outcomes following TBI, which is rescued by treatment with exogenous sAPPα (the secreted ectodomain of APP generated by α-secretase cleavage). Two neuroprotective regions were identified in sAPPα, the N and C-terminal domains D1 and D6a/E2 respectively. As both D1 and D6a/E2 contain heparin binding activity it was hypothesised that this is responsible for the neuroprotective activity. In this study we focused on the heparin binding site, encompassed by residues 96-110 in D1, which has previously been shown to have neurotrophic properties. We found that treatment with APP96-110 rescued motor and cognitive deficits in APP-/- mice following focal TBI. APP96-110 also provided neuroprotection in Sprague Dawley rats following diffuse TBI. Treatment with APP96-110 significantly improved functional outcome as well as preserve histological cellular morphology in APP-/- mice following focal controlled cortical impact injury. Furthermore, following administration of APP96-110 in rats after diffuse impact acceleration TBI, motor and cognitive outcomes were significantly improved and axonal injury reduced. These data define the heparin binding site in the D1 domain of sAPPα, represented by the sequence APP96-110, as the neuroprotective site to confer neuroprotection following TBI. This article is protected by copyright. All rights reserved.Journal of Neurochemistry 08/2013; DOI:10.1111/jnc.12391 · 4.24 Impact Factor
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- "Moreover, binding of heparin to E1eE2 domains also promotes APP dimerization (Dahms et al., 2010; Gralle et al., 2006). Providing a physiological consequence for these interactions, APP dimerization was shown to affect its processing by secretases (Kaden et al., 2008; Lefort et al., 2012; Munter et al., 2007; Scheuermann et al., 2001). Because the extracellular portion of APP contains various structural domains, it is not surprising that several putative physiological ligands have been identified for APP. "
ABSTRACT: A role of amyloid β (Aβ) peptide aggregation and deposition in Alzheimer's disease (AD) pathogenesis is widely accepted. Significantly, abnormalities induced by aggregated Aβ have been linked to synaptic and neuritic degeneration, consistent with the "dying-back" pattern of degeneration that characterizes neurons affected in AD. However, molecular mechanisms underlying the toxic effect of aggregated Aβ remain elusive. In the last 2 decades, a variety of aggregated Aβ species have been identified and their toxic properties demonstrated in diverse experimental systems. Concurrently, specific Aβ assemblies have been shown to interact and misregulate a growing number of molecular effectors with diverse physiological functions. Such pleiotropic effects of aggregated Aβ posit a mayor challenge for the identification of the most cardinal Aβ effectors relevant to AD pathology. In this review, we discuss recent experimental evidence implicating amyloid β precursor protein (APP) as a molecular target for toxic Aβ assemblies. Based on a significant body of pathologic observations and experimental evidence, we propose a novel pathologic feed-forward mechanism linking Aβ aggregation to abnormalities in APP processing and function, which in turn would trigger the progressive loss of neuronal connectivity observed early in AD.Neurobiology of aging 05/2013; 34(11). DOI:10.1016/j.neurobiolaging.2013.04.021 · 4.85 Impact Factor
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- "Yet, one study has shown that the addition of a synthetic loop peptide that prevents AβPP ectodomain dimerization at the GFLD region also decreases Aβ levels. Since the relative decrease in Aβ corresponded to a decrease in AβPPsβ levels generated from β-secretase processing, this study suggests that the loop peptide may have impaired AβPP dimerization, which in turn impaired β-secretase cleavages . Also, it is generally accepted that following α-and β-secretase cleavages, γ-secretase sequentially cleaves the remaining AβPP C-terminal fragment (CTF) at multiple sites starting at the C-terminus from amino acids 715 to 710 to generate Aβ peptides of various lengths from Aβ 39 to 42 [18, 34]. "
ABSTRACT: Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder marked by memory impairment and cognitive deficits. A major component of AD pathology is the accumulation of amyloid plaques in the brain, which are comprised of amyloid beta (Aβ) peptides derived from the amyloidogenic processing of the amyloid precursor protein (AβPP) by β- and γ-secretases. In a subset of patients, inheritance of mutations in the AβPP gene is responsible for altering Aβ production, leading to early onset disease. Interestingly, many of these familial mutations lie within the transmembrane domain of the protein near the GxxxG and GxxxA dimerization motifs that are important for transmembrane interactions. As AβPP dimerization has been linked to changes in Aβ production, it is of interest to know whether familial AβPP mutations affect full-length APP dimerization. Using bimolecular fluorescence complementation (BiFC), blue native gel electrophoresis, and live cell chemical cross-linking, we found that familial Alzheimer's disease (FAD) mutations do not affect full-length AβPP dimerization in transfected HEK293 and COS7 cells. It follows that changes in AβPP dimerization are not necessary for altered Aβ production, and in FAD mutations, changes in Aβ levels are more likely a result of alternative proteolytic processing.01/2013; 2(1):15-28.