Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

Novel zinc-binding site in the E2 domain regulates amyloid precursor-like protein 1 (APLP1) oligomerization.

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438-452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

Peptides based on the presenilin-APP binding domain inhibit APP processing and Aß production through interfering with the APP transmembrane domain.

Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.

Novel APP/Aß mutation K16N produces highly toxic heteromeric Aß oligomers.

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-ß (Aß) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aß. First, due to the K16N mutation APP secretion is affected and a higher amount of Aß peptides is being produced. Second, Aß peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aß42 K16N inhibits fibril formation of Aß42 wild-type. Even more, Aß42 K16N peptides are protected against clearance activity by the major Aß-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease.

Metal binding dictates conformation and function of the amyloid precursor protein (APP) E2 domain.

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aß are key players in the development of Alzheimer's disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6-2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively to copper and zinc at physiological concentrations. Metal-specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in Alzheimer's disease.

The amyloid precursor protein and its homologues: structural and functional aspects of native and pathogenic oligomerization.

Over the last 25 years, remarkable progress has been made not only in identifying key molecules of Alzheimer's disease but also in understanding their meaning in the pathogenic state. One hallmark of Alzheimer pathology is the amyloid plaque. A major component of the extracellular deposit is the amyloid-ß (Aß) peptide which is generated from its larger precursor molecule, i.e., the amyloid precursor protein (APP) by consecutive cleavages. Processing is exerted by two enzymes, i.e., the ß-secretase and the γ-secretase. We and others have found that the self-association of the amyloid peptide and the dimerization and oligomerization of these proteins is a key factor under native and pathogenic conditions. In particular, the Aß homodimer represents a nidus for plaque formation and a well defined therapeutic target. Further, dimerization of the APP was reported to increase generation of toxic Aß whereas heterodimerization with its homologues amyloid precursor like proteins (APLP1 and APLP2) decreased Aß formation. This review mainly focuses on structural features of the homophilic and heterophilic interactions among APP family proteins. The proposed contact sites are described and the consequences of protein dimerization on their functions and in the pathogenesis of Alzheimer's disease are discussed.

Disturbed copper bioavailability in Alzheimer's disease.

Recent data from in vitro, animal, and human studies have shed new light on the positive roles of copper in many aspects of AD. Copper promotes the non-amyloidogenic processing of APP and thereby lowers the Aß production in cell culture systems, and it increases lifetime and decreases soluble amyloid production in APP transgenic mice. In a clinical trial with Alzheimer patients, the decline of Aß levels in CSF, which is a diagnostic marker, is diminished in the verum group (8 mg copper/day), indicating a beneficial effect of the copper treatment. These observations are in line with the benefit of treatment with compounds aimed at normalizing metal levels in the brain, such as PBT2. The data reviewed here demonstrate that there is an apparent disturbance in metal homeostasis in AD. More research is urgently needed to understand how this disturbance can be addressed therapeutically.

Aberrant amyloid precursor protein (APP) processing in hereditary forms of Alzheimer disease caused by APP familial Alzheimer disease mutations can be rescued by mutations in the APP GxxxG motif.

The identification of hereditary familial Alzheimer disease (FAD) mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1) corroborated the causative role of amyloid-beta peptides with 42 amino acid residues (Abeta42) in the pathogenesis of AD. Although most FAD mutations are known to increase Abeta42 levels, mutations within the APP GxxxG motif are known to lower Abeta42 levels by attenuating transmembrane sequence dimerization. Here, we show that aberrant Abeta42 levels of FAD mutations can be rescued by GxxxG mutations. The combination of the APP-GxxxG mutation G33A with APP-FAD mutations yielded a constant 60% decrease of Abeta42 levels and a concomitant 3-fold increase of Abeta38 levels compared with the Gly(33) wild-type as determined by ELISA. In the presence of PS1-FAD mutations, the effects of G33A were attenuated, apparently attributable to a different mechanism of PS1-FAD mutants compared with APP-FAD mutants. Our results contribute to a general understanding of the mechanism how APP is processed by the gamma-secretase module and strongly emphasize the potential of the GxxxG motif in the prevention of sporadic AD as well as FAD.

Subcellular localization and dimerization of APLP1 are strikingly different from APP and APLP2.

The molecular association between APP and its mammalian homologs has hardly been explored. In systematically addressing this issue, we show by live cell imaging that APLP1 mainly localizes to the cell surface, whereas APP and APLP2 are mostly found in intracellular compartments. Homo- and heterotypic cis interactions of APP family members could be detected by FRET and co-immunoprecipitation analysis and occur in a modular mode. Only APLP1 formed trans interactions, supporting the argument for a putative specific role of APLP1 in cell adhesion. Deletion mutants of APP family members revealed two highly conserved regions as important for the protein crosstalk. In particular, the N-terminal half of the ectodomain was crucial for APP and APLP2 interactions. By contrast, multimerization of APLP1 was only partially dependent on this domain but strongly on the C-terminal half of the ectodomain. We further observed that coexpression of APP with APLP1 or APLP2 leads to diminished generation of Abeta42. The current data suggest that this is due to the formation of heteromeric complexes, opening the way for novel therapeutic strategies targeting these complexes.

Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect beta-secretase cleavage of APP.

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.

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42.

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.

Overexpression of phage-type RNA polymerase RpoTp in tobacco demonstrates its role in chloroplast transcription by recognizing a distinct promoter type.

Plant cells possess three DNA-containing compartments, the nucleus, the mitochondria and the plastids. Accordingly, plastid gene regulation is fairly complex. Albeit plastids retained their own genome and prokaryotic-type gene expression system by a plastid-encoded RNA polymerase (PEP), they need a second nuclear-encoded plastid transcription activity, NEP. Candidate genes for putative NEP catalytic subunits have been cloned in Arabidopsis thaliana (AtRpoTp) and Nicotiana sylvestris (NsRpoTp). To provide evidence for RpoTp as a gene encoding a NEP catalytic subunit, we introduced the AtRpoTp and NsRpoTp cDNAs into the tobacco nucleus under the control of the strong constitutive CaMV 35S promoter. Analysis of transcription from NEP and PEP promoters in these transgenic plants using primer extension assays revealed enhanced transcription from typical type I NEP promoters as PatpB-289 in comparison with the wild type. These data provide direct evidence that RpoTp is a catalytic subunit of NEP and involved in recognition of a distinct subset of type I NEP promoters.