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  • 1
    Keywords: PEPTIDE ; CELL ; Germany ; IN-VIVO ; SYSTEM ; DISEASE ; RISK ; SITE ; PROTEIN ; MECHANISM ; IMPACT ; BIOLOGY ; SEQUENCE ; CLEAVAGE ; MUTATION ; etiology ; MUTATIONS ; BETA ; PEPTIDES ; FUNCTIONAL DOMAINS ; AD ; ECTODOMAIN ; DIMERIZATION ; CYTOTOXICITY ; SENILE PLAQUES ; E-cadherin ; MOLECULAR-MECHANISM ; RESIDUES ; INCREASE ; LEADS ; SUBSTRATE ; LEVEL ; RISK-FACTOR ; PRECURSOR ; amyloid A beta ; amyloid precursor protein (APP) ; BETA-PROTEIN ; DIMERIZATION MOTIF ; FAMILIAL ALZHEIMERS-DISEASE ; GAMMA-SECRETASE ACTIVITY ; GxxxG ; HELIX ASSOCIATION ; INTRAMEMBRANE CLEAVAGE ; secretase
    Abstract: Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (A beta) peptides with varying lengths. Particularly A beta 42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease ( AD). However, the precise molecular mechanism of A beta 42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Ab 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 A beta 42, leave the level of A beta 40 unaffected, but increase A beta 38 and shorter Ab 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 A beta 42 production
    Type of Publication: Journal article published
    PubMed ID: 17332749
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  • 2
    Keywords: CAENORHABDITIS-ELEGANS ; APOLIPOPROTEIN-E ; NEURITE OUTGROWTH ; OXIDATIVE STRESS ; HYDROGEN-PEROXIDE ; PICHIA-PASTORIS ; BETA-PROTEIN ; A-BETA ; CEREBROSPINAL-FLUID LIPOPROTEINS ; PRIMARY NEURONAL CULTURES
    Abstract: The amyloid precursor protein (APP) of Alzheimer's disease (AD) has a copper binding domain (CuBD) located in the N-terminal cysteine-rich region that can strongly bind copper(II) and reduce it to Cu(I) in vitro. The CuBD sequence is similar among the APP family paralogs [amyloid precursor-like proteins (APLP1 and APLP2)] and its orthologs (including Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans), suggesting an overall conservation in its function or activity. The APP CuBD is involved in modulating Cu homeostasis and amyloid beta peptide production. In this paper, we demonstrate for the first time that Cu-metallated full-length APP ectodomain induces neuronal cell death in vitro. APP Cu neurotoxicity can be induced directly or potentiated through Cu(I)-mediated oxidation of low-density lipoprotein, a finding that may have important implications for the role of lipoproteins and membrane cholesterol composition in AD. Cu toxicity induced by human APP, Xenopus APP, and APLP2 CuBDs is dependent on conservation of histidine residues at positions corresponding to 147 and 151 of human APP. Intriguingly, APP orthologs with different amino acid residues at these positions had dramatically altered Cu phenotypes. The corresponding C. elegans APL-1 CuBD, which has tyrosine and lysine residues at positions 147 and 151, respectively, strongly protected against Cu-mediated lipid peroxidation and neurotoxicity in vitro. Replacement of histidines 147 and 151 with tyrosine and lysine residues conferred this neuroprotective Cu phenotype to human APP, APLP2, and Xenopus APP CuBD peptides. Moreover, we show that the toxic and protective CuBD phenotypes are associated with differences in Cu binding and reduction. These studies identify a significant evolutionary change in the function of the CuBD in modulating Cu metabolism. Our findings also suggest that targeting of inhibitors to histidine residues at positions 147 and 151 of APP could significantly alter the oxidative potential of APP.
    Type of Publication: Journal article published
    PubMed ID: 11784781
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  • 3
    Keywords: CELLS ; SITES ; CLONING ; PROTEIN ; DOMAIN ; BINDING ; CANDIDATE GENE ; ENCODES ; MUTATION ; TRAFFICKING ; MUTATIONS ; MAMMALIAN-CELLS ; AUSTRALIA ; ATX1 METALLOCHAPERONE ; FUNCTIONAL EXPRESSION ; SURFACE-PLASMON RESONANCE ; TRANSPORTING ATPASE ; WILSON DISEASE GENE
    Abstract: Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two- hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper- dependent chaperone-MBS interactions were determined to be 10(-5)-10(-6) M for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs
    Type of Publication: Journal article published
    PubMed ID: 12679332
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  • 4
    Keywords: brain ; PEPTIDE ; CELLS ; INHIBITOR ; CELL ; Germany ; human ; DISEASE ; SITE ; PROTEIN ; TISSUE ; COMPLEX ; COMPLEXES ; MECHANISM ; FAMILY ; primary ; CONTRAST ; mechanisms ; BIOLOGY ; MOLECULAR-BIOLOGY ; CLEAVAGE ; FORM ; MUTANT ; IDENTIFICATION ; DIFFERENCE ; PLASMA ; MEMBRANE ; inactivation ; SURFACE ; STABILITY ; LIPID RAFTS ; AMYLOID PRECURSOR PROTEIN ; protease ; ACQUIRE ; MOLECULAR-MECHANISM ; IMPAIRMENT ; ALZHEIMERS BETA-SECRETASE ; ASPARTYL PROTEASE ; CONVERTING-ENZYME
    Abstract: beta-Site APP-cleaving enzyme (BACE) is a membranebound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity
    Type of Publication: Journal article published
    PubMed ID: 15247262
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  • 5
    Keywords: CELLS ; EXPRESSION ; CELL ; Germany ; SYSTEM ; SYSTEMS ; PROTEIN ; BINDING ; ALZHEIMERS-DISEASE ; MASS-SPECTROMETRY ; glycosylation ; OXYGEN ; reactive oxygen species ; PRION PROTEIN ; REACTIVE OXYGEN ; function ; manganese ; physiological function ; prion ; PRP ; METALS ; superoxide dismutase ; copper ; Pichia pastoris ; SCRAPIE ; SOD-1 ACTIVITY
    Abstract: The prion protein (PrP) is the key protein implicated in transmissible spongiform encephalopathies. It is a metalloprotein that binds manganese and copper. The latter is involved in the physiological function of the protein. We have previously found that PrP expression in Pichia pastoris affects intracellular metal ion concentrations and that formation of protease-resistant PrP is induced by additional copper and/or manganese. In this study, we show that heterologously expressed PrP is post-translationally modified and transported to the cell wall. We found by combining three different test systems that PrP itself had gained superoxide dismutase-like activity in P. pastoris. However, this activity could not be inhibited by KCN and depended on additional copper in the medium. Thus, this study defines the conditions under which PrP exhibits superoxide dismutase-like activity by showing that copper must be present for the protein to participate in scavenging and detoxification of reactive oxygen species
    Type of Publication: Journal article published
    PubMed ID: 17263729
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  • 6
    Keywords: PEPTIDE ; IN-VITRO ; Germany ; MODEL ; GENERATION ; DISEASE ; antibodies ; antibody ; OLIGOMERS ; BETA ; PRECURSOR PROTEIN ; SECONDARY STRUCTURE ; DIMER ; FIBRILS ; SENILE PLAQUES
    Type of Publication: Journal article published
    PubMed ID: 12840025
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  • 7
    Keywords: PEPTIDE ; Germany ; IN-VIVO ; NEW-YORK ; SITE ; SITES ; PROTEIN ; TIME ; DOMAIN ; IONS ; BINDING ; BIOLOGY ; MOLECULAR-BIOLOGY ; SEQUENCE ; ACID ; MOUSE ; ASSAY ; REGION ; REGIONS ; SURFACE ; PEPTIDES ; Jun ; KINETICS ; AMYLOID PRECURSOR PROTEIN ; AFFINITY ; DOMAINS ; LACKING ; BINDS ; PRION PROTEIN ; biosensor ; molecular biology ; molecular ; CHEMISTRY ; RECOMBINANT ; CONSTANTS ; AGGREGATION ; RESIDUES ; INCREASE ; AMINO-ACID ; REAL-TIME ; SUPEROXIDE-DISMUTASE ; analysis ; NUCLEAR ; BINDING-SITE ; USA ; surface plasmon resonance ; manganese ; INCREASES ; prion ; PRP ; RESONANCE ; FULL-LENGTH ; binding affinity ; COORDINATION ; copper ; COBALT EXPOSURE ; COPPER-BINDING ; S-ADENOSYLHOMOCYSTEINE HYDROLASE
    Abstract: The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60-91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91-230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II)
    Type of Publication: Journal article published
    PubMed ID: 17345106
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  • 8
    Keywords: TRANSGENIC MICE ; CEREBROSPINAL-FLUID ; TRACE-ELEMENTS ; BINDING-SITE ; AMYLOID-PRECURSOR-PROTEIN ; A-BETA ; NEURODEGENERATIVE DISORDERS ; VENTRICULAR FLUID ; PLAQUE-FORMATION ; SECRETED FORMS
    Abstract: Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.
    Type of Publication: Journal article published
    PubMed ID: 12086681
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  • 9
    Keywords: DISEASE ; MECHANISM ; REDUCTION ; OXIDATIVE STRESS ; HYDROGEN-PEROXIDE ; CHAPERONE ; FREE-RADICALS ; METAL-IONS ; ZINC SUPEROXIDE-DISMUTASE ; ALZHEIMER A-BETA
    Abstract: The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.
    Type of Publication: Journal article published
    PubMed ID: 12135352
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  • 10
    ISSN: 0300-9084
    Keywords: APP ; heparin binding site ; processing of APP ; zinc binding site ; βA4 amyloid formation
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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