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1.
J Biol Chem ; : 107510, 2024 Jun 27.
Article in English | MEDLINE | ID: mdl-38944120

ABSTRACT

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant ß-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aß to produce the C-terminally truncated non-toxic Aß fragment Aß34 which is an indicator of amyloid clearance. Here, we analyzed effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aß producing) and amyloidolytic (Aß degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aß42 and Aß40 levels. The concomitant decrease in Aß34 and secreted APPß levels suggested that the elevated Aß42 and Aß40 levels were due to the attenuated Aß degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aß degrading activity of BACE1 was more sensitive to inhibition than its Aß producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aß producing and degrading activities of BACE1, i.e., significantly increased Aß42/Aß40 ratio and decreased Aß34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aß peptides and found that BACE1 has higher affinity for Aß substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aß production without decreasing Aß clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

2.
Angew Chem Int Ed Engl ; 60(8): 3882-3904, 2021 02 19.
Article in English | MEDLINE | ID: mdl-32589355

ABSTRACT

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.


Subject(s)
DNA/chemistry , Polyelectrolytes/chemistry , Proteins/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Survival/drug effects , DNA/metabolism , Drug Carriers/chemistry , Glycosaminoglycans/chemistry , Glycosaminoglycans/metabolism , Humans , Polyelectrolytes/metabolism , Protein Binding , Proteins/metabolism , Thermodynamics
3.
J Biol Chem ; 293(21): 8020-8031, 2018 05 25.
Article in English | MEDLINE | ID: mdl-29636413

ABSTRACT

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.


Subject(s)
Neuroblastoma/prevention & control , Prion Proteins/chemistry , Prion Proteins/metabolism , Protein Multimerization , Scrapie/prevention & control , Animals , HeLa Cells , Humans , Mice , Mice, Transgenic , Neuroblastoma/pathology , Protein Transport , Scrapie/pathology , Tumor Cells, Cultured
4.
J Biol Chem ; 292(32): 13258-13270, 2017 08 11.
Article in English | MEDLINE | ID: mdl-28637867

ABSTRACT

The ß-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aß peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Copper/metabolism , Cytosol/metabolism , Models, Molecular , Alanine/chemistry , Amino Acid Substitution , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/genetics , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Cysteine/chemistry , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Point Mutation , Protein Conformation , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
5.
Biochemistry ; 55(12): 1839-49, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-26900939

ABSTRACT

Alzheimer's disease is characterized by deposition of the amyloid ß-peptide (Aß) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aß peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aß solution. We find that sulindac sulfide induced Aß aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a ß-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aß peptide in the aggregate.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Peptide Fragments/metabolism , Protein Aggregates/physiology , Sulindac/analogs & derivatives , Amino Acid Sequence , Amyloid beta-Peptides/toxicity , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Molecular Sequence Data , Peptide Fragments/toxicity , Protein Aggregates/drug effects , Sulindac/pharmacology
6.
J Biol Chem ; 290(48): 28737-45, 2015 Nov 27.
Article in English | MEDLINE | ID: mdl-26416887

ABSTRACT

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-ß (Aß) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aß populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aß fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two ß-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp(23)-Lys(28) salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly(33), a residue involved in Met(35) oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides/chemistry , Sulindac/analogs & derivatives , Amyloid beta-Peptides/metabolism , Anti-Inflammatory Agents, Non-Steroidal , Humans , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Protein Binding , Protein Structure, Secondary , Sulindac/chemistry , Sulindac/therapeutic use
7.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 3): 494-504, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25760599

ABSTRACT

Beyond the pathology of Alzheimer's disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Šresolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2)2-(heparin)2 complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Heparin/chemistry , Humans , Protein Structure, Secondary , Protein Structure, Tertiary
8.
Biomacromolecules ; 15(5): 1910-9, 2014 May 12.
Article in English | MEDLINE | ID: mdl-24725062

ABSTRACT

Copper (Cu) is a cofactor of various metalloenzymes and has a role in neurodegenerative diseases with disturbed Cu homeostasis, for example, in Alzheimer's disease (AD) and Menkes disease. To address Cu imbalances, we synthesized two different dendritic nanoparticles (NP) for the transport of Cu(II) ions across the blood-brain barrier (BBB). The synthesized NPs show low toxicity and high water solubility and can stabilize high amounts of Cu(II). The Cu(II)-laden NPs crossed cellular membranes and increased the cellular Cu level. A human brain microvascular endothelial cell (HBMEC) model was established to investigate the permeability of the NPs through the BBB. By comparing the permeability × surface area product (PSe) of reference substances with those of NPs, we observed that NPs crossed the BBB model two times more effectively than (14)C-sucrose and sodium fluorescein (NaFl) and up to 60× better than Evans Blue labeled albumin (EBA). Our results clearly indicate that NPs cross the BBB model effectively. Furthermore, Cu was shielded by the NPs, which decreased the Cu toxicity. The novel design of the core-shell NP enabled the complexation of Cu(II) in the outer shell and therefore facilitated the pH-dependent release of Cu in contrast to core-multishell NPs, where the Cu(II) ions are encapsulated in the core. This allows a release of Cu into the cytoplasm. In addition, by using a cellular detection system based on a metal response element with green fluorescent protein (MRE-GFP), we demonstrated that Cu could also be released intracellularly from NPs and is accessible for biological processes. Our results indicate that NPs are potential candidates to rebalance metal-ion homeostasis in disease conditions affecting brain and neuronal systems.


Subject(s)
Blood-Brain Barrier/metabolism , Copper/metabolism , Drug Carriers/metabolism , Models, Biological , Nanoparticles/metabolism , Biological Transport , Bone Marrow Cells/chemistry , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Line , Cell Proliferation/drug effects , Copper/administration & dosage , Copper/pharmacology , Dose-Response Relationship, Drug , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Carriers/pharmacology , Endothelial Cells/chemistry , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Structure-Activity Relationship
9.
J Biol Chem ; 289(3): 1540-50, 2014 Jan 17.
Article in English | MEDLINE | ID: mdl-24225948

ABSTRACT

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aß peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aß and in particular Aß42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aß production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aß40 and Aß42 and an increase in shorter Aß peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aß51 to Aß31/Aß30 and from Aß49 to Aß30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aß48 to Aß38 compared with Aß49 to Aß37. These findings correlate with an increase in the Aß42/40 ratio. GSMs caused a decrease in Aß40 and Aß42 and an increase in Aß37 and Aß38 paralleled by an increase of the intermediates Aß40-38 and Aß42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aß production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aß generation.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/biosynthesis , Gene Expression Regulation , Genetic Diseases, Inborn/metabolism , Mutation , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/genetics , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/pathology , HEK293 Cells , Humans
10.
J Am Chem Soc ; 135(51): 19354-61, 2013 Dec 26.
Article in English | MEDLINE | ID: mdl-24304299

ABSTRACT

The ß-secretase or ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-ß peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.


Subject(s)
Amyloid Precursor Protein Secretases/chemistry , Copper/analysis , Models, Biological , Sulfur/chemistry , Amino Acid Motifs , Animals , Circular Dichroism , Colorimetry , Humans , Mice , Rats , Sequence Alignment , Spectroscopy, Fourier Transform Infrared
11.
Chembiochem ; 14(15): 1943-8, 2013 Oct 11.
Article in English | MEDLINE | ID: mdl-24115334

ABSTRACT

Wobbly backbone: The backbone dynamics of the amyloid precursor protein (APP) transmembrane helix was compared to those of other transmembrane domains. In contrast to expectation, no above-average backbone dynamics was found for the APP transmembrane helix; the dynamics thus appears not to be optimized for cleavage.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Cell Membrane/metabolism , Proteolysis , Humans , Molecular Dynamics Simulation , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary
13.
PLoS One ; 8(3): e58837, 2013.
Article in English | MEDLINE | ID: mdl-23520537

ABSTRACT

A key event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of amyloid-ß (Aß) species in the brain, derived from the sequential cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. Based on a systems biology study to repurpose drugs for AD, we explore the effect of lansoprazole, and other proton-pump inhibitors (PPIs), on Aß production in AD cellular and animal models. We found that lansoprazole enhances Aß37, Aß40 and Aß42 production and lowers Aß38 levels on amyloid cell models. Interestingly, acute lansoprazole treatment in wild type and AD transgenic mice promoted higher Aß40 levels in brain, indicating that lansoprazole may also exacerbate Aß production in vivo. Overall, our data presents for the first time that PPIs can affect amyloid metabolism, both in vitro and in vivo.


Subject(s)
2-Pyridinylmethylsulfinylbenzimidazoles/pharmacology , Alzheimer Disease/drug therapy , Amyloid beta-Peptides/biosynthesis , Enzyme Inhibitors/pharmacology , Proton Pump Inhibitors , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , Disease Models, Animal , Female , Humans , Lansoprazole , Mice , Mice, Knockout
15.
Chembiochem ; 13(18): 2657-60, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23161824

ABSTRACT

Aggregation of amyloid ß (Aß(1-42)), causing toxicity, is a critical step in Alzheimer's disease (AD). AD studies are difficult to compare because Aß(1-42) aggregation is poorly controllable under physiological conditions. To control aggregation and toxicity, we engineered light-switchable Aß(1-42) analogues that enable controllable conversion of nontoxic fibrils into toxic oligomers simply by illumination.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/toxicity , Light , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Protein Engineering , Protein Multimerization/radiation effects , Amino Acid Sequence , Cell Line, Tumor , Humans , Molecular Sequence Data , Protein Structure, Secondary/radiation effects
16.
J Biol Chem ; 287(52): 43765-76, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23115236

ABSTRACT

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrP(Sc)) and amyloid ß peptide (Aß) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrP(Sc)- or Aß-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrP(Sc) or Aß. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrP(Sc)- and Aß-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrP(Sc) and Aß. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrP(Sc)- or Aß-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically.


Subject(s)
Amyloid beta-Peptides/metabolism , Cell Membrane/metabolism , HSP72 Heat-Shock Proteins/metabolism , Heat-Shock Response , PrPSc Proteins/metabolism , Amyloid beta-Peptides/genetics , Animals , CHO Cells , Cell Membrane/genetics , Clusterin/genetics , Clusterin/metabolism , Cricetinae , Cricetulus , HSP72 Heat-Shock Proteins/genetics , Humans , PrPSc Proteins/genetics
17.
J Biol Chem ; 287(40): 33304-13, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22879596

ABSTRACT

The amyloid ß (Aß) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin ß cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin ß can also process APP in a manner reminiscent of ß-secretase. We identified cleavage sites of meprin ß in the amyloid ß sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aß variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin ß than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin ß on APP and Aß generation was also observed in the absence of BACE1/2 activity using a ß-secretase inhibitor and BACE knock-out cells, indicating that meprin ß acts independently of ß-secretase.


Subject(s)
Amyloid beta-Peptides/chemistry , Metalloendopeptidases/metabolism , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid Precursor Protein Secretases/metabolism , Brain/metabolism , Catalysis , HEK293 Cells , Humans , Hydroxamic Acids/chemistry , Kinetics , Metalloproteases/chemistry , Molecular Sequence Data , Mutation , Peptides/chemistry , Protein Isoforms , Protein Structure, Tertiary , Proteomics/methods
18.
FASEB J ; 26(9): 3765-78, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22661005

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/metabolism , Membrane Proteins/metabolism , Peptides/metabolism , Presenilins/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , HEK293 Cells , HeLa Cells , Humans , Microscopy, Confocal , Molecular Sequence Data , Surface Plasmon Resonance
19.
EMBO Mol Med ; 4(7): 647-59, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22514144

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Sequence , Amino Acid Substitution , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Peptides/genetics , Cell Line, Tumor , HEK293 Cells , Humans , Molecular Sequence Data , Mutation , Neprilysin/metabolism , Peptide Fragments/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transfection
20.
J Mol Biol ; 416(3): 438-52, 2012 Feb 24.
Article in English | MEDLINE | ID: mdl-22245578

ABSTRACT

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.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Copper/chemistry , Zinc/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Histidine/chemistry , Humans , Molecular Sequence Data , Protein Conformation
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