Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems.

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.

Dimerization of the cellular prion protein inhibits propagation of scrapie prions.

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.

Full-length cellular ß-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

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.

Sulindac Sulfide Induces the Formation of Large Oligomeric Aggregates of the Alzheimer's Disease Amyloid-ß Peptide Which Exhibit Reduced Neurotoxicity.

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.

Copper transport mediated by nanocarrier systems in a blood-brain barrier in vitro model.

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.

Light-controlled toxicity of engineered amyloid ß-peptides.

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.

The metalloprotease meprin ß generates amino terminal-truncated amyloid ß peptide species.

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.

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.

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms.

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.

Is there a malignant progression associated with a linear change in protein expression levels from normal canine mammary gland to metastatic mammary tumors?

The molecular mechanisms of the development of canine mammary tumors are still incompletely understood. In the present study we hypothesized that there is a malignant progression from normal gland to malignant carcinomas that is associated with a linear change in protein expression. To this end, the proteome of canine normal mammary gland, adenomas, nonmetastatic carcinomas, and metastatic carcinomas was compared. Application of 2D-DIGE and MALDI-TOF-MS identified 48 proteins with significant changes (fold change >|1.5|; p < 0.05) in expression levels at the different stages of malignant progression. Forty-two of these followed three major stepwise but not linear expression patterns. Thirteen proteins showed the adenoma pattern characterized by a change in protein expression levels during progression from normal gland to adenomas which persisted on the same level at the subsequent stages of malignancy. Nine proteins followed the carcinoma pattern with an up- or down-regulation between adenomas and carcinomas. The majority of 20 proteins followed the metastasis pattern with a significant change of protein expression levels between nonmetastatic and metastatic carcinomas. The present study therefore shows that differences in malignancy are associated with a stepwise but not linear change in protein expression levels, which does not finally confirm or disapprove the existence of a malignant progression in canine mammary tumors. In addition, the acquisition of metastatic potential seems to be associated with the strongest changes in protein expression levels.

Proteome of metastatic canine mammary carcinomas: similarities to and differences from human breast cancer.

Mammary tumors are a major health threat to women and female dogs. In both, metastasis of the primary tumor to distant organs is the most common cause of tumor-related death. Nevertheless, the molecular mechanisms of tumor metastasis are far from being understood, and it is still unknown why some human and canine carcinomas metastasize and others do not. Using 2D-DIGE and MALDI-TOF-MS we identified 21 proteins with significant changes (fold change >1.5; p < 0.05) in protein expression between metastasizing (n = 6) and nonmetastasizing (n = 6) canine mammary carcinomas. Quantitative RT-PCR was used to identify transcriptional or post-transcriptional regulation of protein expression. Up-regulated proteins in metastatic carcinomas included proliferating cell nuclear antigen, ferritin light chain, bomapin, tropomyosin 3, thioredoxin-containing domain C5, adenosin, ornithine aminotransferase, coronin 1A, RAN-binding protein 1,3-phosphoglycerate dehydrogenase, and eukaryotic translation elongation factor 1. Down-regulated proteins in metastatic carcinomas included calretinin, myosin, light chain 2, peroxiredoxin 6, maspin, ibrinogen beta chain, vinculin, isocitrate dehydrogenase 1, tropomyosin 1, annexin A5, and Rho GTPase activating protein 1. Interestingly, 19 of these 21 proteins have been described with a malignancy-associated expression in human breast cancer and other human cancer types before. Further investigations are now necessary to test whether these markers are of prognostic value for canine mammary carcinomas and whether their expression is directly involved in canine mammary carcinogenesis or represent solely a secondary reactive phenotype.

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis.

N-terminally truncated Aß peptides starting with pyroglutamate (AßpE3) represent a major fraction of all Aß peptides in the brain of Alzheimer disease (AD) patients. AßpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aß. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AßpE3 and studied the potential involvement of oligomeric AßpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aß plaque load and AßpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AßpE3 oligomers.

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.

Insulin-like growth factor-1 (IGF-1)-induced processing of amyloid-beta precursor protein (APP) and APP-like protein 2 is mediated by different metalloproteinases.

alpha-Secretase cleavage of the amyloid precursor protein (APP) is of great interest because it prevents the formation of the Alzheimer-linked amyloid-beta peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas APLP2 shedding is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP alpha-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate whether the difference in the regulation of APLP2 shedding compared with APP shedding could be due to involvement of different processing enzymes. We focused on the two major alpha-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with small interfering RNAs targeted against TACE. The results clearly demonstrate that different alpha-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.

Role of amyloid-beta glycine 33 in oligomerization, toxicity, and neuronal plasticity.

The aggregation of the amyloid-beta (Abeta) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Abeta there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Abeta. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Abeta sequence is important for the aggregation dynamics of the peptide. Abeta peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Abeta(42) wild type (WT), in vitro and in vivo. Also, whereas Abeta(42) WT is known to inhibit LTP, Abeta(42) G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Abeta on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Abeta. Thus, a specific toxic conformation of Abeta exists, which represents a promising target for therapeutic interventions.

Cellular copper import by nanocarrier systems, intracellular availability, and effects on amyloid beta peptide secretion.

Studies in animals have reported that normalized or elevated Cu levels can inhibit or even remove Alzheimer's disease-related pathological plaques and exert a desirable amyloid-modifying effect. We tested engineered nanocarriers composed of diverse core-shell architectures to modulate Cu levels under physiological conditions through bypassing the cellular Cu uptake systems. Two different nanocarrier systems were able to transport Cu across the plasma membrane of yeast or higher eukaryotic cells, CS-NPs (core-shell nanoparticles) and CMS-NPs (core-multishell nanoparticles). Intracellular Cu levels could be increased up to 3-fold above normal with a sublethal dose of carriers. Both types of carriers released their bound guest molecules into the cytosolic compartment where they were accessible for the Cu-dependent enzyme SOD1. In particular, CS-NPs reduced Abeta levels and targeted intracellular organelles more efficiently than CMS-NPs. Fluorescently labeled CMS-NPs unraveled a cellular uptake mechanism, which depended on clathrin-mediated endocytosis in an energy-dependent manner. In contrast, the transport of CS-NPs was most likely driven by a concentration gradient. Overall, nanocarriers depending on the nature of the surrounding shell functioned by mediating import of Cu across cellular membranes, increased levels of bioavailable Cu, and affected Abeta turnover. Our studies illustrate that Cu-charged nanocarriers can achieve a reasonable metal ion specificity and represent an alternative to metal-complexing agents. The results demonstrate that carrier strategies have potential for the treatment of metal ion deficiency disorders.

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.

IGF-1-induced processing of the amyloid precursor protein family is mediated by different signaling pathways.

The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.