Amyloid Precursor Protein Dimerization Research Articles

APP family member dimeric complexes are formed predominantly in synaptic compartments

BackgroundThe amyloid precursor protein (APP), a key player in Alzheimer’s disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions.ResultsWe investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls.ConclusionsTogether, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Characterization of the role of Kunitz-type protease inhibitor domain in dimerization of amyloid precursor protein.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo- and hetero-dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo-dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero-dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP-APP interactions using coarse-grained models with the Martini force field. Though the model initial ternary complexes (KPI-E1, KPI-KPI, KPI-E2, E1-E1, E2-E2, and E1-E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane-bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo-dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.

Molecular dynamical analysis of potential dimers produced by C55 portion of the amyloid precursor protein

Alzheimer’s disease is a neurodegenerative disease that affects 5.5 million people in the US alone. Agglomerations of amyloid beta (Aβ) peptides are theorized to be one of the causes of the disease. Aβ is created through cleavage of the amyloid precursor protein (APP), a transmembrane receptor protein, by Gamma-secretase. The cleavage leaves the part of the protein, approximately the C55 portion, in the lipid membrane. The dimerization of APP is theorized to limit the production of Aβ by preventing the binding of Gamma secretase.

The C55 Peptide Portion of the Amyloid Precurosr Protein can Form Stable Dimers

Alzheimer's disease (AD) is a neurodegenerative disease of profound cost in terms of economic impact and human suffering that is becoming prevalent as life expectancy in the world's population rises. An important step in the pathology of Alzheimer's disease is the production of toxic amyloid β peptides (Aβ) by the processive cleavage of the amyloid precursor protein (APP). APP is cleaved by β-secretase producing C99 peptide which is processivity cleaved by γ-secretase to produce Aβ. There is no consensus on the effect APP dimerization has on γ-secretase cleavage and Aβ production, as studies have shown that APP dimers inhibits cleavage and others show APP dimers promote cleavage by γ-secretase. To further illuminate the effect of APP dimerization on its cleavage by γ-secretase, the structure of the APP dimer is needed. Using coarse grained enhanced sampling by the molecular dynamics engine, Upside (Jumper et. al. PLOS Computational Biology 2018), we created a large ensemble structure of C55 (the intermembrane and Aβ part of C99) dimers. After using principle component analysis (PCA) and k-means clustering we chose 10 starting conformations of the C55 dimer, which were then equilibrated for 100 nanoseconds in an explicit all-atom POPC membrane. PCA of these trajectories showed 7 possible stable dimer conformations. Dimers were mainly stabilized by interactions of hydrophobic residues and residues in the GxxxG zipper motif. The G37/38 helix kink and curvature of the helices observed experimentally in C99, were not observed in our conformations. initial binding free energy calculations for these dimers show strong interactions which are indicative of the viability, and stability, for these structures. These conformations can be used to assess the effect APP dimerization has on its cleavage mechanism of γ-secretase and the production of Aβ.

Cholesterol-lowering drugs reduce APP processing to Aβ by inducing APP dimerization.

Amyloid beta (Aβ) is a major component of amyloid plaques, which are a key pathological hallmark found in the brains of Alzheimer’s disease (AD) patients. We show that statins are effective at reducing Aβ in human neurons from nondemented control subjects, as well as subjects with familial AD and sporadic AD. Aβ is derived from amyloid precursor protein (APP) through sequential proteolytic cleavage by BACE1 and γ-secretase. While previous studies have shown that cholesterol metabolism regulates APP processing to Aβ, the mechanism is not well understood. We used iPSC-derived neurons and bimolecular fluorescence complementation assays in transfected cells to elucidate how altering cholesterol metabolism influences APP processing. Altering cholesterol metabolism using statins decreased the generation of sAPPβ and increased levels of full-length APP (flAPP), indicative of reduced processing of APP by BACE1. We further show that statins decrease flAPP interaction with BACE1 and enhance APP dimerization. Additionally, statin-induced changes in APP dimerization and APP-BACE1 are dependent on cholesterol binding to APP. Our data indicate that statins reduce Aβ production by decreasing BACE1 interaction with flAPP and suggest that this process may be regulated through competition between APP dimerization and APP cholesterol binding.

Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growthfactor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and invitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

The Effect of Amyloid Precursor Protein Dimerization on its Conformation and Cleavage

Alzheimer's disease (AD) is an important, increasingly prevalent neurological disorder. Aggregation of Amyloid β (Aβ) is important in etiology of AD. Aβ arises from a specific cut of the Amyloid precursor protein (APP). The C99 peptide part of APP is the intermediary that contains the toxic Aβ1-40 and Aβ1-42 peptides (among other products). A potentially significant feature of C99 is its dimerization behavior that may affect its recognition by the γ-secretase enzyme for cleavage. To understand the mechanisms and effects of dimerization, we simulate the C55 part of C99 peptide (the intermembrane part) at monomeric and dimeric levels using POPC and DMPC lipids. To accelerate the membrane contribution to the conformational changes of C55 without the loss of atomistic detail, we also used the novel high mobility mimetic membrane (HMMM) models of POPC and DMPC. Our large-scale molecular dynamics simulations showed that at the monomeric level the structure displayed dependence on the membrane lipid composition. While the transmembrane helix remained stable, the G37/38 kink observed experimentally did not manifest under different lipid compositions. The extracellular part (residues 1-23) develops β-conformation and the juxta-membrane helix disappeared rather quickly. At the dimeric level, we built four conformations: we clustered our monomer simulations and chose the representatives of the largest clusters as the basis for two initial dimer configurations. For the other two we used structures from Sanders' experimentally determined C99 structure (PDB: 2LP1). We observed that when the N-terminal residues (50-55) of the monomers are in close proximity, the dimer stabilizes. Similarly, interaction of extramembrane residues (1-23) help stabilize the dimer, especially with the presence of β-strands in this region. To better explore the possible conformations of C55-dimers, we are supplementing these observations with enhanced sampling methods.

The crystal structure of amyloid precursor-like protein 2 E2 domain completes the amyloid precursor protein family.

The amyloid precursor-like protein 2 (APLP2) molecule is a type I transmembrane protein that is crucial for survival, cell-cell adhesion, neuronal development, myelination, cancer metastasis, modulation of metal, and glucose and insulin homeostasis. Moreover, the importance of the amyloid precursor protein (APP) family in biology and disease is very well known because of its central role in Alzheimer disease. In this study, we determined the crystal structure of the independently folded E2 domain of APLP2 and compared that with its paralogues APP and APLP2, demonstrating high overall structural similarities. The crystal structure of APLP2 E2 was solved as an antiparallel dimer, and analysis of the protein interfaces revealed a distinct mode of dimerization that differs from the previously reported dimerization of either APP or APLP1. Analysis of the APLP2 E2 metal binding sites suggested it binds zinc and copper in a similar manner to APP and APLP1. The structure of this key protein might suggest a relationship between the distinct mode of dimerization and its biologic functions.-Roisman, L. C., Han, S., Chuei, M. J., Connor, A. R., Cappai, R. The crystal structure of amyloid precursor-like protein 2 E2 domain completes the amyloid precursor protein family.

Amyloid Precursor Protein Dimerisation Reduces Neurite Outgrowth

The amyloid precursor protein (APP) undergoes extensive metabolism, and its transport and proteolytic processing can be modulated by its ability to form a homodimer. We have investigated the functional consequences of stabilised APP dimer expression in cells by studying the engineered dimerisation of the APPL17C (residue 17 in Aβ sequence) construct, which is associated with a 30% increase in APP dimer expression, on APP's neurite outgrowth promoting activity. Overexpression of APPL17C in SH-SY5Y cells decreased neurite outgrowth upon retinoic acid differentiation as compared to overexpressing APPWT cells. The APPL17C phenotype was rescued by replacing the APPL17C media with conditioned media from APPWT cells, indicating that the APPL17C mutant is impairing the secretion of a neuritogenic promoting factor. APPL17C had altered transport and was localised in the endoplasmic reticulum. Defining the molecular basis of the APPL17C phenotype showed that RhoA GTPase activity, a negative regulator of neurite outgrowth, was increased in APPL17C cells. RhoA activity was decreased after APPWT conditioned media rescue. Moreover, treatment with the RhoA inhibitor, Y27632, restored a wild-type morphology to the APPL17C cells. Small RNAseq analysis of APPL17C and APPWT cells identified several differentially expressed miRNAs relating to neurite outgrowth. Of these, miR-34a showed the greatest decrease in expression. Lentiviral-mediated overexpression of miR-34a rescued neurite outgrowth in APPL17C cells to APPWT levels and changed RhoA activation. This study has identified a novel link between APP dimerisation and its neuritogenic activity which is mediated by miR-34a expression.

A new etiologic model for Alzheimers Disease

This etiologic model proposes that Alzheimers Disease (AD) arises when an unusually rapid increase in ventricle volume triggers axon stretch that culminates in the physical separation of trans-synaptic proteins. As a result, these proteins, such as neurexin, neuroligin, N-Cadherin and Amyloid Precursor Protein (APP), experience a change in the configuration of their cytoplasmic tail, so that instead of transmitting signals to create and maintain synaptic structure they activate enzymes, and generate molecules, that stimulate neurite growth; for example, the transformation of the N-Cadherin tail dissolves its complex with presenilin and β-catenin triggering activation of glycogen synthase kinase 3 beta (GSK3β) and cytoskeletal disruption. This disruption leads to an increase in pro nerve growth factor (proNGF), a molecule that stimulates neurite growth via the p75 neurotrophin receptor (p75). GSK3β contributes to this growth by increasing microtubule instability through the phosphorylation of tau. Separation of trans-synaptic APPs leads to their cis dimerization and this stimulates production of amyloid beta (Aβ), an autocrine growth factor that interacts with both the p75 and alpha 7 nicotinic acetylcholine receptors. Cis dimerization of APPs may also allow the autophosphorylation of Y682 and T668 in the APP cytoplasmic tail, triggering activation of c-Jun N terminal kinase, Abelson kinase and cyclin dependent kinase 5, all of which play a role in neurite growth. ProNGF, Aβ and the kinase cascades work together to transform synapses into growth cones and stimulate sprouting of neuropil threads in an attempt to reconnect axons and dendrites. Neurofibrillary tangles, located in neural cell soma, consist of neurofilaments and microtubules needed to fuel this renewal of neurite growth. The model suggests that the best way to treat AD is to prevent synaptic separation by identifying individuals experiencing unusually high rates of ventricle growth and reducing this to more normal levels by shunting or some other technique.

Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases is a determining factor in Alzheimer's disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aβ production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aβ production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.

In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain (TM) Structure and Dimerization in Native E. Coli Membranes

Cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases leads to generation of amyloid peptide (Aβ), which is the primary constituent of Alzheimer's disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment.

Palmitoylated APP Forms Dimers, Cleaved by BACE1.

A major rate-limiting step for Aβ generation and deposition in Alzheimer’s disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8–10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein

Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer's disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C9923-55 monomer and homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarse-grained models. We demonstrate that the C9923-55 homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C991-55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-β protein, on membrane thickness and lipid composition.

Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aβ Oligomers in Cells.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G29XXXG33 and G38XXXA42 motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.

Extension of a protein docking algorithm to membranes and applications to amyloid precursor protein dimerization.

Novel adjustments are introduced to the docking algorithm, DOCK/PIERR, for the purpose of predicting structures of transmembrane protein complexes. Incorporating knowledge about the membrane environment is shown to significantly improve docking accuracy. The extended version of DOCK/PIERR is shown to perform comparably to other leading docking packages. This membrane version of DOCK/PIERR is applied to the prediction of coiled-coil homodimer structures of the transmembrane region of the C-terminal peptide of amyloid precursor protein (C99). Results from MD simulation of the C99 homodimer in POPC bilayer and docking are compared. Docking results are found to capture key aspects of the homodimer ensemble, including the existence of three topologically distinct conformers. Furthermore, the extended version of DOCK/PIERR is successful in capturing the effects of solvation in membrane and micelle. Specifically, DOCK/PIERR reproduces essential differences in the homodimer ensembles simulated in POPC bilayer and DPC micelle, where configurational entropy and surface curvature effects bias the handedness and topology of the homodimer ensemble.

Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing

Alzheimer’s disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

Independent relationship between amyloid precursor protein (APP) dimerization and γ-secretase processivity.

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.

Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain.

The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer's disease (AD). In addition, many physiological functions have been described for APP, including a role in cell-cell- and cell-ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.

Copper enhances APP dimerization and promotes Aβ production

Alzheimer's disease (AD) is characterized by the deposition of amyloid-β (Aβ) plaques, senile plaque. The Aβ peptide is cleaved from amyloid precursor protein (APP) by β-secretase and γ-secretase. Until now, many literatures have documented that the high concentration of copper is present in Aβ plaques and enhances aggregation of. The APP copper binding domain (CuBD) is located in the N-terminal next to the growth factor-like domain that gets involved in APP homodimerization. Importantly, dimerization of APP has profound effect on Aβ production. We investigated whether copper alters the state of APP dimerization and how it affects APP metabolism. Here, we demonstrate that copper enhanced APP dimerization and increased extracellular release of Aβ. Moreover, copper chelator, d-penicillamine, suppressed APP dimerization and decreased extracellular release of Aβ. These results suggest that the action of copper may be profoundly associated with the pathway of Aβ production in AD pathogenesis.