Interaction Of Amyloid Precursor Protein Research Articles

Role of APP Interactions with Heterotrimeric G Proteins: Physiological Functions and Pathological Consequences.

Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth via adenylyl cyclase/PKA-dependent pathways. These reports offer the intriguing perspective that G protein switching might modulate APP-dependent responses in a context-dependent manner. In this review, we provide an up-to-date perspective on the model that APP plays a variety of roles as an atypical G protein-coupled receptor in both the developing and adult nervous system, and we discuss the hypothesis that disruption of these normal functions might contribute to the progressive neuropathologies that typify AD.

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration.

Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimer's disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL-Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin-APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin-APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD. Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained controversial. This research provides new evidence that members of the Contactin family function as authentic ligands for APP and its orthologs, and that this evolutionarily conserved class of membrane-attached proteins regulates key aspects of APP-dependent migration and outgrowth in the embryonic nervous system. By defining the normal role of Contactin-APP signaling during development, these studies also provide the framework for investigating how the misregulation of Contactin-APP interactions might contribute to neuronal dysfunction in the context of both normal aging and neurodegenerative conditions, including Alzheimer's disease.

Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.

BackgroundThe metalloprotease meprin β cleaves the Alzheimer’s Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.ResultsHerein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).ConclusionConcluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0084-5) contains supplementary material, which is available to authorized users.

The Amyloid Precursor Protein-A Novel Player within the Molecular Array of Presynaptic Nanomachines.

More than 20 years ago the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease (AD). The pathophysiology of AD, characterized by a massive loss of synapses, cognitive decline, and behavioral changes was in principle attributed to the accumulation of Aβ. Within the last decades, much effort has gone into understanding the molecular basis of the progression of AD. However, little is known about the actual physiological function of APPs. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone (PAZ) highlights APP as a hitherto unknown player within the setting of the presynapse. The molecular array of presynaptic nanomachines comprising the life cycle of synaptic vesicles, exo- and endocytosis, cytoskeletal rearrangements, and mitochondrial activity provides a balance between structural and functional maintenance and diversity. The generation of genetically designed mouse models further deciphered APP as an essential player in synapse formation and plasticity. Deletion of APP causes an age-dependent phenotype: while younger mice revealed almost no physiological impairments, this condition was changed in the elderly mice. Interestingly, the proteomic composition of neurotransmitter release sites already revealed substantial changes at young age. These changes point to a network that incorporates APP into a cluster of nanomachines. Currently, the underlying mechanism of how APP acts within these machines is still elusive. Within the scope of this review, we shall construct a network of APP interaction partners within the PAZ. Furthermore, we intend to outline how deletion of APP affects this network during space and time leading to impairments in learning and memory. These alterations may provide a molecular link to the pathogenesis of AD and the physiological function of APP in the central nervous system.

Cholesterol-induced astrocyte activation is associated with increased amyloid precursor protein expression and processing.

Cholesterol is essential for maintaining lipid raft integrity and has been regarded as a crucial regulatory factor for amyloidogenesis in Alzheimer's disease (AD). The vast majority of studies on amyloid precursor protein (APP) metabolism and amyloid β-protein (Aβ) production have focused on neurons. The role of astrocytes remains largely unexplored, despite the presence of activated astrocytes in the brains of most patients with AD and in transgenic models of the disease. The role of cholesterol in Aβ production has been thoroughly studied in neurons and attributed to the participation of lipid rafts in APP metabolism. Thus, in this study, we analyzed the effect of cholesterol loading in astrocytes and analyzed the expression and processing of APP. We found that cholesterol exposure induced astrocyte activation, increased APP content, and enhanced the interaction of APP with BACE-1. These effects were associated with an enrichment of ganglioside GM1-cholesterol patches in the astrocyte membrane and with increased ROS production. GLIA 2015;63:2010-2022.

α(2A) adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction.

Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer's disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α(2A) adrenergic receptor (α(2A)AR). Genetic deficiency of the α(2A)AR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α(2A)AR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α(2A)AR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α(2A)AR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α(2A)AR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α(2A)AR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α(2A)AR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α(2A)R antagonists would be an effective therapeutic strategy for AD.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Specific antibody binding to the APP672-699 region shifts APP processing from α- to β-cleavage.

Alzheimer's disease (AD), a progressive neurodegenerative disorder that is the most common cause of dementia in the elderly, is characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles, as well as a progressive loss of synapses and neurons in the brain. The major pertinacious component of amyloid plaques is Aβ, a variably sized peptide derived from the integral membrane protein amyloid precursor protein (APP). The Aβ region of APP locates partly within its ecto- and trans-membrane domains. APP is cleaved by three proteases, designated as α-, β-, and γ-secretases. Processing by β- and γ-secretase cleaves the N- and C-terminal ends of the Aβ region, respectively, releasing Aβ, whereas α-secretase cleaves within the Aβ sequence, releasing soluble APPα (sAPPα). The γ-secretase cleaves at several adjacent sites to yield Aβ species containing 39–43 amino acid residues. Both α- and β-cleavage sites of human wild-type APP are located in APP672–699 region (ectodomain of β-C-terminal fragment, ED-β-CTF or ED-C99). Therefore, the amino acid residues within or near this region are definitely pivotal for human wild-type APP function and processing. Here, we report that one ED-C99-specific monoclonal antibody (mAbED-C99) blocks human wild-type APP endocytosis and shifts its processing from α- to β-cleavage, as evidenced by elevated accumulation of cell surface full-length APP and β-CTF together with reduced sAPPα and α-CTF levels. Moreover, mAbED-C99 enhances the interactions of APP with cholesterol. Consistently, intracerebroventricular injection of mAbED-C99 to human wild-type APP transgenic mice markedly increases membrane-associated β-CTF. All these findings suggest that APP672–699 region is critical for human wild-type APP processing and may provide new clues for the pathogenesis of sporadic AD.

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein

Ca2+ dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca2+ homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca2+ homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca2+ upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca2+ in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca2+ store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca2+ release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca2+ homeostasis affect APP protein interactions. Our results suggest that Ca2+ and Homers play a significant role in the development of Alzheimer's disease pathology.

Sortilin and SorLA Display Distinct Roles in Processing and Trafficking of Amyloid Precursor Protein

The development and progression of Alzheimer's disease is linked to excessive production of toxic amyloid-β peptide, initiated by β-secretase cleavage of the amyloid precursor protein (APP). In contrast, soluble APPα (sAPPα) generated by the α-secretase is known to stimulate dendritic branching and enhance synaptic function. Regulation of APP processing, and the shift from neurotrophic to neurotoxic APP metabolism remains poorly understood, but the cellular localization of APP and its interaction with various receptors is considered important. We here identify sortilin as a novel APP interaction partner. Like the related APP receptor SorLA, sortilin is highly expressed in the CNS, but whereas SorLA mainly colocalizes with APP in the soma, sortilin interacts with APP in neurites. The presence of sortilin promotes α-secretase cleavage of APP, unlike SorLA, which inhibits the generation of all soluble products. Also, sortilin and SorLA both bind and mediate internalization of sAPP but to different cellular compartments. The interaction involves the 6A domain of APP, present in both neuronal and non-neuronal APP isoforms. This is important as sAPP receptors described so far only bind the non-neuronal isoforms, leaving SorLA and sortilin as the only receptors for sAPP generated by neurons. Together, our findings establish sortilin, as a novel APP interaction partner that influences both production and cellular uptake of sAPP.

Retromer Regulates Postendocytic Sorting of β‐Secretase in Polarized Madin–Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.

Interactome of the Amyloid Precursor Protein APP in Brain Reveals a Protein Network Involved in Synaptic Vesicle Turnover and a Close Association with Synaptotagmin-1

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.

P4‐071: Involvement of X11/X11L in Alzheimer's‐related beta‐amyloid generation and epileptogenesis

Background: Amyloid beta (Ab), originating from cleavage of APP (amyloid precursor protein), is a hallmark of Alzheimer’s disease. P75NTR is known to regulate Abeta deposition in the brain but its role in APP trafficking and processing is not known. Methods: We use FRET (fluorescence resonance energy transfer) analysis to characterize the nature of the APPp75NTR interaction in HEK293 cells which were transfected with p75NTR-CFP and APP-YFP plasmids and controls. These cells were subjected to a range of neurotrophins, and Ab, at differing concentrations. Co-immunoprecipitation and fractionation were used to examine the interaction between p75NTR and APP. Results: The FRET efficiency of APP-p75NTR was significantly higher than the negative control. All neurotrophins triggered an increase in the FRETefficiency between p75NTR and APP. Interestingly, Ab at all concentrations triggered a dramatic increase in FRET efficiency.The interaction increased dramatically within the first 1-3 minutes of the addition of Ab to the cells. Co-IP data support that Abeta enhanced the interaction between APP and p75NTR. When a phosphorylation inhibitor KT5720 was added to transfected cells with added Ab, FRET efficiency dropped. Reciprocally, when a phosphorylation activator 8-BrcAMP was added, FRET efficiency increased significantly. This data seems to support the idea that p75NTR-APP interaction is under the influence of a phosphorylation event. Furthermore FRET efficiency dropped further by blocking endogenous Ab with an antibody 6E10, indicating endogenous Ab may increase the interaction of APP and p75NTR. These results could suggest that Ab may have a positive feed forward mechanism that increases an output of Ab via the interaction among Abeta, p75NTR and APP. Conclusions: We conclude that p75NTR ligands Ab and neurotrophins enhance the interaction of p75NTR-APP in a phosphorylation dependent manner, and the increased interaction of p75NTR-APPmay participate in the pathogenesis of AD.

LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer’s disease

BackgroundThe Aβ peptide that accumulates in Alzheimer’s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by β- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aβ production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aβ production.ResultsIn this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aβ, while knockdown of LRP10 expression increases Aβ production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP β-cleavage into Aβ. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.ConclusionsThe present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer’s disease.

RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing

Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

Erratum to: Selective Interaction of Amyloid Precursor Protein with Different Isoforms of Neural Cell Adhesion Molecule

Erratum to: Selective Interaction of Amyloid Precursor Protein with Different Isoforms of Neural Cell Adhesion Molecule

Relationships between the amyloid precursor protein and its various proteolytic fragments and neuronal systems.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and in its familial form is associated with mutations in the amyloid precursor protein (APP) and the presenilins (PSs). Much data regarding the interactions of APP, its proteolytic fragments and PS have been generated, expanding our understanding of the roles of these proteins in mechanisms underlying cognitive function and revealing many complex relationships with wide ranging cellular systems. In this review, we examine the multiple interactions of APP and its proteolytic fragments with other neuronal systems in terms of feedback loops and use these relationships to build a map. We highlight the complexity involved in the APP proteolytic system and discuss alternative perspectives on the roles of APP and its proteolytic fragments in dynamic processes associated with disease progression in AD. We highlight areas where data are missing and suggest potential confounding factors. We suggest that a systems biology approach enhances representations of the data and may be more useful in modelling both normal cognition and disease processes.

Structural Studies on the Similarity and Potential Interaction Between Aβ42 and Prion Peptides

The Aβ42 peptide associated with Alzheimer's Disease is derived by proteolysis of the amyloid precursor protein (APP). Within the Aβ sequence are three consecutive GxxxG motifs that mediate both helix interactions in APP and β-sheet interactions in Aβ42. The GxxxG motif was originally shown to mediate helix interactions in the TM domain of glycophorin A. More recent studies have shown that amyloid fibrils formed from glycophorin A fragments contain parallel and in-register β-strands, as in Aβ42, and that the β-sheets within glycophorin A fibrils are stabilized by a central ridge of methionine residues packing against grooves created by glycines of the GxxxG motif. Strikingly, sequence analysis of the human prion protein reveals three consecutive GxxxG motifs in an amyloidogenic region from 118-135.In this investigation, 13C solid-state NMR spectroscopy is used to probe the structure of amyloid fibrils formed from the human PrP(118-135) peptide. We show that fibrils of PrP(118-135) have a parallel and in-register architecture where β-sheet-to-β-sheet packing is facilitated by side chain interactions involving Met129 packing against either Gly127 or Gly131 of a GxxxG motif. Peptide inhibitors, designed with a GxFxGxF framework and previously shown to block Aβ fibril formation, were tested on PrP(118-135) and found to inhibit fibril formation and block inter-sheet packing. The data show both Aβ and PrP(118-135) form similar fibrils and can be inhibited using the same generically designed peptide.Interestingly, both Aβ and PrP form β-sheet rich aggregates that cause neurodegenerative disease. Lauren et al. (Nature 457, 1128) proposed Aβ42 might exert toxicity on neuronal cells by binding to human prion protein at residues 95-110. We have carried out FTIR and NMR studies to characterize interactions between PrP (95-136) and Aβ42, and the possible role of GxxxG sequences.

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.

Cellular effects of APOE4: Implications for Alzheimer's disease

The dominant risk factor for Alzheimer's disease (AD) is the epsilon-4 allele of apolipoprotein E (ApoE4). This allele confers increased risk for sporadic as well as familial AD disease. Individuals with two copies of the ApoE e4 allele have an approximately eight-fold increased risk of AD and have a significantly lower age of onset compared to AD patients not carrying this allele. Recent data indicate that the greater risk of AD associated with the ApoE4 isoform might relate to ApoE's susceptibility to proteolysis and neurotoxicity. However, the exact molecular mechanisms underlying ApoE and amyloid precursor protein (APP) interactions sub serving the risk-factor effect of ApoE4 remain unclear. The amyloid precursor protein, APP, has been shown to function as a molecular switch: cleavage at the beta, gamma, and caspase sites results in the production of four peptides-sAPPb (from which N-APP is derived), Ab, Jcasp, and C31-that mediate neurite retraction, synaptic reorganization, and ultimately programmedcell death. In contrast, cleavage at the alpha site produces the trophic peptide sAPPa and the inhibitor of APP gamma-site cleavage, aCTF. The decision between these two proteolytic pathways is governed at least in part by ligand binding: interaction with the axon guidance and trophic factor netrin-1 increases a-site cleavage, whereas interaction with the anti-trophin Ab inhibits alpha-site cleavage and increases net production of the four neurite-retractive peptides. Since this system exhibits positive feedback (e.g., Ab begets more Ab via APP processing, whereas aCTF inhibits Ab production), it is by definition prionic. Therefore, it is of interest to determine whether ApoE isoforms impact this trophic-anti-trophic peptide balance differentially, and, if so, by what mechanism. HEK-293T cells, B103-APP (rat neuroblastoma cells stably expressing human wild-type APP), 7W CHO cells (stably transfected with human APP751) and A172 human glioblastoma cells were used to determine whether ApoE, and in particular ApoE4, triggered alternative cellular APP processing that would provide a basis for understanding the cellular toxicity that has been reported previously. Transient transfection of cells was performed with Lipofectamine 2000. Whole-cell extracts were prepared and typically, 100mg of cell extract was loaded for SDS-PAGE and Western blot analysis. A total of 150-200 μg protein was also subjected to immunoprecipitation using specific antibodies to pull down the desired protein. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. The sAPPa, sAPPb, and Ab levels were obtained using the PerkinElmer (PE) AlphaLisa assay, and measurements were performed on an AlphaLisa reader. Immunocytochemistry was performed on cells 24 hrs after transfection with APP, ApoE, or the two in combination. The ApoE expression constructs were a kind gift from Dr. Yadong Huang of the Gladstone Institute. Cells were probed with primary antibodies and stained with an AlexaFluor secondary antibody (either 488 or 555). APP transcriptional activity was performed using the Gal4-APP system with a luciferase readout. Cells were harvested 48 h after transfection and the luciferase and b-galactosidase activities were determined with the Promega luciferase assay kit and the Promega b-galactosidase assay kit, respectively. ApoE3, ApoE4, and its proteolytic product ApoE4delta (ApoEdelta272) co-immunoprecipitated with full-length APP both in cell lysates and conditioned media. Interaction of APP with ApoE4 or ApoE4delta was accompanied by decreased sAPPa secretion and reduced sAPPa/Aß and sAPPa/sAPPß ratios as compared to ApoE3. Both ApoE4 and ApoE4δ reduced interaction between APP and Fe65 and also suppressed Fe65-mediated intracellular APPs.