The Large Ectodomain of APP Prevents APP from being Directly Cleaved by γ-Secretase.

BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of amyloid-ß peptide (Aß) in the brain. Aß is produced by sequential ß- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting ß- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aß production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aß generation without inhibiting any enzyme. METHODS: An APP mutant mimicking secreted APPγ was overexpressed in cells to test ß-cleavage and Aß production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms. RESULTS: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aß. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aß, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase. CONCLUSIONS: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aß. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy.

Cell-free protein production of a gamma secretase homolog.

Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

A light at the end of the tunnel - from mutation identification to a potential treatment for Alzheimer's disease.

Recent advances have driven the development of immunotherapies that act by either promoting or suppressing a patient's immune system to treat inflammation, autoimmune disease, cardiovascular disease, infectious diseases, and several cancers. In addition, research conducted over the past 25 years has identified therapeutic targets and indicated that immunotherapy could be used to treat Alzheimer's disease (AD). Despite a number of setbacks, this approach has now led to the development of the first disease-modifying treatments for this devastating disease. A key neuropathological feature of AD is the accumulation of a ~40-amino acid peptide known as amyloid ß (Aß) in the brain and cerebrovasculature. Our detection of an Aß precursor protein mutation that caused early-onset AD in a Swedish family by enhancing Aß protofibril formation sharpened the focus on soluble Aß aggregates (oligomers and protofibrils) as viable therapeutic targets. Initial studies developed and tested a mouse monoclonal antibody (mAb158) with specific conformation-dependent binding to these soluble Aß aggregates. Treatment with mAb158 selectively reduced Aß protofibrils in the brain and cerebrospinal fluid of a transgenic mouse model of AD. A humanized version of mAb158 (lecanemab) subsequently entered clinical trials. Based on promising Phase 2 data showing plaque clearance and reduced cognitive decline, a Phase 3 trial found that lecanemab slowed decline on the primary cognitive endpoint by 27% over 18 months and also produced positive effects on secondary clinical endpoints and key biomarkers. In July 2023, the FDA granted lecanemab a full approval, and this therapeutic antibody will be marketed as Leqembi®. This represents a significant advance for patients with AD, although many challenges remain. In particular, it is now more important than ever to identify individuals who are vulnerable to AD, so that treatment can be initiated at an early stage in the disease process.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of ß-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

[Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies].

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-ß peptides (Aß), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aß generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Comprehensive Peptide Cyclization Examination Yields Optimized APP Scaffolds with Improved Affinity toward Mint2.

Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.

Impact of A2T and D23N mutations on C99 homodimer conformations.

The proteolytic cleavage of C99 by γ-secretase is the last step in the production of amyloid-ß (Aß) peptides. Previous studies have shown that membrane lipid composition, cholesterol concentration, and mutation in the transmembrane helix modified the structures and fluctuations of C99. In this study, we performed atomistic molecular dynamics simulations of the homodimer of the 55-residue congener of the C-terminal domain of the amyloid protein precursor, C99(1-55), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-cholesterol lipid bilayer and compared the conformational ensemble of wild-type (WT) sequence to those of the A2T and D23N variants. These mutations are particularly interesting as the protective Alzheimer's disease (AD) A2T mutation is known to decrease Aß production, whereas the early onset AD D23N mutation does not affect Aß production. We found noticeable differences in the structural ensembles of the three sequences. In particular, A2T varies from both WT and D23N by having long-range effects on the population of the extracellular juxtamembrane helix, the interface between the G29xxx-G33xxx-G37 motifs, and the fluctuations of the transmembrane helical topologies.

Live Cell Fluorescence Imaging Shows Neurotransmitter Activation Promotes Aggregation of the Intracellular Domain of Amyloid Precursor Protein.

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCß signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCß1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCß reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

Mechanistic regulation of γ-secretase by their substrates.

γ-Secretase (GS) is a transmembrane (TM) enzyme that plays important roles in the processing of approximately 90 substrates. The amyloid precursor protein (APP) is one of these substrates, and the peptides derived from their processing are related with the development of Alzheimer's disease. However, the mechanistic process involved in the GS substrate processing and regulation remains elusive. In this work, we employed extensive atomistic molecular dynamics simulations, reduction dimensionality, and network analysis to understand the dynamic behavior of GS in its apo form and bound to transmembrane fragments of APP-C99, APP-C83, and Notch. An evaluation of the global conformation of the enzyme revealed that GS and GS-C83 systems display extended and compact conformations. However, systems with long extracellular N-terminal substrates, such as APP-C99 and Notch, preferred compact conformations. Interestingly, our network analysis revealed that the NCT-lobule (residues 223-248) plays a crucial role in the communication and the dynamics between the extracellular and TM components of the enzyme, impacting the catalytic site. In our GS-C99 simulated system, the interaction paths of the substrate processing region encompass the ε-site and ζ-site, leading to more imprecise positioning of the catalytic residue Asp385. Conversely, our GS-C83 simulated system shows more stability at the ε-site. Our observations shed light on the important mechanics of the fascinating GS architecture and may contribute to propose new GS modulators able to impact on the Alzheimer's disease treatment.

Identification of the probable structure of the sAPPα-GABABR1a complex and theoretical solutions for such cases.

Amyloid precursor protein (APP) is the core of the pathogenesis of Alzheimer's disease (AD). Existing studies have shown that the soluble secreted APP (sAPPα) fragment obtained from the hydrolysis of APP by α-secretase has a synaptic function. Thereinto, a nine-residue fragment (APP9mer) of the extension domain region of sAPPα can bind directly and selectively to the N-terminal sushi1 domain (SD1) of the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a) protein, which can influence synaptic transmission and plasticity by changing the GABABR1a conformation. APP9mer is a highly flexible, disordered region, and as such it is difficult to experimentally determine the optimal APPmer-SD1 binding complex. In this study we constructed two types of APP9mer-SD1 complexes through molecular docking and molecular dynamics simulation, aiming to explore the recognition function and mechanism of the specific binding of APP9mer with SD1, from which the most probable APPmer-SD1 model conformation is predicted. All the data from the analyses of RMSD, RMSF, PCA, DCCM and MM/PBSA binding energy as well as comparison with the experimental dissociation constant Kd suggest that 2NC is the most likely conformation to restore the crystal structure of the experimental APP9mer-SD1 complex. Of note, the key recognition residues of APP9mer are D24, D25, D27, W29 and W30, which mainly act on the 9-45 residue domain of SD1 (consisting of two loops and three short ß-chains at the N-terminus of SD1). The mini-model with key residues identified establishes the molecular basis with deep insight into the interaction between APP and GABABR1a and provides a target for the development of therapeutic strategies for modulating GABABR1a-specific signaling in neurological and psychiatric disorders. More importantly, the study offers a theoretical solution for how to determine a biomolecular structure with a highly flexible, disordered fragment embedded within. The flexible fragment involved in a protein structure has to be deserted usually during the structural determination with experimental methods (e.g. X-ray crystallography, etc.).

An internal docking site stabilizes substrate binding to γ-secretase: Analysis by molecular dynamics simulations.

Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid ß (Aß) peptides of different lengths. Longer Aß peptides are associated with the formation of neurotoxic plaques related to Alzheimer's disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aß fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.

γ-Secretase in Alzheimer's disease.

Alzheimer's disease (AD) is caused by synaptic and neuronal loss in the brain. One of the characteristic hallmarks of AD is senile plaques containing amyloid ß-peptide (Aß). Aß is produced from amyloid precursor protein (APP) by sequential proteolytic cleavages by ß-secretase and γ-secretase, and the polymerization of Aß into amyloid plaques is thought to be a key pathogenic event in AD. Since γ-secretase mediates the final cleavage that liberates Aß, γ-secretase has been widely studied as a potential drug target for the treatment of AD. γ-Secretase is a transmembrane protein complex containing presenilin, nicastrin, Aph-1, and Pen-2, which are sufficient for γ-secretase activity. γ-Secretase cleaves >140 substrates, including APP and Notch. Previously, γ-secretase inhibitors (GSIs) were shown to cause side effects in clinical trials due to the inhibition of Notch signaling. Therefore, more specific regulation or modulation of γ-secretase is needed. In recent years, γ-secretase modulators (GSMs) have been developed. To modulate γ-secretase and to understand its complex biology, finding the binding sites of GSIs and GSMs on γ-secretase as well as identifying transiently binding γ-secretase modulatory proteins have been of great interest. In this review, decades of findings on γ-secretase in AD are discussed.

Structure and mechanism of the γ-secretase intramembrane protease complex.

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.

Physiology and pharmacology of amyloid precursor protein.

Amyloid precursor protein (APP) is an evolutionarily conserved transmembrane protein and a well-characterized precursor protein of amyloid-beta (Aß) peptides, which accumulate in the brains of individuals with Alzheimer's disease (AD)-related pathologies. Aß has been extensively investigated since the amyloid hypothesis in AD was proposed. Besides Aß, previous studies on APP and its proteolytic cleavage products have suggested their diverse pathological and physiological functions. However, their roles still have not been thoroughly understood. In this review, we extensively discuss the evolutionarily-conserved biology of APP, including its structure and processing pathway, as well as recent findings on the physiological roles of APP and its fragments in the central nervous system and peripheral nervous system. We have also elaborated upon the current status of APP-targeted therapeutic approaches for AD treatment by discussing inhibitors of several proteases participating in APP processing, including α-, ß-, and γ-secretases. Finally, we have highlighted the future perspectives pertaining to further research and the potential clinical role of APP.

Isolation and Characterization of Heparan Sulfate Containing Amyloid Precursor Protein Degradation Products.

Numerous studies indicate that heparan sulfate proteoglycans (HSPGs) participate in a network of complex molecular events involving amyloid precursor protein (APP) processing and formation, oligomerization, intracellular targeting, clearance, and propagation of amyloid ß in Alzheimer's disease (AD). A mutual functional interplay between recycling glypican-1 and APP processing has been demonstrated where the HS released from glypican-1 by a Cu/NO-ascorbate-dependent reaction forms a conjugate with APP degradation products and undergoes an endosome-nucleus-autophagosome co-trafficking. HS has been shown to display contradictory and dual effects in AD involving both prevention and promotion of amyloid ß formation. It is therefore important to identify the source, detailed structural features as well as factors that favor formation of the neuroprotective forms of HS. Here, a method for isolation and identification of HS-containing APP degradation products has been described. The method is based on isolation of radiolabeled HS followed by identification of accompanying APP degradation products by SDS-PAGE and Western blotting.

Members of the vertebrate contactin and amyloid precursor protein families interact through a conserved interface.

Contactins (CNTNs) are neural cell adhesion molecules that encode axon-target specificity during the patterning of the vertebrate visual and olfactory systems. Because CNTNs are tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, they lack an intracellular region to communicate across the membrane. Instead, they form coreceptor complexes with distinct transmembrane proteins to transmit signals inside the cell. In particular, a complex of CNTN4 and amyloid precursor protein (APP) is known to guide the assembly of specific circuits in the visual system. Here, using in situ hybridization in zebrafish embryos, we show that CNTN4, CNTN5, and the APP homologs, amyloid beta precursor like protein 1 and amyloid beta precursor like protein 2, are expressed in olfactory pits, suggesting that these receptors may also function together in the organization of olfactory tissues. Furthermore, we use biochemical and structural approaches to characterize interactions between members of these two receptor families. In particular, APP and amyloid beta precursor like protein 1 interact with CNTN3-5, whereas amyloid beta precursor like protein 2 only binds to CNTN4 and CNTN5. Finally, structural analyses of five CNTN-amyloid pairs indicate that these proteins interact through a conserved interface involving the second fibronectin type III repeat of CNTNs and the copper-binding domain of amyloid proteins. Overall, this work sets the stage for analyzing CNTN-amyloid-mediated connectivity in vertebrate sensory circuits.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by ß and γ secretases leads to the generation of Aß40 (non-amyloidogenic) and Aß42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aß42:Aß40 ratio and the accumulation of toxic Aß42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aß42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aß42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction.

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimer's disease is cut processively by γ-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis-composed of a helical peptide inhibitor linked to a transition-state analogue-that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of the substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both the exosite and the active site, as demonstrated by inhibitor cross-competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.

The amyloid precursor protein is a conserved Wnt receptor.

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.