Specific interaction from different Aß42 peptide fragments to α7nAChR-A study of molecular dynamics simulation.

CONTEXT: Existing researches confirmed that ß amyloid (Aß) has a high affinity for the α7 nicotinic acetylcholine receptor (α7nAChR), associating closely to Alzheimer's disease. The majority of related studies focused on the experimental reports on the neuroprotective role of Aß fragment (Aßx), however, with a lack of investigation into the most suitable binding region and mechanism of action between Aß fragment and α7nAChR. In the study, we employed four Aß1-42 fragments Aßx, Aß1-16, Aß10-16, Aß12-28, and Aß30-42, of which the first three were confirmed to play neuroprotective roles upon directly binding, to interact with α7nAChR. METHODS: The protein-ligand docking server of CABS-DOCK was employed to obtain the α7nAChR-Aßx complexes. Only the top α7nAChR-Aßx complexes were used to perform all-atom GROMACS dynamics simulation in combination with Charmm36 force field, by which α7nAChR-Aßx interactions' dynamic behavior and specific locations of these different Aßx fragments were identified. MM-PBSA calculations were also done to estimate the binding free energies and the different contributions from the residues in the Aßx. Two distinct results for the first three and fourth Aßx fragments in binding site, strength, key residue, and orientation, account for why the fourth fails to play a neuroprotective role at the molecular level.

Distinctive contribution of two additional residues in protein aggregation of Aß42 and Aß40 isoforms.

Amyloid-ß (Aß) is one of the amyloidogenic intrinsically disordered proteins (IDPs) that self-assemble to protein aggregates, incurring cell malfunction and cytotoxicity. While Aß has been known to regulate multiple physiological functions, such as enhancing synaptic functions, aiding in the recovery of the blood-brain barrier/brain injury, and exhibiting tumor suppression/antimicrobial activities, the hydrophobicity of the primary structure promotes pathological aggregations that are closely associated with the onset of Alzheimer's disease (AD). Aß proteins consist of multiple isoforms with 37-43 amino acid residues that are produced by the cleavage of amyloid-ß precursor protein (APP). The hydrolytic products of APP are secreted to the extracellular regions of neuronal cells. Aß 1-42 (Aß42) and Aß 1-40 (Aß40) are dominant isoforms whose significance in AD pathogenesis has been highlighted in numerous studies to understand the molecular mechanism and develop AD diagnosis and therapeutic strategies. In this review, we focus on the differences between Aß42 and Aß40 in the molecular mechanism of amyloid aggregations mediated by the two additional residues (Ile41 and Ala42) of Aß42. The current comprehension of Aß42 and Aß40 in AD progression is outlined, together with the structural features of Aß42/Aß40 amyloid fibrils, and the aggregation mechanisms of Aß42/Aß40. Furthermore, the impact of the heterogeneous distribution of Aß isoforms during amyloid aggregations is discussed in the system mimicking the coexistence of Aß42 and Aß40 in human cerebrospinal fluid (CSF) and plasma. [BMB Reports 2024; 57(6): 263-272].

Accurate and highly sensitive detection of Alzheimer's disease-related extracellular vesicles via förster resonance energy transfer.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-ß (1-42) (Aß42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aß42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aß42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL-1 and the linear range was from 104 to 108 particles mL-1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.

Single-Molecule Maps of Membrane Insertion by Amyloid-ß Oligomers Predict Their Toxicity.

The interaction of small Amyloid-ß (Aß) oligomers with the lipid membrane is an important component of the pathomechanism of Alzheimer's disease (AD). However, oligomers are heterogeneous in size. How each type of oligomer incorporates into the membrane, and how that relates to their toxicity, is unknown. Here, we employ a single molecule technique called Q-SLIP (Quencher-induced Step Length Increase in Photobleaching) to measure the membrane insertion of each monomeric unit of individual oligomers of Aß42, Aß40, and Aß40-F19-Cyclohexyl alanine (Aß40-F19Cha), and correlate it with their toxicity. We observe that the N-terminus of Aß42 inserts close to the center of the bilayer, the less toxic Aß40 inserts to a shallower depth, and the least toxic Aß40-F19Cha has no specific distribution. This oligomer-specific map provides a mechanistic representation of membrane-mediated Aß toxicity and should be a valuable tool for AD research.

Molecular mechanism of substrate recognition and cleavage by human γ-secretase.

Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-ß (Aß) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aß49, Aß46, and Aß43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a ß-strand that forms a hybrid ß-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate ß-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new ß-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.

A Practical Guide to All-Atom and Coarse-Grained Molecular Dynamics Simulations Using Amber and Gromacs: A Case Study of Disulfide-Bond Impact on the Intrinsically Disordered Amyloid Beta.

Intrinsically disordered proteins (IDPs) pose challenges to conventional experimental techniques due to their large-scale conformational fluctuations and transient structural elements. This work presents computational methods for studying IDPs at various resolutions using the Amber and Gromacs packages with both all-atom (Amber ff19SB with the OPC water model) and coarse-grained (Martini 3 and SIRAH) approaches. The effectiveness of these methodologies is demonstrated by examining the monomeric form of amyloid-ß (Aß42), an IDP, with and without disulfide bonds at different resolutions. Our results clearly show that the addition of a disulfide bond decreases the ß-content of Aß42; however, it increases the tendency of the monomeric Aß42 to form fibril-like conformations, explaining the various aggregation rates observed in experiments. Moreover, analysis of the monomeric Aß42 compactness, secondary structure content, and comparison between calculated and experimental chemical shifts demonstrates that all three methods provide a reasonable choice to study IDPs; however, coarse-grained approaches may lack some atomistic details, such as secondary structure recognition, due to the simplifications used. In general, this study not only explains the role of disulfide bonds in Aß42 but also provides a step-by-step protocol for setting up, conducting, and analyzing molecular dynamics (MD) simulations, which is adaptable for studying other biomacromolecules, including folded and disordered proteins and peptides.

Protective effect of trehalose sugar on amyloid-membrane interactions using BLM electrophysiology.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia and memory loss in the elderly population. The amyloid-ß peptide (Aß) is one of the main pathogenic factors in AD and is known to cause damage to neuronal cellular membranes. There is no cure currently available for AD, and new approaches, including preventive strategies, are highly desirable. In this work, we explore the possibility of protecting neuronal membranes from amyloid-induced damage with naturally existing sugar trehalose. Trehalose has been shown to protect plant cellular membranes in extreme conditions and modify Aß misfolding. We hypothesize that trehalose can protect the neuronal membrane from amyloid toxicity. In this work, we studied the protective effect of trehalose against Aß1-42-induced damage in model lipid membranes (DPPC/POPC/cholesterol) using atomic force microscopy and black lipid membrane electrophysiology. Our results demonstrate that Aß1-42 damaged membranes and led to ionic current leakage across these membranes due to the formation of various defects and pores. The presence of trehalose reduced the ion current across membranes caused by Aß1-42 peptide damage, thus efficiently protecting the membranes. These findings suggest that the trehalose sugar can potentially be useful in protecting neuronal membranes against amyloid toxicity in AD.

Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aß1-40 by NMR.

This study delves into the early aggregation process of the Aß1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAß1-40, a modified Aß1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aß1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Glycomacropeptide: A comprehensive understanding of its major biological characteristics and purification methodologies.

Glycomacropeptide (GMP) is a bioactive peptide derived from whey protein, consisting of 64 amino acids. It is a phenylalanine-free peptide, making it a beneficial dietary option for individuals dealing with phenylketonuria (PKU). PKU is an inherited metabolic disorder characterized by high levels of phenylalanine in the bloodstream, resulting from a deficiency of phenylalanine dehydrogenase in affected individuals. Consequently, patients with PKU require lifelong adherence to a low-phenylalanine diet, wherein a significant portion of their protein intake is typically sourced from a phenylalanine-free amino acid formula. GMP has several nutritional values, numerous bioactivity properties, and therapeutic effects in various inflammatory disorders. Despite all these features, the purification of GMP is an imperative requirement; however, there are no unique methods for achieving this goal. Traditionally, several methods have been used for GMP purification, such as thermal or acid treatment, alcoholic precipitation, ultrafiltration (UF), gel filtration, and membrane separation techniques. However, these methods have poor specificity, and the presence of large amounts of impurities can interfere with the analysis of GMP. More efficient and highly specific GMP purification methods need to be developed. In this review, we have highlighted and summarized the current research progress on the major biological features and purification methodologies associated with GMP, as well as providing an extensive overview of the recent developments in using charged UF membranes for GMP purification and the influential factors.

In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aß. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aß. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA-Aß40 interaction: prednisone favors HSA-Aß interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.

Structural characterization of E22G Aß1-42 fibrils via1H detected MAS NMR.

Amyloid fibrils have been implicated in the pathogenesis of several neurodegenerative diseases, the most prevalent example being Alzheimer's disease (AD). Despite the prevalence of AD, relatively little is known about the structure of the associated amyloid fibrils. This has motivated our studies of fibril structures, extended here to the familial Arctic mutant of Aß1-42, E22G-Aß1-42. We found E22G-AßM0,1-42 is toxic to Escherichia coli, thus we expressed E22G-Aß1-42 fused to the self-cleavable tag NPro in the form of its EDDIE mutant. Since the high surface activity of E22G-Aß1-42 makes it difficult to obtain more than sparse quantities of fibrils, we employed 1H detected magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments to characterize the protein. The 1H detected 13C-13C methods were first validated by application to fully protonated amyloidogenic nanocrystals of GNNQQNY, and then applied to fibrils of the Arctic mutant of Aß, E22G-Aß1-42. The MAS NMR spectra indicate that the biosynthetic samples of E22G-Aß1-42 fibrils comprise a single conformation with 13C chemical shifts extracted from hCH, hNH, and hCCH spectra that are very similar to those of wild type Aß1-42 fibrils. These results suggest that E22G-Aß1-42 fibrils have a structure similar to that of wild type Aß1-42.

Apo and Aß46-bound γ-secretase structures provide insights into amyloid-ß processing by the APH-1B isoform.

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.

Copper(II), Nickel(II) and Zinc(II) Complexes of Peptide Fragments of Tau Protein.

Copper(II), nickel(II) and zinc(II) complexes of various peptide fragments of tau protein were studied by potentiometric and spectroscopic techniques. All peptides contained one histidyl residue and represented the sequences of tau(91-97) (Ac-AQPHTEI-NH2), tau(385-390) (Ac-KTDHGA-NH2) and tau(404-409) (Ac-SPRHLS-NH2). Imidazole-N donors of histidine were the primary metal binding sites for all peptides and all metal ions, but in the case of copper(II) and nickel(II), the deprotonated amide groups were also involved in metal binding by increasing pH. The most stable complexes were formed with copper(II) ions, but the presence of prolyl residues resulted in significant changes in the thermodynamic stability and speciation of the systems. It was also demonstrated that nickel(II) and especially zinc(II) complexes have relatively low thermodynamic stability with these peptides. The copper(II)-catalyzed oxidation of the peptides was also studied. In the presence of H2O2, the fragmentation of peptides was detected in all cases. In the simultaneous presence of H2O2 and ascorbic acid, the fragmentation of the peptide is less preferred, and the formation of 2-oxo-histidine also occurs.

Insights into the baicalein-induced destabilization of LS-shaped Aß42 protofibrils using computer simulations.

Amyloid-ß (Aß) peptides aggregate spontaneously into various aggregating species comprising oligomers, protofibrils, and mature fibrils in Alzheimer's disease (AD). Disrupting ß-sheet rich neurotoxic smaller soluble Aß42 oligomers formed at early stages is considered a potent strategy to interfere with AD pathology. Previous experiments have demonstrated the inhibition of the early stages of Aß aggregation by baicalein; however, the molecular mechanism behind inhibition remains largely unknown. Thus, in this work, molecular dynamics (MD) simulations have been employed to illuminate the molecular mechanism of baicalein-induced destabilization of preformed Aß42 protofibrils. Baicalein binds to chain A of the Aß42 protofibril through hydrogen bonds, π-π interactions, and hydrophobic contacts with the central hydrophobic core (CHC) residues of the Aß42 protofibril. The binding of baicalein to the CHC region of the Aß42 protofibril resulted in the elongation of the kink angle and disruption of K28-A42 salt bridges, which resulted in the distortion of the protofibril structure. Importantly, the ß-sheet content was notably reduced in Aß42 protofibrils upon incorporation of baicalein with a concomitant increase in the coil content, which is consistent with ThT fluorescence and AFM images depicting disaggregation of pre-existing Aß42 fibrils on the incorporation of baicalein. Remarkably, the interchain binding affinity in Aß42 protofibrils was notably reduced in the presence of baicalein leading to distortion in the overall structure, which agrees with the structural stability analyses and conformational snapshots. This work sheds light on the molecular mechanism of baicalein in disrupting the Aß42 protofibril structure, which will be beneficial to the design of therapeutic candidates against disrupting ß-sheet rich neurotoxic Aß42 oligomers in AD.

An N-terminal acidic ß-sheet domain is responsible for the metal-accumulation properties of amyloid-ß protofibrils: a molecular dynamics study.

The influence of metal ions on the structure of amyloid- ß (Aß) protofibril models was studied through molecular dynamics to explore the molecular mechanisms underlying metal-induced Aß aggregation relevant in Alzheimer's disease (AD). The models included 36-, 48-, and 188-mers of the Aß42 sequence and two disease-modifying variants. Primary structural effects were observed at the N-terminal domain, as it became susceptible to the presence of cations. Specially when ß-sheets predominate, this motif orients N-terminal acidic residues toward one single face of the ß-sheet, resulting in the formation of an acidic region that attracts cations from the media and promotes the folding of the N-terminal region, with implications in amyloid aggregation. The molecular phenotype of the protofibril models based on Aß variants shows that the AD-causative D7N mutation promotes the formation of N-terminal ß-sheets and accumulates more Zn2+, in contrast to the non-amyloidogenic rodent sequence that hinders the ß-sheets and is more selective for Na+ over Zn2+ cations. It is proposed that forming an acidic ß-sheet domain and accumulating cations is a plausible molecular mechanism connecting the elevated affinity and concentration of metals in Aß fibrils to their high content of ß-sheet structure at the N-terminal sequence.

Protection of Si Nanowires against Aß Toxicity by the Inhibition of Aß Aggregation.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid beta (Aß) plaques in the brain. Aß1-42 is the main component of Aß plaque, which is toxic to neuronal cells. Si nanowires (Si NWs) have the advantages of small particle size, high specific surface area, and good biocompatibility, and have potential application prospects in suppressing Aß aggregation. In this study, we employed the vapor-liquid-solid (VLS) growth mechanism to grow Si NWs using Au nanoparticles as catalysts in a plasma-enhanced chemical vapor deposition (PECVD) system. Subsequently, these Si NWs were transferred to a phosphoric acid buffer solution (PBS). We found that Si NWs significantly reduced cell death in PC12 cells (rat adrenal pheochromocytoma cells) induced by Aß1-42 oligomers via double staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescein diacetate/propyl iodide (FDA/PI). Most importantly, pre-incubated Si NWs largely prevented Aß1-42 oligomer-induced PC12 cell death, suggesting that Si NWs exerts an anti-Aß neuroprotective effect by inhibiting Aß aggregation. The analysis of Fourier Transform Infrared (FTIR) results demonstrates that Si NWs reduce the toxicity of fibrils and oligomers by intervening in the formation of ß-sheet structures, thereby protecting the viability of nerve cells. Our findings suggest that Si NWs may be a potential therapeutic agent for AD by protecting neuronal cells from the toxicity of Aß1-42.

Aß Oligomer Dissociation Is Catalyzed by Fibril Surfaces.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aß peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aß42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

Systematic Assessment of Deep Learning-Based Predictors of Fragmentation Intensity Profiles.

In recent years, several deep learning-based methods have been proposed for predicting peptide fragment intensities. This study aims to provide a comprehensive assessment of six such methods, namely Prosit, DeepMass:Prism, pDeep3, AlphaPeptDeep, Prosit Transformer, and the method proposed by Guan et al. To this end, we evaluated the accuracy of the predicted intensity profiles for close to 1.7 million precursors (including both tryptic and HLA peptides) corresponding to more than 18 million experimental spectra procured from 40 independent submissions to the PRIDE repository that were acquired for different species using a variety of instruments and different dissociation types/energies. Specifically, for each method, distributions of similarity (measured by Pearson's correlation and normalized angle) between the predicted and the corresponding experimental b and y fragment intensities were generated. These distributions were used to ascertain the prediction accuracy and rank the prediction methods for particular types of experimental conditions. The effect of variables like precursor charge, length, and collision energy on the prediction accuracy was also investigated. In addition to prediction accuracy, the methods were evaluated in terms of prediction speed. The systematic assessment of these six methods may help in choosing the right method for MS/MS spectra prediction for particular needs.

Neuroprotective azaphilones from a deep-sea derived fungus Penicillium sp. SCSIO41030.

Ten azaphilones including one pair of new epimers and three new ones, penineulones A-E (1-5) with the same structural core of angular deflectin, were obtained from a deep-sea derived Penicillium sp. SCSIO41030 fermented on a liquid medium. Their structures including absolute configurations were elucidated using chiral-phase HPLC analysis, extensive NMR spectroscopic and HRESIMS data, ECD and NMR calculations, and by comparing NMR data with literature data. Biological assays showed that the azaphilones possessed no antitumor and anti-viral (HSV-1/2) activities at concentrations of 5.0 µM and 20 µM, respectively. In addition, azaphilones 8 and 9 showed neuroprotective effects against Aß25-35-induced neurotoxicity in primary cultured cortical neurons at a concentration of 10 µM. Azaphilones 8 and 9 dramatically promoted axonal regrowth against Aß25-35-induced axonal atrophy. Our study indicated that azaphilones could be promising lead compounds for neuroprotection.

Profiling of the Proteins Interacting with Amyloid Beta Peptides in Clinical Samples by PACTS-TPP.

The accumulation of amyloid beta (Aß1-42) results in neurotoxicity and is strongly related to neurodegenerative disorders, especially Alzheimer's disease (AD), but the underlying molecular mechanism is still poorly understood. Therefore, there is an urgent need for researchers to discover the proteins that interact with Aß1-42 to determine the molecular basis. Previously, we developed peptide-ligand-induced changes in the abundance of proTeinS (PACTS)-assisted thermal proteome profiling (TPP) to identify proteins that interact with peptide ligands. In the present study, we applied this technique to analyze clinical samples to identify Aß1-42-interacting proteins. We detected 115 proteins that interact with Aß1-42 in human frontal lobe tissue. Pathway enrichment analysis revealed that the differentially expressed proteins were involved mainly in neurodegenerative diseases. Further orthogonal validation revealed that Aß1-42 interacted with the AD-associated protein mitogen-activated protein kinase 3 (MAPK3), and knockdown of the Aß1-42 amyloid precursor protein (APP) inhibited the MAPK signaling pathway, suggesting potential functional roles for Aß1-42 in interacting with MAPK3. Overall, this study demonstrated the application of the PACTS-TPP in clinical samples and provided a valuable data source for research on neurodegenerative diseases.