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1.
Int J Mol Sci ; 25(12)2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38928405

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Disulfides , Intrinsically Disordered Proteins , Molecular Dynamics Simulation , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Disulfides/chemistry , Intrinsically Disordered Proteins/chemistry , Humans , Protein Structure, Secondary , Peptide Fragments/chemistry , Protein Conformation
2.
Anal Chim Acta ; 1314: 342779, 2024 Jul 25.
Article in English | MEDLINE | ID: mdl-38876518

ABSTRACT

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.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Extracellular Vesicles , Fluorescence Resonance Energy Transfer , Gold , Metal Nanoparticles , Tetraspanin 30 , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Extracellular Vesicles/chemistry , Animals , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/blood , Mice , Humans , Tetraspanin 30/metabolism , Gold/chemistry , Metal Nanoparticles/chemistry , Peptide Fragments/analysis , Peptide Fragments/blood , Peptide Fragments/chemistry , Polymers/chemistry , Indoles/chemistry , Limit of Detection
3.
J Phys Chem Lett ; 15(24): 6292-6298, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38855822

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Amyloid beta-Peptides/chemistry , Lipid Bilayers/chemistry , Peptide Fragments/chemistry , Humans , Alzheimer Disease/metabolism , Single Molecule Imaging/methods
4.
Science ; 384(6700): 1091-1095, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38843321

ABSTRACT

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.


Subject(s)
Amyloid Precursor Protein Secretases , Amyloid beta-Peptides , Amyloid beta-Protein Precursor , Presenilin-1 , Humans , Amyloid beta-Peptides/chemistry , Amyloid beta-Protein Precursor/chemistry , Amyloid Precursor Protein Secretases/chemistry , Crystallography, X-Ray , Models, Molecular , Peptide Fragments/metabolism , Peptide Fragments/chemistry , Presenilin-1/chemistry , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Proteolysis , Substrate Specificity
5.
BMB Rep ; 57(6): 263-272, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38835114

ABSTRACT

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].


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Protein Isoforms , Animals , Humans , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Peptide Fragments/metabolism , Peptide Fragments/chemistry , Protein Aggregates/physiology , Protein Aggregation, Pathological/metabolism , Protein Isoforms/metabolism
6.
J Mol Model ; 30(7): 233, 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38937296

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptide Fragments , Protein Binding , alpha7 Nicotinic Acetylcholine Receptor , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , alpha7 Nicotinic Acetylcholine Receptor/chemistry , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Humans , Binding Sites , Ligands
7.
Molecules ; 29(9)2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38731472

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Nanowires , Neuroprotective Agents , Silicon , Animals , Rats , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/toxicity , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Cell Survival/drug effects , Nanowires/chemistry , Neuroprotective Agents/pharmacology , Neuroprotective Agents/chemistry , PC12 Cells , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Peptide Fragments/pharmacology , Protein Aggregates/drug effects , Silicon/chemistry
8.
J Biol Inorg Chem ; 29(4): 407-425, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38811408

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Molecular Dynamics Simulation , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/genetics , Protein Conformation, beta-Strand , Humans , Zinc/metabolism , Zinc/chemistry , Alzheimer Disease/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/genetics , Metals/metabolism , Metals/chemistry
9.
J Phys Chem B ; 128(19): 4655-4669, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38700150

ABSTRACT

Protein misfolding, aggregation, and fibril formation play a central role in the development of severe neurological disorders, including Alzheimer's and Parkinson's diseases. The structural stability of mature fibrils in these diseases is of great importance, as organisms struggle to effectively eliminate amyloid plaques. To address this issue, it is crucial to investigate the early stages of fibril formation when monomers aggregate into small, toxic, and soluble oligomers. However, these structures are inherently disordered, making them challenging to study through experimental approaches. Recently, it has been shown experimentally that amyloid-ß 42 (Aß42) and α-synuclein (α-Syn) can coassemble. This has motivated us to investigate the interaction between their monomers as a first step toward exploring the possibility of forming heterodimeric complexes. In particular, our study involves the utilization of various Amber and CHARMM force-fields, employing both implicit and explicit solvent models in replica exchange and conventional simulation modes. This comprehensive approach allowed us to assess the strengths and weaknesses of these solvent models and force fields in comparison to experimental and theoretical findings, ensuring the highest level of robustness. Our investigations revealed that Aß42 and α-Syn monomers can indeed form stable heterodimers, and the resulting heterodimeric model exhibits stronger interactions compared to the Aß42 dimer. The binding of α-Syn to Aß42 reduces the propensity of Aß42 to adopt fibril-prone conformations and induces significant changes in its conformational properties. Notably, in AMBER-FB15 and CHARMM36m force fields with the use of explicit solvent, the presence of Aß42 significantly increases the ß-content of α-Syn, consistent with the experiments showing that Aß42 triggers α-Syn aggregation. Our analysis clearly shows that although the use of implicit solvent resulted in too large compactness of monomeric α-Syn, structural properties of monomeric Aß42 and the heterodimer were preserved in explicit-solvent simulations. We anticipate that our study sheds light on the interaction between α-Syn and Aß42 proteins, thus providing the atom-level model required to assess the initial stage of aggregation mechanisms related to Alzheimer's and Parkinson's diseases.


Subject(s)
Amyloid beta-Peptides , Molecular Dynamics Simulation , Peptide Fragments , Solvents , alpha-Synuclein , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , Solvents/chemistry , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Multimerization , Humans
10.
Molecules ; 29(9)2024 May 06.
Article in English | MEDLINE | ID: mdl-38731652

ABSTRACT

Bovine milk is an essential supplement due to its rich energy- and nutrient-rich qualities. Caseins constitute the vast majority of the proteins in milk. Among these, ß-casein comprises around 37% of all caseins, and it is an important type of casein with several different variants. The A1 and A2 variants of ß-casein are the most researched genotypes due to the changes in their composition. It is accepted that the A2 variant is ancestral, while a point mutation in the 67th amino acid created the A1 variant. The digestion derived of both A1 and A2 milk is BCM-7. Digestion of A2 milk in the human intestine also forms BCM-9 peptide molecule. The opioid-like characteristics of BCM-7 are highlighted for their potential triggering effect on several diseases. Most research has been focused on gastrointestinal-related diseases; however other metabolic and nervous system-based diseases are also potentially triggered. By manipulating the mechanisms of these diseases, BCM-7 can induce certain situations, such as conformational changes, reduction in protein activity, and the creation of undesired activity in the biological system. Furthermore, the genotype of casein can also play a role in bone health, such as altering fracture rates, and calcium contents can change the characteristics of dietary products. The context between opioid molecules and BCM-7 points to a potential triggering mechanism for the central nervous system and other metabolic diseases discussed.


Subject(s)
Caseins , Endorphins , Humans , Animals , Caseins/chemistry , Caseins/metabolism , Caseins/genetics , Endorphins/chemistry , Endorphins/metabolism , Milk/chemistry , Milk/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/genetics , Opioid Peptides/chemistry , Opioid Peptides/metabolism , Cattle
11.
ACS Chem Neurosci ; 15(11): 2296-2307, 2024 06 05.
Article in English | MEDLINE | ID: mdl-38785363

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Peptide Fragments , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/chemistry , Humans , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Amyloid/metabolism , Amyloid/chemistry , Alzheimer Disease/metabolism , Kinetics
12.
Phys Chem Chem Phys ; 26(23): 16674-16686, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38809059

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Flavanones , Molecular Dynamics Simulation , Peptide Fragments , Flavanones/chemistry , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Hydrophobic and Hydrophilic Interactions , Hydrogen Bonding , Humans , Protein Conformation, beta-Strand
13.
J Proteome Res ; 23(6): 1983-1999, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38728051

ABSTRACT

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.


Subject(s)
Deep Learning , Humans , Peptide Fragments/chemistry , Peptide Fragments/analysis , Tandem Mass Spectrometry/methods , Tandem Mass Spectrometry/statistics & numerical data , Proteomics/methods , Proteomics/statistics & numerical data
14.
Compr Rev Food Sci Food Saf ; 23(3): e13370, 2024 May.
Article in English | MEDLINE | ID: mdl-38783570

ABSTRACT

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.


Subject(s)
Caseins , Caseins/chemistry , Peptide Fragments/analysis , Peptide Fragments/chemistry , Humans , Phenylketonurias
15.
Phys Chem Chem Phys ; 26(20): 14664-14674, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38715538

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Peptide Fragments , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Amyloid/chemistry , Amyloid/metabolism , Nuclear Magnetic Resonance, Biomolecular , Escherichia coli/genetics , Escherichia coli/metabolism , Mutation , Humans
16.
Int J Mol Sci ; 25(9)2024 May 02.
Article in English | MEDLINE | ID: mdl-38732194

ABSTRACT

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.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Protein Binding , Serum Albumin, Human , Humans , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/chemistry , Ligands , Serum Albumin, Human/metabolism , Serum Albumin, Human/chemistry , Alzheimer Disease/metabolism , Molecular Weight , Binding Sites , Peptide Fragments/metabolism , Peptide Fragments/chemistry
17.
Biophys J ; 123(12): 1690-1704, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38751113

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides , Lipid Bilayers , Peptide Fragments , Trehalose , Trehalose/pharmacology , Trehalose/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/chemistry , Peptide Fragments/metabolism , Peptide Fragments/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Cell Membrane/metabolism , Cell Membrane/drug effects , Electrophysiological Phenomena/drug effects
18.
Biomolecules ; 14(5)2024 May 15.
Article in English | MEDLINE | ID: mdl-38785993

ABSTRACT

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.


Subject(s)
Amyloid beta-Peptides , Proteolysis , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/chemistry , Humans , Proteolysis/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptide Fragments/metabolism , Peptides/chemistry , Peptides/pharmacology , Cell Line, Tumor
19.
Int J Nanomedicine ; 19: 4299-4317, 2024.
Article in English | MEDLINE | ID: mdl-38766654

ABSTRACT

Background: Inhibition of amyloid ß protein fragment (Aß) aggregation is considered to be one of the most effective strategies for the treatment of Alzheimer's disease. (-)-Epigallocatechin-3-gallate (EGCG) has been found to be effective in this regard; however, owing to its low bioavailability, nanodelivery is recommended for practical applications. Compared to chemical reduction methods, biosynthesis avoids possible biotoxicity and cumbersome preparation processes. Materials and Methods: The interaction between EGCG and Aß42 was simulated by molecular docking, and green tea-conjugated gold nanoparticles (GT-Au NPs) and EGCG-Au NPs were synthesized using EGCG-enriched green tea and EGCG solutions, respectively. Surface active molecules of the particles were identified and analyzed using various liquid chromatography-tandem triple quadrupole mass spectrometry methods. ThT fluorescence assay, circular dichroism, and TEM were used to investigate the effect of synthesized particles on the inhibition of Aß42 aggregation. Results: EGCG as well as apigenin, quercetin, baicalin, and glutathione were identified as capping ligands stabilized on the surface of GT-Au NPs. They more or less inhibited Aß42 aggregation or promoted fibril disaggregation, with EGCG being the most effective, which bound to Aß42 through hydrogen bonding, hydrophobic interactions, etc. resulting in 39.86% and 88.50% inhibition of aggregation and disaggregation effects, respectively. EGCG-Au NPs were not as effective as free EGCG, whereas multiple thiols and polyphenols in green tea accelerated and optimized heavy metal detoxification. The synthesized GT-Au NPs conferred the efficacy of diverse ligands to the particles, with inhibition of aggregation and disaggregation effects of 54.69% and 88.75%, respectively, while increasing the yield, enhancing water solubility, and decreasing cost. Conclusion: Biosynthesis of nanoparticles using green tea is a promising simple and economical drug-carrying approach to confer multiple pharmacophore molecules to Au NPs. This could be used to design new drug candidates to treat Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides , Catechin , Gold , Metal Nanoparticles , Molecular Docking Simulation , Peptide Fragments , Tea , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Catechin/chemistry , Catechin/pharmacology , Catechin/analogs & derivatives , Tea/chemistry , Metal Nanoparticles/chemistry , Metal Nanoparticles/administration & dosage , Gold/chemistry , Ligands , Peptide Fragments/chemistry , Peptide Fragments/antagonists & inhibitors , Humans , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Protein Aggregates/drug effects
20.
Molecules ; 29(10)2024 May 07.
Article in English | MEDLINE | ID: mdl-38792033

ABSTRACT

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.


Subject(s)
Coordination Complexes , Copper , Nickel , Peptide Fragments , Zinc , tau Proteins , Nickel/chemistry , Copper/chemistry , Zinc/chemistry , tau Proteins/chemistry , Coordination Complexes/chemistry , Peptide Fragments/chemistry , Oxidation-Reduction , Histidine/chemistry , Hydrogen-Ion Concentration , Hydrogen Peroxide/chemistry , Thermodynamics
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