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Amyloid aggregates play a major role in diseases as well as in normal physiological function. Receptor-interacting protein kinases 1 and 3 (RIP1/RIP3) aggregates complexes in cellular necroptosis is one example of protein aggregation in normal cellular function. Although recently there have been several studies on full kinase proteins aggregation, the aggregation potential of small peptide sequences of RIP1/RIP3, the physicochemical properties, and the potential in biomedical applications have not been explored. Hence, in this paper, we study the aggregation propensity of peptides consisting of four and twelve amino acid sequences in the RHIM region of RIP1/RIP3 proteins that are known to drive the beta-sheet formation and the subsequent aggregation. The aggregation kinetics, physicochemical characterization, mechanosensitive properties, cellular effects, and potential as a cancer drug depot have been investigated. The results show that the number and concentration of amino acids play a role in amyloid-like aggregates' properties. Further, the aggregates when formulated with cisplatin-induced significant lung cancer cell toxicity compared to an equal amount of cisplatin with and without ultrasound. The study would serve as a platform for further investigation on RIP1/RIP3 peptide and protein aggregates, their role in multiple cellular functions and diseases, and their potential as drug depots. [ABSTRACT FROM AUTHOR]

Alpha-Synuclein is a presynaptic neuronal protein whose masses are involved in Parkinson's disease, a chronic and progressive neurodegenerative disorder of the central nervous system. It has been observed that upon exposure to anionic surfactants, fluorinated alcohols and membranes in vitro, α -synuclein adopts alpha helix structure and can be fibrillated, while in aqueous solution, the protein is intrinsically disordered and the fibrillation is undesirable. Solvent is one of the most critical environmental parameters in biological studies as well as computer simulations. In this paper, we have investigated the effect of solvent composition on the conformation of α -synuclein protein using molecular dynamics (MD) simulations. For this purpose, the effect of different combinations of water and trifluoroethanol (TFE) solvents on the secondary structure of a fragment of α -synuclein was investigated. Moreover, thermodynamic calculations were performed through a coarse-grained (CG) model of solutions with different concentrations of α -synuclein in the aqueous phase and in TFE medium. The potential of mean force (PMF) calculations was performed as a function of separation distance between the centers of masses of two peptides. The results indicated that in the presence of TFE, the secondary structures have a regular arrangement of amino acids stabilized by hydrogen bonding patterns. In addition, decreasing the TFE concentration reduces the stability of α -helix structures of the protein. It seems that in diluted TFE solutions, high concentrations of water in the medium lead to the breakage of peptide–TFE hydrogen bonds. However, the hydrophobic nature of TFE and α -synuclein molecules prevents further aggregation in the concentrated solutions. The α-synuclein can be fibrillated in the presence of Trifluoroethanol (TFE). Stability of α-helix structure of peptide reduces as TFE concentration decreases. Hydrophobic nature of TFE and α-synuclein molecules prevents further aggregation. [ABSTRACT FROM AUTHOR]

Amyloids are implicated in many diseases, and disruption of lipid membrane structures is considered as one possible mechanism of pathology. In this paper we investigate interactions between an aggregating peptide and phospholipid membranes, focusing on the nanometer-scale structures of the aggregates formed, as well as on the effect on the aggregation process. As a model system, we use the small amyloid-forming peptide named NACore, which is a fragment of the central region of the protein α-synuclein that is associated with Parkinson's disease. We find that phospholipid vesicles readily associate with the amyloid fibril network in the form of highly distorted and trapped vesicles that also may wet the surface of the fibrils. This effect is most pronounced for model lipid systems containing only zwitterionic lipids. Fibrillation is found to be retarded by the presence of the vesicles. At the resolution of our measurements, which are based mainly on cryogenic transmission electron microscopy (cryo-TEM), X-ray scattering, and circular dichroism (CD) spectroscopy, we find that the resulting aggregates can be well fitted as linear combinations of peptide fibrils and phospholipid bilayers. There are no detectable effects on the cross-β packing of the peptide molecules in the fibrils, or on the thickness of the phospholipid bilayers. This suggests that while the peptide fibrils and lipid bilayers readily co-assemble on large length-scales, most of them still retain their separate structural identities on molecular length-scales. Comparison between this relatively simple model system and other amyloid systems might help distinguish aspects of amyloid-lipid interactions that are generic from aspects that are more protein specific. Finally, we briefly consider possible implications of the obtained results for in-vivo amyloid toxicity. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are potentially powerful alternatives to conventional antibiotics in combating multidrug resistance, given their broad spectrum of activity. They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures, resulting in permeability and destruction of target microorganism membranes. Our earlier work showed that two leading AMPs, MSI-78 (4-20) and pardaxin (1-22), had potent antimicrobial activity against a range of bacteria. It is known that the attachment of moderate-length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer, inducing membrane curvature, destabilization, and potential leakage. Thus, in this work, we aimed to investigate the antimicrobial activity, oligomerization propensity, and lipidmembrane binding interactions of a range of N-terminal lipidated analogs of MSI-78 (4-20) and pardaxin (1-22). Molecular modeling results suggest that aggregation of the N-lipidated AMPs may impart greater structural stability to the peptides in solution and a greater depth of lipid bilayer insertion for the N-lipidated AMPs over the parental peptide. Our experimental and computational findings provide insights into how N-terminal lipidation of AMPs may alter their conformations, with subsequent effects on their functional properties in regard to their self-aggregation behavior, membrane interactions, and antimicrobial activity. [ABSTRACT FROM AUTHOR]

Amyloids were conventionally referred to as extracellular and intracellular accumulation of Aβ42 peptide, which causes the formation of plaques and neurofibrillary tangles inside the brain leading to the pathogenesis in Alzheimer's disease. Subsequently, amyloid-like deposition was found in the etiology of prion diseases, Parkinson's disease, type II diabetes, and cancer, which was attributed to the aggregation of prion protein, α-Synuclein, islet amyloid polypeptide protein, and p53 protein, respectively. Hence, traditionally amyloids were considered aggregates formed exclusively by proteins or peptides. However, since the last decade, it has been discovered that other metabolites, like single amino acids, nucleobases, lipids, glucose derivatives, etc., have a propensity to form amyloid-like toxic assemblies. Several studies suggest direct implications of these metabolite assemblies in the patho-physiology of various inborn errors of metabolisms like phenylketonuria, tyrosinemia, cystinuria, and Gaucher's disease, to name a few. In this review, we present a comprehensive literature overview that suggests amyloid-like structure formation as a common phenomenon for disease progression and pathogenesis in multiple syndromes. The review is devoted to providing readers with a broad knowledge of the structure, mode of formation, propagation, and transmission of different extracellular amyloids and their implications in the pathogenesis of diseases. We strongly believe a review on this topic is urgently required to create awareness about the understanding of the fundamental molecular mechanism behind the origin of diseases from an amyloid perspective and possibly look for a common therapeutic strategy for the treatment of these maladies by designing generic amyloid inhibitors. [ABSTRACT FROM AUTHOR]

Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the β-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation. [ABSTRACT FROM AUTHOR]

Liquid sheared amyloid-β (Aβ) initiates amyloid cascade reactions, producing unstable, potentially toxic oligomers. There is a need for new analytical tools with which to study these oligomers. A very small bore capillary flow system is proposed as a tool for studying the effects of liquid shear in amyloid research. This simple system consists of injecting a short cylindrical liquid sample plug containing dissolved amyloid into a liquid mobile phase flowing through an empty, very small internal diameter capillary tube. For liquid samples containing a single protein sample, under conditions in which there is laminar flow and limited sample protein molecular diffusion, chromatograms monitoring the optical protein absorbance of capillary effluent contain either one or two peaks, depending on the mobile phase flow rate. By controlling the sample diffusion times through changes in flow rate and/or capillary diameter, this tool can be used to generate aliquot samples with precise, reproducible amounts of shear for exploring the effects of variable shear on amyloid systems. The tool can be used for producing in-capillary stopped flow spectra of shear-stressed Aβ monomers as well as for kinetic studies of Aβ dimer- and oligomer-forming reactions between shear stressed Aβ monomers. Many other experiments are suggested using this experimental tool for studying the effects of shear on different Aβ and other amyloid systems, including testing for potentially serious amyloid sampling errors in spinal tap quantitative analysis. The technique has potential as both a laboratory research and a clinical tool. [ABSTRACT FROM AUTHOR]

Aggregates of amyloidogenic peptides are involved in the pathogenesis of several degenerative disorders. Herein, an iridium(III) complex, Ir-1, is reported as a chemical tool for oxidizing amyloidogenic peptides upon photoactivation and subsequently modulating their aggregation pathways. Ir-1 was rationally designed based on multiple characteristics, including 1) photoproperties leading to excitation by low-energy radiation; 2) generation of reactive oxygen species responsible for peptide oxidation upon photoactivation under mild conditions; and 3) relatively easy incorporation of a ligand on the IrIII center for specific interactions with amyloidogenic peptides. Biochemical and biophysical investigations illuminate that the oxidation of representative amyloidogenic peptides (i.e., amyloid-β, α-synuclein, and human islet amyloid polypeptide) is promoted by light-activated Ir-1, which alters the conformations and aggregation pathways of the peptides. Additionally, their potential oxidation sites are identified as methionine, histidine, or tyrosine residues. Overall, our studies on Ir-1 demonstrate the feasibility of devising metal complexes as chemical tools suitable for elucidating the nature of amyloidogenic peptides at the molecular level, as well as controlling their aggregation. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-β (Aβ) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aβ. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aβ interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aβ aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aβ-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells. [ABSTRACT FROM AUTHOR]

Gelsolin amyloidosis (AGel) is characterized by multiple systemic and ophthalmic features resulting from pathological tissue deposition of the gelsolin (GSN) protein. To date, no cure is available for the treatment of any form of AGel. More than ten single-point substitutions in the GSN gene are responsible for the occurrence of the disease and, among them, D187N/Y is the most widespread variant. These substitutions undergo an aberrant proteolytic cascade, producing aggregation-prone peptides of 5 and 8 kDa, containing the Gelsolin Amyloidogenic Core, spanning residues 182–192 (GAC182–192). Following a structure-based approach, we designed and synthesized three novel sequence-specific peptidomimetics (LB-5, LB-6, and LB-7) built on a piperidine-pyrrolidine unnatural amino acid. LB-5 and LB-6, but not LB-7, efficiently inhibit the aggregation of the GAC182–192 amyloidogenic peptides at sub-stoichiometric concentrations. These peptidomimetics resulted also effective in vivo, in a C. elegans-based assay, in counteracting the proteotoxicity of aggregated GAC182–192. These data pave the way to a novel pharmacological strategy against AGel and also validate a toolbox exploitable in other amyloidogenic diseases. [ABSTRACT FROM AUTHOR]

The present work deals with the micellar state study of sodium cholate and sodium deoxycholate in the aqueous solution of a bioactive peptide, namely glycyl dipeptide, having different concentrations through conductivity and fluorescence methods at different temperatures. The data obtained from conductivity is plotted against the concentration of Bile salts, and CMC (critical micelle concentration) values are calculated. The results realized have been elucidated with reference to Glycyl dipeptide–bile salts hydrophobic/hydrophilic interactions existing in solution. In addition, the CMC values converted to mole fraction (X c m c) values have been used to evaluate the standard thermodynamic factors of micellization viz., enthalpy H , free energy Δ G m 0 , and entropy (Δ S m 0) which extract information regarding thermodynamic feasibility of micellar state, energy alteration, and the assorted interactions established in the existing (bile salts–water–glycyl dipeptide) system. Furthermore, the pyrene fluorescence spectrum has also been utilized to study the change in micro polarity induced by the interactions of bile salts with glycyl dipeptide and the aggregation action of bile salts. The decrease in modification in the ratio of intensities of first and third peaks i.e., (I1/I3) for the pyrene molecules in aqueous bile salts solution by the addition of dipeptide, demonstrates that the micelle polarity is affected by glycyl dipeptide. This ratio has also been utilized to determine CMC values for the studied system, and the results have been found to be in good correlation with observations made in conductivity studies. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is characterized by the appearance of neurofibrillary tangles comprising of the Tau protein and aggregation of amyloid-b peptides (Aβ 1–40 and Aβ 1–42). A concomitant loss of the ribosomal population is also observed in AD-affected neurons. Our studies demonstrate that, similarly to Tau protein aggregation, in vitro aggregation of Aβ peptides in the vicinity of the yeast 80S ribosome can induce co-aggregation of ribosomal components. The RNA-stimulated aggregation of Aβ peptides and the TauK18 variant is dependent on the RNA:protein stoichiometric ratio. A similar effect of stoichiometry is also observed on the ribosome–protein coaggregation process. Polyphenolic inhibitors of amyloid aggregation, such as rosmarinic acid and myricetin, inhibit RNA-stimulated Aβ and Tau-K18 aggregation and can mitigate the co-aggregation of ribosomal components. [ABSTRACT FROM AUTHOR]

Short peptides are potential drug candidates for pharmaceutical and biotech industries. Short peptides are natural ligands for numerous G-protein coupled receptors (GPCR) and hence constitute a large number of drug candidates. Synthetic short peptides are also extensively developed as agonistic or antagonistic ligands that function in a similar manner to antibodies, soluble receptors and protein ligands. Characterization of the peptides in solution is often performed in the presence of organic solvents, which can presumably generate the structure bound to the target surface and also enhance the solubility of the peptides. Analytical ultracentrifugation (AUC) technique should provide information on the state of self-association of the peptide in solution. Its application for short peptides has been far less than the applications for proteins. We believe that AUC should be used to show the associated state of the peptides, as reviewed in this paper. [ABSTRACT FROM AUTHOR]

Abstract: This paper provides a brief overview of the effects of protein hydrolysis on aggregation and gel forming properties of protein systems. Among the food globular proteins, whey proteins and soy proteins are the most extensively studied for their ability to form different textures upon proteolysis. Recent studies were focused on identifying aggregating peptides and on mechanisms of aggregation and gelation. [Copyright &y& Elsevier]

Metal dyshomeostasis plays a critical role in the reactive oxygen species (ROS) formation and protein misfolding and aggregation; hence, contributing to neurodegeneration. Tau protein plays a key role in normal cellular function by maintaining microtubule formation in brain. The role of metal ions on tau protein biochemistry has not been systematically evaluated, but earlier reports indicated that metal ions modulate the complex biochemistry of this protein and its peptides. Herein, we evaluated interactions of biologically-relevant Cu(II) ions with the four repeat peptides of tau protein (R1 through R4) and their role on the formation of ROS, Cu(II) to Cu(I) reduction, and ultimately, peptide aggregation. The role of R peptides on ROS formation was characterized in the absence and presence of biological reducing agent, ascorbate by using UV–Vis and fluorescence spectroscopy. In the presence of the reducing agent, all Cu(II)-peptide complexes reduced hydroxyl radical (OH·), while only Cu(II)-R3 complex depleted the hydrogen peroxide (H2O2). In the absence of a reducing agent, only Cu(II)-R2 and Cu(II)-R3 complexes, which contain Cys and His residues, produced OH· and H2O2. Only R2 and R3 peptides, but not R1 and R4, reduced Cu(II) to Cu(I). The aggregation propensities of R peptides were modulated by Cu(II) and ascorbate, and were imaged by transmission electron microscopy. All metallo-peptides were characterized predominantly as singly charged mononuclear complexes by mass spectrometry. The data indicate that Cu(II)-peptide complexes may act as pro-oxidants or antioxidants and exhibit unique aggregation propensities under specific environmental conditions, with implications in the biological setting. [ABSTRACT FROM AUTHOR]

Three elastin peptides derived from a peculiar elastin sequence (exon 30) were investigated by Infra-red spectroscopy (IRTF), differential scanning calorimetry (DSC) and dielectric spectroscopy (DDS) to clarify the relationship between structural organization and physical properties of these peptides in the solid state. If a great majority of elastin derived peptides form organized structures, only few are able to coacervate, and only one, that is encoded by Exon 30, gives rise to an irreversible precipitation into amyloid fibers. The peptides studied in this work are constituted by 17, 18 or 22 amino acids whose sequences are contained in the longer exon 30. They all contain the XGGZG sequence (where X, Z = V, L) previously suspected to be responsible for amyloid formation in elastin peptides. Two of them gave rise to amyloid fibers while the other one was able to coacervate. In this work we attempted to correlate vibrational, thermal and dielectric behavior of these peptides in the solid state with the propensity to lead to reversible or irreversible aggregation in vivo. [ABSTRACT FROM AUTHOR]

Amyloids are implicated in many diseases, and disruption of lipid membrane structures is considered as one possible mechanism of pathology. In this paper we investigate interactions between an aggregating peptide and phospholipid membranes, focusing on the nanometer-scale structures of the aggregates formed, as well as on the effect on the aggregation process. As a model system, we use the small amyloid-forming peptide named NACore, which is a fragment of the central region of the protein α-synuclein that is associated with Parkinson’s disease. We find that phospholipid vesicles readily associate with the amyloid fibril network in the form of highly distorted and trapped vesicles that also may wet the surface of the fibrils. This effect is most pronounced for model lipid systems containing only zwitterionic lipids. Fibrillation is found to be retarded by the presence of the vesicles. At the resolution of our measurements, which are based mainly on cryogenic transmission electron microscopy (cryo-TEM), X-ray scattering, and circular dichroism (CD) spectroscopy, we find that the resulting aggregates can be well fitted as linear combinations of peptide fibrils and phospholipid bilayers. There are no detectable effects on the cross-β packing of the peptide molecules in the fibrils, or on the thickness of the phospholipid bilayers. This suggests that while the peptide fibrils and lipid bilayers readily co-assemble on large length-scales, most of them still retain their separate structural identities on molecular length-scales. Comparison between this relatively simple model system and other amyloid systems might help distinguish aspects of amyloid-lipid interactions that are generic from aspects that are more protein specific. Finally, we briefly consider possible implications of the obtained results for in-vivo amyloid toxicity.

RADA 16-I is a synthetic amphiphilic peptide which self-assembles into nanofibers and scaffolds in favor of cell growth, hemostasis and tissue engineering. However, it is still unclear in which conditions the peptide maintains its stability and structural consistency in aqueous solutions during storage time. This study investigates dynamic behavior of RADA 16-I using circular dichroism, so as to monitor changes in conformation of the peptides dispersed in water (pH 5) as well as 0.003 M (pH 4) and 0.02 M (pH 3) acetic acid solutions for various incubation times (0.5, 60 and 120 days), concentrations (0.1, 0.3 and 0.5%) and temperatures (4 and 25 °C). The results showed that the peptides exhibit a predominantly helical structure immediately after making their solutions. However, it was seen that, when exposed to solutions with pH 3 and 4, the peptides started to lose the helical structure, with increased amounts of aggregation at these two acidic pH values; this was attributed to increased hydrophobicity. The stable RADA 16-I peptides were identified at pH 5, 0.1% solution and 4 °C, with their secondary structure remained mostly unchanged during the 120-day test period. Furthermore, statistical analysis showed that, the concentration had little effect while pH and temperature had significant effects on the peptide stability, while acidic pH enhanced aggregation. These results may serve as a scientific basis for the processing and application of peptide. [ABSTRACT FROM AUTHOR]

Amyloid fibrils are highly ordered, β-sheet rich forms of aggregated peptides and proteins that are associated with a variety of pathological human disorders, including Alzheimer's and Parkinson's diseases. Amyloid fibril-forming peptides may be functionally related to antimicrobial peptides, despite differing significantly in sequence and structure. Specifically, their interaction with lipid membranes has mechanistic similarities. The 17-amino acid peptide uperin 3.5 (U3.5) from an Australian amphibian is antimicrobial and amyloidogenic. Using a quartz crystal microbalance, we investigated the interaction of U3.5 with artificial membranes and found that (i) the membrane interaction of U3.5 is independent of the peptide's aggregation state, (ii) the presence of cholesterol in the membrane dramatically alters peptide–membrane interaction leading to a transmembrane pore-like arrangement of U3.5, and (iii) electrostatic interaction is important for the membrane activity of U3.5 whereby removal of the positive charge at position 7 of U3.5 enhanced its fibrillar aggregation and ablated its membrane interaction, i.e. there is an inverse relationship between the antimicrobial and amyloidogenic properties of U3.5. [ABSTRACT FROM AUTHOR]

The misfolding and aggregation of proteins and peptides into amyloid fibrils are believed to be responsible for the dysfunction and death of neuron cells in many neurodegenerative diseases. Resolving the atomic structures of amyloid peptides at different aggregation stages by molecular simulations has opened new ways to probe the molecular mechanisms of amyloid aggregation, toxicity, and inhibition, as well as to validate computational data with available experimental ones. In this review article, we summarize some recent and important findings on: 1) a number of atomic structures of amyloid oligomers with typical β-sheet-rich conformations, related to amyloid aggregation; 2) different amyloid peptide-induced membrane-disruption mechanisms, related to amyloid toxicity; and 3) rational design of different amyloid inhibitors capable of preventing amyloid aggregation and toxicity, related to amyloid inhibition. All these findings will provide some mechanistic implications for molecular mechanisms of amyloid aggregation, toxicity, and inhibition, which are fundamentally and practically important for the treatment of amyloid diseases. [ABSTRACT FROM AUTHOR]

A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286, is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a 'semen-derived enhancer of virus infection' (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into β-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L−1. Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03 μmol L−1), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs. [ABSTRACT FROM AUTHOR]

Amyloid β-peptide (Aβ) fibrillation is a major hallmark of Alzheimer's disease (AD). Inhibition of Aβ fibrillation is thus considered to be an effective strategy for AD prevention and treatment. Here we show that para-sulfonatocalix[ n]arenes (SC[ n]A, n=4, 6, 8), a class of amphiphilic calixarene derivatives, can bind to Aβ42 through nonspecific and multipoint hydrophobic interactions. Their binding leads to a pronounced delay in β-sheet adoption and formation of multiple secondary structures of the peptide, accompanied by changes at the level of the fibrillary architecture. Furthermore, the ζ-potential value of Aβ42 incubated with SC[6/8]A decreased, which correlated with the reduction of amyloid cytotoxicity. Overall, the SC[ n]A effectively inhibits Aβ42 fibrillation and reduces amyloid cytotoxicity, and SC[8]A showed the best performance among the three macrocycles, possibly owing to its having the strongest interactions with Aβ42. [ABSTRACT FROM AUTHOR]

Current theories on the origin of life reveal significant gaps in our understanding of the mechanisms that allowed simple chemical precursors to coalesce into the complex polymers that are needed to sustain life. The volcanic gas carbonyl sulfide (COS) is known to catalyze the condensation of amino acids under aqueous conditions, but the reported di-, tri-, and tetra-peptides are too short to support a regular tertiary structure. Here, we demonstrate that alanine and valine, two of the proteinogenic amino acids believed to have been among the most abundant on a prebiotic earth, can polymerize into peptides and subsequently assemble into ordered amyloid fibers comprising a cross-β-sheet quaternary structure following COS-activated continuous polymerization of as little as 1 m m amino acid. Furthermore, this spontaneous assembly is not limited to pure amino acids, since mixtures of glycine, alanine, aspartate, and valine yield similar structures. [ABSTRACT FROM AUTHOR]

Objectives: The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Results: Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Conclusions: Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins. [ABSTRACT FROM AUTHOR]

The biological activity of antibiotic peptaibols has been linked to their ability to aggregate, but the structure-activity relationship for aggregation is not well understood. Herein, we report a systematic study of a class of synthetic helical oligomer (foldamer) composed of aminoisobutyric acid (Aib) residues, which mimic the folding behavior of peptaibols. NMR spectroscopic analysis was used to quantify the dimerization constants in solution, which showed hydrogen-bond donors at the N terminus promoted aggregation more effectively than similar modifications at the C terminus. Elongation of the peptide chain also favored aggregation. The geometry of aggregation in solution was investigated by means of titrations with [D6]DMSO and 2D NOE NMR spectroscopy, which allowed the NH protons most involved in intermolecular hydrogen bonds in solution to be identified. X-ray crystallography studies of two oligomers allowed a comparison of the inter- and intramolecular hydrogen-bonding interactions in the solid state and in solution and gave further insight into the geometry of foldamer-foldamer interactions. These solution-based and solid-state studies indicated that the preferred geometry for aggregation is through head-to-tail interactions between the N and C termini of adjacent Aib oligomers. [ABSTRACT FROM AUTHOR]

In the present work, a new electrochemical strategy for the sensitive and specific detection of soluble β-amyloid Aβ(1-40/1-42) peptides in a rat model of Alzheimer's disease (AD) is described. In contrast to previous antibody-based methods, β-amyloid(1-40/1-42) was quantified based on its binding to gelsolin, a secretory protein present in the cerebrospinal fluid (CSF) and plasma. The level of soluble β-amyloid peptides in the CSF and various brain regions were found with this method to be lower in rats with AD than in normal rats. [ABSTRACT FROM AUTHOR]

Formation of an asymmetric dimer by the EGFR (epidermal growth factor receptor) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, andwe sought to understand the basis for this. PeptideCwas found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C probably underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregationmediated mechanism. [ABSTRACT FROM AUTHOR]

Aggregates of amyloid beta (Aβ) peptides are believed to be responsible for the neuropathology of Alzheimer's disease. In this work, ferrocene (Fc) is attached to the aggregating core of the Aβ peptides, KLVFFAE. Inhibition of Fc-KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC-electrochemical detection (HPLC-EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc-KLVFFAE in its monomeric form. We demonstrate that tagging Fc to KLVFFAE affords a cost-effective and electroactive mimicry of Aβ(142) and HPLC-EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of Aβ peptides. [ABSTRACT FROM AUTHOR]

Aggregation of amyloid β-peptide (Aβ) is closely related to the pathogenesis of Alzheimer's disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aβ1-42, high doses are needed for the inhibition of Aβ aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo-Aβ β-sheet structures can work as an aggregation inhibitor against Aβ. To further test this design strategy, we constructed protein analogues that mimic Aβ β-sheet structures of amyloids by using insulin-like growth factor 2 receptor domain 11 (IGF2R-d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aβ-like β sheets, can bind more preferentially to oligomeric Aβ1-42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aβ1-42 efficiently, even though lower concentrations of IG11KK than Aβ were used. The aggregation kinetics of Aβ in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aβ aggregation owing to its favorable binding to oligomeric structures of Aβ. The design strategy using β-barrel proteins such as IGF2R-d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation. [ABSTRACT FROM AUTHOR]

Self-assembly is a desired property in supramolecular chemistry, but extensive aggregation may be counterproductive. Rigid systems typically have better organization, but are inherently less dynamic. This work shows that ion transport by amphiphilic heptapeptides (synthetic anion transporters or SATs) is affected by aggregation of the monomers in the bulk aqueous phase to which they are added and within the bilayer. Ion transport was assessed for all compounds by assay of Cl− release from liposomes. The mechanism of ion transport was confirmed by planar bilayer conductance studies for two compounds at opposite ends of the efficacy scale. Dynamic light scattering, the Langmuir trough, transmission electron microscopy, ion release from liposomes, and planar bilayer conductance studies were used to assess the importance of self-assembly versus aggregation in ion transport. Generally, greater aggregation was has an adverse effect on the transport, although at least dimerization is required for amphiphilic heptapeptides to readily transport Cl−. Anion transport in these systems was found to be sensitive to changes in the C-terminal portion of the (Gly)3Pro(Gly)3 sequence. Moreover, a significant difference in transport efficacy was apparent when L-Trp was replaced by D-Trp in the same position. [ABSTRACT FROM AUTHOR]

Genetic, biochemical and pathological evidence support that self-assembly of amyloid-beta (Aβ) peptide into toxic aggregates is implicated as the cause of Alzheimer's disease. An attractive therapeutic strategy for the treatment of AD is to prevent or interfere with Aβ aggregation. A systematic investigation of the effects of proline-, glycine-, arginine- and lysine- containing peptides (PGKLVYA, KKLVFFARRRRA and KKLVFFA) on the beta-amyloid aggregation was made using FTIR, circular dichroism, ANS binding, ThT binding and TEM analysis. These peptides are based on the central hydrophobic region of Aβ (residues 16-20), which is believed to be crucial in Aβ self-association. There is increasing evidence to suggest that protein aggregation, including amyloid fibril formation results from the strong self-association tendency of the partially folded intermediates. Addition of PGKLVYA and KKLVFFARRRRA resulted in increase in ANS fluorescence intensity, suggesting enhanced exposure of hydrophobic surface area. As observed by ThT and TEM analysis PGKLVYA and KKLVFFARRRRA promote non-fibrillar ensembles, while peptide KKLVFFA accelerated the fibrillization of Aβ peptide by stabilizing intermolecular interactions. Circular dichroism and FTIR data showed that PGKLVYA and KKLVFFARRRRA effectively prevented amyloid-beta (Aβ) peptide adopting the beta-sheet secondary structure correlated with fibrillogenesis. This result indicates that PGKLVYA and KKLVFFARRRRA might have triggered another mechanism of Aβ assembly. [ABSTRACT FROM AUTHOR]

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an α-helical conformation or aggregated β-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to β-sheet formation and subsequent aggregation. [ABSTRACT FROM AUTHOR]

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer’s disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ42 fibrillization and initiate formation of non-fibrillar Aβ42 aggregates, and that the inhibitory effect of Zn(II) (IC50 = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ42 aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ42 fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ42. H13A and H14A mutations in Aβ42 reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ42 aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ42 aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer’s disease is discussed in this light. [ABSTRACT FROM AUTHOR]

Molecular dynamics (MD) simulations of amyloid beta peptide have been performed in aqueous solutions of trifluoroethanol with different concentrations. The amount of α-helical secondary structure increases when going from pure water to trifluoroethanol-rich solutions. The conformation obtained in 40% (v/v) trifluoroethanol solution is very similar to the experimental observations of beta peptide in sodium dodecyl sulfate micelle. In this solution, the peptide has two helical segments connected through a looped region. The C-terminal helix of beta peptide unfolds in pure water. The effect of trifluoroethanol on peptide's secondary structure has been explained using the properties calculated from MD trajectories. [ABSTRACT FROM AUTHOR]

We report the development of a high-level bacterial expression system for the Alzheimer’s disease-associated amyloid β-peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Aβ(1–40) and Met-Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss. [ABSTRACT FROM AUTHOR]

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins [ABSTRACT FROM AUTHOR]

In order to provide insights into the misfolding mechanism and the subsequent aggregate formation which cause what are known as the neurodegenerative polyglutamine diseases, we have simulated a 10-residue polyglutamine (poly-Q) chain in vacuum and in solvent by multicanonical method, which enabled us to study the system in a wide temperature range and discuss thermodynamic properties. It is understood that the system in vacuum shows two phase transitions, first of them occur at high temperature that is the well-known helix-coil transition and the second one is a solid-solid transition. However, the poly-Q chain in solvent is in a random coil state at higher temperatures, goes through a conformational change at T = 200 K and assumes predominantly a mixture of anti-parallel β-sheet and α-helix structures at low temperatures. One-residue glutamine dipeptide is also simulated and low-energy stable conformations are identified. [ABSTRACT FROM AUTHOR]

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1–R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by 1H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1–R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed. [ABSTRACT FROM PUBLISHER]

NMR studies of amyloid β-peptides (Aβ) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer’s disease-related conformational polymorphism of Aβ. In the aqueous medium, neither of the polypeptides Aβ(1–40)ox or Aβ(1–42)ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Aβ(1–40)ox and Aβ(1–42)ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Aβ(1–42)ox when compared to Aβ(1–40)ox. The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Aβ(1–42)ox in aqueous solution has much higher plaque-competence than Aβ(1–40)ox. [ABSTRACT FROM AUTHOR]

This review is focused on the biosensing assay based on AuNPs (AuNPs) modified by proteins, peptides and nucleic acid aptamers. The unique physical properties of AuNPs allow their modification by proteins, peptides or nucleic acid aptamers by chemisorption as well as other methods including physical adsorption and covalent immobilization using carbodiimide chemistry or based on strong binding of biotinylated receptors on neutravidin, streptavidin or avidin. The methods of AuNPs preparation, their chemical modification and application in several biosensing assays are presented with focus on application of nucleic acid aptamers for colorimetry assay for determination of antibiotics and bacteria in food samples. [ABSTRACT FROM AUTHOR]

Amyloid proteins are linked to the pathogenesis of several diseases including Alzheimer's disease, but at the same time a range of functional amyloids are physiologically important in humans. Although the disease pathogenies have been associated with protein aggregation, the mechanisms and factors that lead to protein aggregation are not completely understood. Paradoxically, unique characteristics of amyloids provide new opportunities for engineering innovative materials with biomedical applications. In this review, we discuss not only outstanding advances in biomedical applications of amyloid peptides, but also the mechanism of amyloid aggregation, factors affecting the process, and core sequences driving the aggregation. We aim with this review to provide a useful manual for those who engineer amyloids for innovative medicine solutions. [ABSTRACT FROM AUTHOR]

We have found that a quartz-crystal microbalance is a facile and useful tool for detecting the specific aggregation of the amyloid- β peptides responsible for Alzheimer's disease. This method greatly reduces the time and samples (corresponds to 1 Hz frequency decrease per 30 pg mass increase on a 4.9 mm2 electrode) that are needed in the aggregation assay, and is also applicable to molecular interactions between smaller amyloid- β fragment peptides such as the pentapeptide KLVFF. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are a diverse group of membrane-active peptides which play a crucial role as mediators of the primary host defense against microbial invasion. Many AMPs are found to be fully or partially disordered in solution and to acquire secondary structure upon interaction with a lipid membrane. Here, we report molecular dynamics simulations studies on the solution behaviour of a specific AMP, bombinin H2. We show that in monomeric form in water solution the peptide is somewhat disordered and preferably adopts a helix-loop-helix conformation. However, when more than a single monomer is placed in the solution, the peptides self-associate in aggregates. Within the aggregate, the peptides provide each other with an amphipathic environment that mimics the water–membrane interface, which allows them to adopt a single-helix structure. We hypothesise that this is the mechanism by which bombinin H2 and, possibly, other small linear AMPs reach the target membrane in a functional folded state and are able to effectively exert their antimicrobial action on it. [ABSTRACT FROM AUTHOR]

The adsorption of amyloidogenic peptides, amyloid beta 1–40 (Aβ1–40), alpha-synuclein (α-syn), and beta 2 microglobulin (β2m), was attempted over the surface of nano-gold colloidal particles, ranging from d = 10 to 100 nm in diameter (d). The spectroscopic inspection between pH 2 and pH 12 successfully extracted the critical pH point (pHo) at which the color change of the amyloidogenic peptide-coated nano-gold colloids occurred due to aggregation of the nano-gold colloids. The change in surface property caused by the degree of peptide coverage was hypothesized to reflect the ΔpHo, which is the difference in pHo between bare gold colloids and peptide coated gold colloids. The coverage ratio (Θ) for all amyloidogenic peptides over gold colloid of different sizes was extracted by assuming Θ = 0 at ΔpHo = 0. Remarkably, Θ was found to have a nano-gold colloidal size dependence, however, this nano-size dependence was not simply correlated with d. The geometric analysis and simulation of reproducing Θ was conducted by assuming a prolate shape of all amyloidogenic peptides. The simulation concluded that a spiking-out orientation of a prolate was required in order to reproduce the extracted Θ. The involvement of a secondary layer was suggested; this secondary layer was considered to be due to the networking of the peptides. An extracted average distance of networking between adjacent gold colloids supports the binding of peptides as if they are "entangled" and enclosed in an interfacial distance that was found to be approximately 2 nm. The complex nano-size dependence of Θ was explained by available spacing between adjacent prolates. When the secondary layer was formed, Aβ1–40 and α-syn possessed a higher affinity to a partially negative nano-gold colloidal surface. However, β2m peptides tend to interact with each other. This difference was explained by the difference in partial charge distribution over a monomer. Both Aβ1–40 and α-syn are considered to have a partial charge (especially δ+) distribution centering around the prolate axis. The β2m, however, possesses a distorted charge distribution. For a lower Θ (i.e., Θ <0.5), a prolate was assumed to conduct a gyration motion, maintaining the spiking-out orientation to fill in the unoccupied space with a tilting angle ranging between 5° and 58° depending on the nano-scale and peptide coated to the gold colloid. [ABSTRACT FROM AUTHOR]

In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin "amyloidogenic" analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3–7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation. [ABSTRACT FROM AUTHOR]

In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time. The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged in triggering aggregation of the entire structure of human insulin under physiological conditions. It was found that the aggregation process occurs through various structural conformers and may favor the formation of a fibrous structure of aggregate. [ABSTRACT FROM AUTHOR]