Aβ Peptide Fragments Research Articles

Interactions of Fibrillar Proteins with Lipids: A Molecular Docking Insight

The aggregation of misfolded proteins into specific ordered aggregates, amyloid fibrils, associated with more than forty human diseases, currently attracts great research attention in biomedical and nanotechnological aspects. These aggregates and their oligomeric intermediates are thought to exert their toxic action predominantly at the level of cell membranes. In addition, membrane lipids were found in many amyloid deposits in vivo suggesting that lipid molecules are able to incorporate into fibril structure affecting their morphology and mechanical properties. However, the biological implications and structural prerequisites of fibril-lipid interactions still remain unclear. In the present study the molecular docking techniques was employed to explore the interactions between the amyloid fibrils and lipids in the model systems containing the fibrillar forms of lysozyme, insulin, Aβ (1-42) peptide and N-terminal (1-83) fragment of apolipoprotein A-I, as a protein component and cholesterol, cardiolipin or phosphatidylcholine as a lipid component. Using the PatchDock web server and BIOVIA Discovery Studio software, the structural peculiarities of fibril-lipid associates were uncovered. The van der Waals and alkyl/π-alkyl interactions were found to prevail in stabilization of all types of fibril-lipid complexes. The analysis of most energetically favorable docking positions revealed a preferable surface location of lipids and partial penetration of acyl chains of cardiolipin and phosphatidylcholine into fibril grooves.

Controlling Amyloid Beta Peptide Aggregation and Toxicity by Protease-Stable Ligands.

Polymerization of soluble amyloid beta (Aβ) peptide into protease-stable insoluble fibrillary aggregates is a critical step in the pathogenesis of Alzheimer's disease (AD). The N-terminal (NT) hydrophobic central domain fragment 16KLVFF20 plays an important role in the formation and stabilization of β-sheets by self-recognition of the parent Aβ peptide, followed by aggregation of Aβ in the AD brain. Here, we analyze the effect of the NT region inducing β-sheet formation in the Aβ peptide by a single amino acid mutation in the native Aβ peptide fragment. We designed 14 hydrophobic peptides (NT-01 to NT-14) by a single mutation at 18Val by using hydrophobic residues leucine and proline in the natural Aβ peptide fragment (KLVFFAE) and analyzed its effect on the formation of Aβ aggregates. Among all these peptides, NT-02, NT-03, and NT-13 significantly affected the Aβ aggregate formation. When the NT peptides were coincubated with the Aβ peptide, a significant reduction in β-sheet formation and increment in random coil content of Aβ was seen, confirmed by circular dichroism spectroscopy and Fourier transform infrared spectroscopy, followed by the reduction of fibril formation measured by the thioflavin-T (ThT) binding assay. The aggregation inhibition was monitored by Congo red and ThT staining and electron microscopic examination. Moreover, the NT peptides protect the PC-12 differentiated neurons from Aβ-induced toxicity and apoptosis in vitro. Thus, manipulation of the Aβ secondary structure with protease-stable ligands that promote the random coil conformation may provide a tool to control the Aβ aggregates observed in AD patients.

Establishing a Type 2 Diabetes phenotype in APPxhQC transgenic mice expressing N‐terminally modified pGlu Aβ peptides.

AbstractBackgroundAlzheimer’s disease (AD) is a debilitating neurodegenerative disease characterized by the accumulation of amyloid beta (Aβ) plaques in the brain. Aβ peptides are commonly subjected to post‐translational modifications like truncation known to play pivotal role in Aβ plaque aggregation. N‐terminally truncated Aβ peptides containing pyroglutamatic acid (pGlu) catalyzed by glutaminyl cyclase (QC) e.g., pGlu3‐Aβ (3‐40/42) are the major Aβ peptide fragments within the core of the neuritic plaques and correlate with disease severity and progression. Type 2 diabetes (T2D) is one of the major risk factors associated with AD and compelling evidence supports the notion that insulin resistance, a key feature of T2D, is involved in AD‐type neurodegeneration. Hence, in this study, we aim to establish a T2D phenotype in APPxhQC transgenic using low‐dose Streptozocin injection in combination with high fat diet (HFD).MethodsAPPxhQC mice, crossbreds of APPSL and hQC mice, were used. APPSL mice express human APP751 with the Swedish and the London mutation on a C57Bl/6RccHsd background. hQC mice on B6CBAF1/J background express human glutaminyl cyclase (QC) enzyme. The cross‐breeding results in an increased generation of N‐terminal modified pGlu Aβ peptides. To establish a T2D phenotype, 8‐9 months old APPxhQC transgenic mice were allocated to 4 treatment groups receiving: standard chow diet, HFD, standard chow diet in combination with streptozocin injections and HFD in combination with streptozocin injections. The intraperitoneal glucose tolerance test (ipGTT) and intraperitoneal insulin tolerance test (ipITT) were performed at 6 and 7 weeks after the initiation of the diet, as a relevant parameter of T2D progression.ResultsStreptozocin‐injected mice maintained on HFD had the least tolerance to injected glucose as shown by ipGTT. Impaired glucose clearance was observed at 15, 30, 60 and 120 mins after intraperitoneal glucose injection (2g/kg). Also, impaired glucose clearance at 15 mins after intraperitoneal insulin injection (0.75U/kg) was observed in the same group.ConclusionCombination of high‐fat diet and streptozocin injections leads to the development of robust T2D symptoms in APPxhQC transgenic mice. Current experiments are focusing on combinational effects of T2D and post‐translationally modified amyloid peptides in AD progression and pathophysiology.

Activated Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Signal and Suppressed Autophagy Participate in Protection Offered by Licochalcone A Against Amyloid-β Peptide Fragment 25–35–Induced Injury in SH-SY5Y Cells

To assess effect of licochalcone A (LicA) on amyloid-β (Aβ) peptide fragment 25-35-induced nerve injury and reveal the potential molecular mechanisms involved. Viability of SH-SY5Y cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay after treatment with Aβ25-35 and/or LicA, following which apoptosis was detected by flow cytometry and Hoechst staining. Then, reactive oxygen species, glutathione, and superoxide dismutase were measured with flow cytometry and spectrophotometry. The ultrastructure of mitochondria was examined by transmission electron microscopy, and the biomarker proteins of autophagy, apoptosis, and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were measured with Western blotting. LicA improved cell viability and decreased lactate dehydrogenase leakage remarkably in Aβ25-35-induced injury in SH-SY5Y cells. After treatment with LicA, reactive oxygen species, glutathione, and superoxide dismutase levels in cells all were significantly decreased, which indicated that LicA has an antioxidative effect on Aβ25-35-induced oxidative injury. LicA could also significantly reduce Aβ25-35-induced autophagy in SH-SY5Y cells. In the cells injured by Aβ25-35, LicA prevented the transformation from light chain protein 3-I to light chain protein 3-II and reduced the levels of proteins GRP78, GRP94, CHOP, and Bax, but increased the levels of antiapoptotic protein and phosphorylation of PI3K, Akt, and mTOR. These effects of LicA were restored or suppressed by mTOR inhibitor rapamycin or PI3K inhibitor LY294002. LicA protects SH-SY5Y cells against Aβ25-35-induced injury, wherein suppressed autophagy and activated PI3K/Akt/mTOR signaling pathway are involved, and mTOR-dependent autophagy at least plays some role.

Absolute Quantitation of Tryptophan-Containing Peptides and Amyloid β-Peptide Fragments by Coulometric Mass Spectrometry.

Isotope-labeled internal standards are routinely used for mass spectrometry (MS)-based absolute quantitation. However, syntheses of isotope-labeled peptides are time-consuming and costly. To tackle this issue, we recently developed a coulometric mass spectrometric (CMS) approach for absolute quantitation without the use of standards, based on the electrochemical oxidation of cysteine or tyrosine-containing peptides followed by mass spectrometric measurement of the oxidation yield. To further expand the utility of this method, herein we present the CMS method for absolute quantitation of peptides based on tryptophan electrochemical oxidation. Several tryptophan-containing peptides, such as WGG, WQPPRARI, WAGGDASGE, RTRPLWVRME, and KVPRNQDWL, were successfully quantified with a quantification error ranging from -4.5 to +4.3%. Furthermore, this quantitation approach is also applicable to protein, in which protein can be digested and a surrogate peptide can be selected for quantification to reflect the amount of the parent protein, as exemplified by CMS analysis of peptide GITWK from cytochrome c. The CMS result agreed well with the traditional isotope dilution method, with only a small difference of 3.5%. In addition, CMS was used to successfully quantify amyloid beta (Aβ) peptide fragments (up to 28 amino acid residues) based on tyrosine oxidation. The validity of the CMS method for peptide and protein absolute quantitation without using isotope-labeled peptide standards would greatly facilitate proteomics research.

Supramolecular Antiparallel β-Sheet Formation by Tetrapeptides Based on Amyloid Sequence.

Self-assembly of short peptides has emerged as an interesting research field for a wide range of applications. Recently, several truncated fragments of long-chain peptides or proteins responsible for different neurodegenerative diseases were studied to understand whether they can mimic the property and function of native peptides or not. It was reported that such a kind of peptide adopts a β-sheet structure in the disease state. It was observed that aromatic amino acid-rich peptide fragments possess a high tendency to adopt a β-sheet conformation. In this article, we are first time reporting the crystal structure of two tetrapeptides: Boc-GAII-OMe (Peptide 1) and Boc-GGVV-OMe (Peptide 2), composed of aliphatic amino acids, and the sequences are similar to the Aβ-peptide fragments Aβ29-32 and Aβ37-40 , respectively. In the solid-state, they are self-assembled in an antiparallel β-sheet fashion. The peptide units are connected by the strong amide hydrogen-bonding (N-H···O) interactions. Apart from that, other noncovalent interactions are also present, which help to stabilize the cross-β-sheet arrangement. Interestingly, in the crystal structure of Peptide 1, noncovalent C···C interaction between the electron-deficient carbonyl carbon, and the electron-rich sp3-carbon atom is observed, which is quite rare in the literature. The calculated torsion angles for these peptides are lying in the β-sheet region of the Ramachandran plot. FT-IR studies also indicate the formation of an antiparallel β-sheet structure in the solid-state. Circular dichroism of the peptides in the aqueous solution also suggests the presence of predominantly β-sheet-like conformation in the aqueous solution. Under cross-polarized light, Congo Red stained both peptides showed green-gold color due to birefringence indicating their amyloidogenic nature. This result indicates that the short peptide composed of aliphatic amino acid is capable of forming a β-sheet structure in the absence of aromatic amino acid and also can mimic the function of the native amyloid peptide.

Partitioning of Aβ Peptide Fragments into Blood-Brain Barrier Mimetic Bilayer.

We used all-atom replica-exchange umbrella sampling molecular dynamics simulations to investigate the partitioning of the charged tetrapeptide KLVF and its neutral apolar counterpart VVIA into the blood-brain barrier (BBB)-mimetic bilayer. Our findings allowed us to reconstruct the partitioning mechanism for these two Aβ peptide fragments. Despite dissimilar sequences, their permeation shares significant common features. Computations of free energies and permeabilities show that partitioning of both peptides is highly unfavorable, ruling out passive transport. The peptides experience multiple rotational transitions within the bilayer and typically cause considerable lipid disorder and bilayer thinning. Near the bilayer midplane, they lose almost entirely their solvation shell and the interactions with the lipid headgroups. The peptides cause complex reorganization within the proximal bilayer region. Upon insertion, they induce striking cholesterol influx reversed by its depletion and the influx of DMPC when the peptides reach the midplane. The differences in partitioning mechanisms are due to the much higher polarity of KLVF peptide, the permeation of which is more unfavorable and which exclusively assumes vertical orientations within the bilayer. In contrast, VVIA positions itself flat between the leaflets, causing minor disorder and even thickening of the BBB-mimetic bilayer. Due to the high density of the cholesterol-rich BBB bilayer, the unfavorable work associated with the peptide insertion provides a significant, but not dominant, contribution to the partition free energy, which is still governed by dehydration and loss of peptide-headgroup interactions. Comparison with experiments indicates that KLVF and VVIA permeation is similar to that of proline tetrapeptide, mannitol, or cimetidine, all of which exhibit no passive transport.

Promotion and Inhibition of Amyloid-β Peptide Aggregation: Molecular Dynamics Studies.

Aggregates of amyloid-β (Aβ) peptides are known to be related to Alzheimer’s disease. Their aggregation is enhanced at hydrophilic–hydrophobic interfaces, such as a cell membrane surface and air-water interface, and is inhibited by polyphenols, such as myricetin and rosmarinic acid. We review molecular dynamics (MD) simulation approaches of a full-length Aβ peptide, Aβ40, and Aβ(16–22) fragments in these environments. Since these peptides have both hydrophilic and hydrophobic amino acid residues, they tend to exist at the interfaces. The high concentration of the peptides accelerates the aggregation there. In addition, Aβ40 forms a β-hairpin structure, and this structure accelerates the aggregation. We also describe the inhibition mechanism of the Aβ(16–22) aggregation by polyphenols. The aggregation of Aβ(16–22) fragments is caused mainly by the electrostatic attraction between charged amino acid residues known as Lys16 and Glu22. Since polyphenols form hydrogen bonds between their hydroxy and carboxyl groups and these charged amino acid residues, they inhibit the aggregation.

Aluminium Binding to Modified Amyloid-β Peptides: Implications for Alzheimer's Disease.

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer’s disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aβ peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aβ(1–16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aβ(1–16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aβ(1–16)A36,13,14 and Aβ(1–16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aβ(9–16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aβ(9–16)G110, (H-9GGEVHHQK16-NH2), Aβ(9–16)G213,14 (H-9GYEVGGQK16-NH2), and Aβ(9–16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al–peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aβ peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.

An agnostic reevaluation of the amyloid cascade hypothesis of Alzheimer's disease pathogenesis: The role of APP homeostasis.

To reassess the role of amyloid beta (Aβ) and the amyloid precursor protein (APP) system in the pathogenesis of Alzheimer's disease (AD). APP is a cell adhesion molecule that has been highly conserved over the course of phylogeny that has critical roles in brain development, synaptic plasticity, and the brain's intrinsic immune system. The amyloid cascade hypothesis describes a relatively linear, deterministic sequence of events triggered by a gain of Aβ peptide fragment toxicity that results in neurodegeneration and cognitive loss, yet well designed immunotherapy and beta secretase inhibitor trials that have successfully targeted Aβ have failed to have any consistent effects on the steady decline of cognition. Mutations of the APP and presenilin genes not only alter the ratio of longer to shorter Aβ fragments (resulting in a gain of Aβ toxicity), but also disrupt the normal homeostatic roles of their respective proteins. The evolutionary history, physiological importance, and complexity of the APP and presenilin systems, as well as other critical components including tau and apolipoprotein E (APOE) imply that altered function of such systems could have severe consequences that include but need not be limited to a gain of Aβ toxicity and would more generally result in altered homeostasis of APP-related functions. Challenges that a loss of APP homeostasis addresses better than the more limited gain of Aβ toxicity model include the topographic mismatches between Aβ and tau pathology, the profile and chronology of cognitive and biomarker changes that precede the clinical expression of mild cognitive impairment and dementia, and the disappointments of Aβ targeted therapeutics among others. The importance of APP, α- and β-secretases, the presenilins and γ-secretase, as well as tau was recognized by the authors of the amyloid cascade hypothesis, and has since led multiple investigators to propose alternative, more balanced hypotheses including reduced homeostasis and frank loss-of-function of key components that include but go beyond the currently envisioned linear model of Aβ toxicity.

Supramolecular β-Sheet Forming Peptide Conjugated with Near-Infrared Chromophore for Selective Targeting, Imaging, and Dysfunction of Mitochondria.

Herein, conjugation of the amyloid-β (Aβ) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aβ16-20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson's correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer -220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

Histidine-Lacked Aβ(1–16) Peptides: pH-Dependent Conformational Changes in Metal Ion Binding

The region 1–16 of the amyloid-β peptides associated with Alzheimer’s disease can be considered as the metal-binding domain, and the residues His6, His13, and His14 have been identified as ligands that coordinate heavy metal ions. To establish the role played by the three histidine residues of the Aβ(1–16) sequence in binding metal ions, two variants of Aβ(1–16) peptide fragment (H-1DAEFRHDSGYEVHHQK16-NH2) were synthesized by Fmoc/tBu Solid Phase Peptide Synthesis strategy and purified by RP-HPLC. The three histidine residues were replaced by either three serine residues to form Ser-Aβ(1–16): H-1DAEFRSDSGYEVSSQK16-NH2 or three alanine ones to form Ala–Aβ(1–16): H-1DAEFRADSGYEVAAQK16-NH2. The three peptides were comparatively characterized by mass spectrometry (MS), circular dichroism spectroscopy (CD) and atomic force microscopy (AFM), being subsequently used to study their interaction with metal ions at various pH values. During our experiments, significant pH-dependent changes in the conformation of these peptides have been observed and reported. AFM images showed dramatic changes in the film morphology of peptides upon binding metal ions. In the MS spectra we have unambiguously identified several metal–peptide molecular ions, thus confirming the affinity of Aβ(1–16) peptides to copper and nickel ions. The newly synthesized peptides may bring new evidence on the relationship of histidine residues in amyloid-β peptides with metal ions. In addition, new directions for research and experimental works are suggested.

The effect of terminal groups and halogenation of KLVFF peptide on its activity as an inhibitor of β-amyloid aggregation.

The aggregation of Aβ peptide into amyloid fibrils in the brain is associated with Alzheimer's disease (AD). Inhibition of Aβ aggregation seemed a potential treatment for AD. It was previously shown that a short fragment of Aβ peptide (KLVFF, 16-20) bound Aβ inhibited its aggregation. In this work, using KLVFF peptide, we synthesized two peptide families and then evaluated their inhibitory capacities by conventional assays such as thioflavin T (ThT) fluorescence spectroscopy, turbidity measurement, and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). The effect of peptide terminal groups on its inhibitory activity was first studied. Subsequently, the influence of halogenated amino acids on peptide anti-aggregation properties was investigated. We found that iodinated peptide with amine in the N and amide in the C termini, respectively, was the best inhibitor of Aβ fibers formation. Halogenated peptides seemed to decrease the number of Aβ fibrils; however, they did not reduce Aβ cytotoxicity. The data obtained in this work seemed promising in developing potential peptide drugs for treatment of AD.

Improving separation optimization in capillary electrophoresis by using a general quality criterion

In this paper we extend the use of the quality criterion t’ to optimize separations in capillary electrophoresis (CE). The theoretical parameter t’ takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T′, for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization. Here, we demonstrate the applicability of T′ using as a variable the electrophoretic mobility (me) for the optimization of pH in the separation of a mixture of amyloid beta (Aβ) peptide fragments. In addition, it is shown the versatility of T′ using other variables related to me, which do not require experimental measurements. This is the case with ionizable compounds as the Aβ peptide fragments, whose charge-to-mass ratios can be calculated if accurate pKa values are available in the literature. The excellent performance of T’ for Aβ peptide fragments is further validated optimizing the pH for the separation of mixtures of harmala alkaloids (HAlks) and quinolone antibiotics.

MicroRNA-25 aggravates Aβ1-42-induced hippocampal neuron injury in Alzheimer's disease by downregulating KLF2 via the Nrf2 signaling pathway in a mouse model.

Recently, numerous microRNAs (miRNAs) have been considered as key players in the regulation of neuronal processes. The purpose of the present study is to explore the effect of miR-25 on hippocampal neuron injury in Alzheimer's disease (AD) induced by amyloid β (Aβ) peptide fragment 1 to 42 (Aβ1-42) via Kruppel-like factor 2 (KLF2) through the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway. A mouse model of AD was established through Aβ1-42 induction. The underlying regulatory mechanisms of miR-25 were analyzed through treatment of miR-25 mimics, miR-25 inhibitors, or small interfering RNA (siRNA) against KLF2 in hippocampal tissues and cells isolated from AD mice. The targeting relationship between miR-25 and KLF2 was predicted using a target prediction program and verified by luciferase activity determination. MTT assay was used to evaluate the proliferative ability and flow cytometry to detect cell cycle distribution and apoptosis. KLF2 was confirmed as a target gene of miR-25. When the mice were induced by Aβ1-42, proliferation was suppressed while apoptosis was promoted in hippocampal neurons as evidenced by lower levels of KLF2, Nrf2, haem oxygenase, glutathione S transferase α1, glutathione, thioredoxin, and B-cell lymphoma-2 along with higher bax level. However, such alternations could be reversed by treatment of miR-25 inhibitors. These findings indicate that miR-25 may inhibit hippocampal neuron proliferation while promoting apoptosis, thereby aggravating hippocampal neuron injury through downregulation of KLF2 via the Nrf2 signaling pathway.

Structural analysis of copper(I) interaction with amyloid β peptide

The N-terminal fragment of Aβ (β = beta) peptide is able to bind essential transition metal ions like, copper, zinc and iron. Metal binding usually occurs via the imidazole nitrogens of the three His residues which play a key role in the coordination chemistry. Among all the investigated systems, the interaction between copper and Amyloid β assume a biological relevance because of the interplay between the two copper oxidation states, Cu(II) and Cu(I), and their involvement in redox reactions. Both copper ions share the ability to bind Amyloid β. A huge number of investigations have demonstrated that Cu(II) anchors to the N-terminal amino and His6, His13/14 imidazole groups, while Cu(I) forms a linear complex by coordinating to the His13 and His14 dyad. In this study we have analyzed Cu(I) interaction with the Amyloid β fragment encompassing the first 16 amino-acids. Our data were obtained by means of NMR spectroscopy which provided relevant structural details of the metal complexes. Our findings are consistent with the involvement of two or three His in the Cu(I) coordination sphere and indicate that His6 effectively participates to the metal binding.

Interaction of Amyloid Aβ(9–16) Peptide Fragment with Metal Ions: CD, FT-IR, and Fluorescence Spectroscopic Studies

Heavy metal ions and those of aluminum may interact with amyloid-β peptides (Αβ) associated with Alzheimer’s disease, contributing to Aβ aggregation and fibrillation that worsen this neuropathology. Nevertheless, the precise residues involved in metal ligation or interaction are yet to be established, although the N-terminal Aβ(1–16) peptide fragment is considered to be the metal-binding domain. However, because the results of the metal ion binding studies using Aβ(1–16) were not very conclusive, we have investigated shorter peptides. Here, we report the synthesis of truncated Aβ(9–16) peptide of the human Aβ(1–40) and Aβ(1–42) peptides, as well as its characterization by nuclear magnetic resonance and mass spectrometry. Furthermore, the Aβ(9–16) peptide interactions with various metal ions like Cu2+, Zn2+, Fe3+, Al3+, and Ni2+ were studied by circular dichroism, Fourier transform-infrared, and fluorescence spectroscopy. Our results show that the newly synthesized peptide sequence, which contains just two histidine and one tyrosine residues, seems to be a key site for metal binding, which was not thoroughly investigated so far. Light scattering spectra of Aβ(9–16) and its complexes with metal ions were measured, whereas some fluorescence experiments were also performed. Our data suggest that Aβ(9–16) peptide fragment coordination by metal ions is dependent on the type of metal, and the amino acid residues involved in metal bonding.

Establishment of Constraints on Amyloid Formation Imposed by Steric Exclusion of Globular Domains

In many disease-related and functional amyloids, the amyloid-forming regions of proteins are flanked by globular domains. When located in close vicinity of the amyloid regions along the chain, the globular domains can prevent the formation of amyloids because of the steric repulsion. Experimental tests of this effect are few in number and non-systematic, and their interpretation is hampered by polymorphism of amyloid structures. In this situation, modeling approaches that use such a clear-cut criterion as the steric tension can give us highly trustworthy results. In this work, we evaluated this steric effect by using molecular modeling and dynamics. As an example, we tested hybrid proteins containing an amyloid-forming fragment of Aβ peptide (17–42) linked to one or two globular domains of GFP. Searching for the shortest possible linker, we constructed models with pseudo-helical arrangements of the densely packed GFPs around the Aβ amyloid core. The molecular modeling showed that linkers of 7 and more residues allow fibrillogenesis of the Aβ-peptide flanked by GFP on one side and 18 and more residues when Aβ-peptide is flanked by GFPs on both sides. Furthermore, we were able to establish a more general relationship between the size of the globular domains and the length of the linkers by using analytical expressions and rigid body simulations. Our results will find use in planning and interpretation of experiments, improvement of the prediction of amyloidogenic regions in proteins, and design of new functional amyloids carrying globular domains.

Binding of Cytotoxic Aβ25-35 Peptide to the Dimyristoylphosphatidylcholine Lipid Bilayer.

Aβ25-35 is a short, cytotoxic, and naturally occurring fragment of the Alzheimer's Aβ peptide. To map the molecular mechanism of Aβ25-35 binding to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer, we have performed replica exchange with solute tempering molecular dynamics simulations using all-atom explicit membrane and water models. Consequences of sequence truncation on the binding mechanism have been measured by utilizing as a control our previous simulations probing binding of the longer peptide Aβ10-40 to the same bilayer. The most intriguing feature of Aβ25-35 binding to the DMPC bilayer is a coexistence of two bound states with strikingly different characteristics: a dominant surface-bound state and a less stable inserted state. In the surface-bound state, the peptide samples extended conformations, in which its unbound C-terminal is pointed away from the bilayer. In contrast, in the inserted state, the C-terminal resides deep in the bilayer hydrophobic core. In both states, the N-terminal remains anchored to the bilayer. Free energy landscape analysis reveals that the two states are separated by a moderate barrier, suggesting that Aβ25-35 monomer may frequently interconvert between them. The net effect of Aβ25-35 binding is a minor impact on the bilayer structure, which contrasts with the considerable bilayer perturbations induced by a longer Aβ10-40 peptide penetrating deep into the bilayer core. Therefore, we conclude that the binding mechanisms of Aβ25-35 and Aβ10-40 peptides are different. Potential implications of our results for Aβ25-35 cytotoxicity are discussed. A comparison of experimental data with our findings reveals a good agreement.

To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for Fibril and Nanofilm Formation of Fragments of Aβ Peptide.

To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aβ peptide, Aβ(16-25), Aβ(31-40), and Aβ(33-42), using the methods of electron microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aβ(31-40) and Aβ(33-42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aβ(16-25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aβ peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloid-related diseases.