Evidence of π‐stacking interactions in the self‐assembly of hIAPP22‐29

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self‐assembly of human islet amyloid polypeptide (hIAPP)_22‐29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe‐23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe‐23 virtually abolish the ability of hIAPP_22‐29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β‐sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the ? C?C? ring mode (ca. 1600 cm^?1) upon aggregation that has previously been shown to be indicative of π‐stacking. While previous work has demonstrated that π‐stacking is not an absolute requirement for fibrillization, our findings indicate that Phe‐23 also contributes to fibril formation through π‐stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self‐assembly of hIAPP_22‐29. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Hexafluoroisopropanol induces self‐assembly of β‐amyloid peptides into highly ordered nanostructures

Deposition of insoluble fibrillar aggregates of β‐amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α‐helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α‐helical to β‐conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring‐like structures. Aβ16–22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α‐helical conformation in Aβ16–22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16–22 are composed of the peptides in β‐conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid‐like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16–22 in HFIP results in the formation of annular amyloid‐like structures. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Docosahexaenoic acid disrupts in vitro amyloid β1‐40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats

We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta_1‐40 (Aβ_1‐40)‐infused Alzheimer’s disease (AD)‐model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Aβ_1‐40, cholesterol and the composition of fatty acids were investigated in the Triton X100‐insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Aβ_1‐40 were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Aβ_1‐40, cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Aβ fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T‐derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed‐fibril Aβ peptides, demonstrating the anti‐amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti‐oligomer‐specific antibody and non‐reducing Tris‐Glycine gradient (4–20%) gel electrophoresis. DHA concentration‐dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA‐induced suppression of in vivo Aβ_1‐40 aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial peptide (Cn‐AMP2) from liquid endosperm of Cocos nucifera forms amyloid‐like fibrillar structure

Cn‐AMP2 is an antimicrobial peptide derived from liquid endosperm of coconut (Cocos nucifera). It consists of 11 amino acid residues and predicted to have high propensity for β‐sheet formation that disposes this peptide to be amyloidogenic. In the present study, we have examined the amyloidogenic propensities of Cn‐AMP2 in silico and then tested the predictions under in vitro conditions. The in silico study revealed that the peptide possesses high amyloidogenic propensity comparable with Aβ. Upon solubilisation and agitation in aqueous buffer, Cn‐AMP2 forms visible aggregates that display bathochromic shift in the Congo red absorbance spectra, strong increase in thioflavin T fluorescence and fibrillar morphology under transmission electron microscopy. All these properties are typical of an amyloid fibril derived from various proteins/peptides including Aβ. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of introducing a short amyloidogenic sequence from the Aβ peptide at the N‐terminus of 18‐residue amphipathic helical peptides

Fibril formation is the hallmark of pathogenesis in Alzheimer's disease and other amyloid disorders caused by conformational alterations leading to the aggregation of soluble monomers. Aβ40 self‐associates to form amyloid fibrils. Its central seven‐residue segment KLVFFAE (Aβ16–22), which is thought to be crucial for fibril formation of the full‐length peptide, forms fibrils even in isolation. Context‐dependent induction of amyloid formation by such sequences in peptides, which otherwise do not have that propensity, is of considerable interest. We have examined the effect of introducing the Aβ16–22 sequence at the N‐terminus of two amphipathic helical 18‐residue peptides Ac‐WYSEMKRNVQRLERAIEE‐am and Ac‐KQLIRFLKRLDRNLWGLA‐am, which have high average hydrophobic moment <μH> values but have net charges of 0 and +4, respectively, at neutral pH. Upon incubation in aqueous buffer, fibril‐like aggregates were discernible by transmission electron microscopy for the peptide with only 0 net charge, which also displayed ThT binding and β‐structure. Although both the sequences have been derived from amphipathic helical segments in globular proteins and possess high average hydrophobic moments, the +4 charge peptide lacks the ability to form fibrils, while the peptide with 0 charge has the tendency to form fibrillar structures. Variation in the net charge and the presence of several glutamic acids in the sequence of the peptide with net charge 0 appear to favor the formation of fibrils when the Aβ16–22 sequence is attached at the N‐terminus. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular mechanism of β-sheet self-organization at water-hydrophobic interfaces

The capacity to form β‐sheet structure and to self‐organize into amyloid aggregates is a property shared by many proteins. Severe neurodegenerative pathologies such as Alzheimer's disease are thought to involve the interaction of amyloidogenic protein oligomers with neuronal membranes. To understand the experimentally observed catalysis of amyloid formation by lipid membranes and other water‐hydrophobic interfaces, we examine the physico‐chemical basis of peptide adsorption and aggregation in a model membrane using atomistic molecular simulations. Blocked octapeptides with simple, repetitive sequences, (Gly‐Ala)₄, and (Gly‐Val)₄, are used as models of β‐sheet‐forming polypeptide chains found in the core of amyloid fibrils. In the presence of an n‐octane phase mimicking the core of lipid membranes, the peptides spontaneously partition at the octane‐water interface. The adsorption of nonpolar sidechains displaces the peptides' conformational equilibrium from a heterogeneous ensemble characterized by a high degree of structural disorder toward a more ordered ensemble favoring β‐hairpins and elongated β‐strands. At the interface, peptides spontaneously aggregate and rapidly evolve β‐sheet structure on a 10 to 100 ns time scale, while aqueous aggregates remain amorphous. Catalysis of β‐sheet formation results from the combination of the hydrophobic effect and of reduced conformational entropy of the polypeptide chain. While the former drives interfacial partition and displaces the conformational equilibrium of monomeric peptides, the planar interface further facilitates β‐sheet organization by increasing peptide concentration and reducing the dimensionality of self‐assembly from three to two. These findings suggest a general mechanism for the formation of β‐sheets on the surface of globular proteins and for amyloid self‐organization at hydrophobic interfaces. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The dye SYPRO orange binds to amylin amyloid fibrils but not pre‐fibrillar intermediates

Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type‐2 diabetes. Amyloid sensitive dyes, particularly thioflavin‐T, are widely used to detect ex‐vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self‐assembly. We show that the dye SYPRO‐orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type‐2 diabetes. No fluorescence enhancement is observed in the presence of pre‐fibrillar species or in the presence of non‐amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO‐orange fluorescence and match the time course determined with thioflavin‐T assays. Thus, SYPRO‐orange offers an alternative to thioflavin‐T assays of amylin amyloid formation. The implications for the interpretation of SYPRO‐orange‐based assays of protein stability and protein‐ligand interactions are discussed.     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Wild‐type,Flemish, and Dutch amyloid‐β exhibit different cytotoxicities depending on Aβ40 to Aβ42 interaction time and concentration ratio

Addition of amyloid β (Aβ) peptide Aβ40 to Aβ42 can delay Aβ42 aggregation, but consequent cytotoxicity has been reported to be enhanced or diminished. In the present study, we found that cytotoxicity was enhanced when human neuroblastoma SH‐SY5Y cells were incubated in a mixture of wt Aβ42 and Aβ40wt at a ratio of 1 : 10–20 (0.1 : 1–2 μM) for 24–36 h, whereas the enhancement was detected in cells incubated for longer times (48–60 h) with the less amyloidogenic Flemish Aβ40 variant or in cells incubated for as short as 12 h with the more amyloidogenic Dutch variant. Reductions in cytotoxicity by Aβ40 were most prominently observed in the Flemish and wt Aβ40/Aβ42 mixture at ratio 1 : 20 incubated for a short time (~12 h). The most cytotoxic Aβ40/Aβ42 mixtures were enriched in Aβ protofibril‐like structures, implying a strong correlation between cytotoxicity and this structure, the formation of which was dependent on amyloidogenic properties and incubation time. The consequences of the interactions were probably because of the different amyloidogenic properties of the Aβ40 variants, rather than to those of Aβ42, because aggregation rates of Aβ40 variants were highly dependent on sequence, whereas those of Aβ42 variants were not. These studies highlight a potential role for Aβ40 in cytotoxicity and provide novel mechanistic insights into the pathogenesis of each familial Alzheimer's disease‐associated Aβ40 variant. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Differential effects of phe19 and phe20 on fibril formation by amyloidogenic peptide Aβ16–22 (Ac‐KLVFFAE‐NH2)

The sequence KLVFFAE (Aβ16–22) in Alzheimer's β‐amyloid is thought to be a core β‐structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in β‐sheet. To elucidate the mechanism of the initial folding process, we undertook combined X‐ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized Aβ16–22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild‐type (WT) Aβ16–22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab‐like β‐crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30‐Å period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side‐chain dispositions for WT and 20CN, but not for 19CN. These X‐ray results and modeling imply that in the assembly of WT Aβ16–22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

β‐sheet assembly in amyloidogenic glutamic acid nanostructures: Insights from X‐ray scattering and infrared nanospectroscopy

Glutamic acid–rich peptides are crucial to a variety of biological processes, including glutamatergic neurotransmission and immunological defense. Glutamic acid sequences often exhibit unusual organization into β₂‐type sheets, where bifurcated H bonds formed between glutamic acid side chains and NH in amide bonds on adjacent β‐strands play a paramount role for stabilizing the molecular assembly. Herein, we investigate the self‐assembly and supramolecular structure of simplified models consisting of alternating glutamic acid/phenylalanine residues. Small‐angle X‐ray scattering and atomic force microscopy show that the aggregation pathway is characterized by the formation of small oligomers, followed by coalescence into nanofibrils and nanotapes. Amyloidogenic features are further demonstrated through fiber X‐ray diffraction, which reveal molecular packing according to cross‐β patterns, where β‐strands appear perpendicularly oriented to the long axis of nanofibrils and nanotapes. Nanoscale infrared spectroscopy from individual nanoparticles on dried samples shows a remarkable decrease of β₂‐sheet content, accompanied by growth of standard β‐sheet fractions, indicating a β₂‐to‐β₁ transition as a consequence of the release of solvent from the interstices of peptide assemblies. Our findings highlight the key role played by water molecules in mediating H‐bond formation in β₂‐sheets commonly found in amyloidogenic glutamic acid–rich aggregates.     

K114 (trans,trans)‐bromo‐2,5‐bis(4‐hydroxystyryl)benzene is an efficient detector of cationic amyloid fibrils

Cationic amyloid fibrils found in human semen enhance the transmission of the human immunodeficiency virus (HIV) and thus, are named semen‐derived enhancer of virus infection (SEVI). The mechanism for the enhancement of transmission is not completely understood but it has been proposed that SEVI neutralizes the repulsion that exists between the negatively charged viral envelope and host cell membrane. Consistent with this view, here we show that the fluorescence of cationic thioflavin T (ThT) in the presence of SEVI is weak, and thus ThT is not an efficient detector of SEVI. On the other hand, K114 ((trans, trans)‐bromo‐2,5‐bis(4‐hydroxystyryl)benzene) forms a highly fluorescent, phenolate‐like species on the cationic surface of SEVI. This species does not form in the presence of amyloid fibrils from insulin and amyloid‐β protein, both of which are efficiently detected by ThT fluorescence. Together, our results show that K114 is an efficient detector of SEVI.     

Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.