Amyloid peptides incorporating a core sequence from the amyloid beta peptide and gamma amino acids: relating bioactivity to self-assembly

A series of heptapeptides comprising the core sequence Ab(16–20), KLVFF, of the amyloid b peptide coupled with paired N-terminal c-amino acids are investigated in terms of cytotoxicity reduction and binding to the full Ab peptide, both pointing to inhibition of fibrillisation for selected compounds. This is related to the self-assembly capacity of the heptapeptides.

Fibrils and nanotubes assembled from a modified amyloid-β peptide fragment differ in the packing of the same β-sheet building blocks

The peptide AAKLVFF assembles into fibrils in water and nanotubes in methanol. Solid-state NMR data are consistent with fibrils constructed from β-sheet bilayers and nanotubes bounded by a wall of offset β-sheet monolayers. Remarkably distinct morphologies are thus traced to subtle differences in the arrangement of the same fundamental building blocks.

Fibrillisation of ring-closed amyloid peptides

Ring-closing olefin metathesis reactions are used to create intramolecularly ring closed peptides or inter-molecularly ring-closed peptide dimers based on a designed amyloid peptide sequence. The uncrosslinked peptide self-assembles into high aspect ratio nanotubes, however ring-closing leads to the formation of fibrillar and twisted/helical ribbon structures.

Control of strand registry by attachment of PEG chains to amyloid peptides influences nanostructure

The self-assembly in aqueous solution of PEG-peptide conjugates comprising a model amyloid peptide sequence FFKLVFF that contains the Ab(16–20) KLVFF motif is investigated. X-ray diffraction reveals different packing motifs dependent on PEG chain length. This is correlated to remarkable differences in self-assembled nanostructures. The control of strand registry points to a subtle interplay between aromatic stacking, electrostatic and amphiphilic interactions.

Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method

We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Young’s modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, a-synuclein, Ab(1–42), insulin, b-lactoglobulin,lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2–4 GPa.

Conductance of amyloid β based peptide filaments: structure–function relations.

Controlling the morphology of self-assembled peptide nanostructures, particularly those based on amyloid peptides, has been the focus of intense research. In order to exploit these structures in electronic applications, further understanding of their electronic behavior is required. In this work, the role of peptide morphology in determining electronic conduction along self-assembled peptide nanofilament networks is demonstrated. The peptides used in this work were based on the sequence AAKLVFF, which is an extension of a core sequence from the amyloid b peptide. We show that the incorporation of a non-natural amino acid, 2-thienylalanine, instead of phenylalanine improves the obtained conductance with respect to that obtained for a similar structure based on the native sequence, which was not the case for the incorporation of 3-thienylalanine. Furthermore, we demonstrate that the morphology of the self-assembled structures, which can be controlled by the solvent used in the assembly process, strongly affects the conductance, with larger conduction obtained for a morphology of long, straight filaments. Our results demonstrate that, similar to natural systems, the assembly and folding of peptides could be of great importance for optimizing their function as components of electronic devices. Hence, sequence design and assembly conditions can be used to control the performance of peptide based structures in such electronic applications.

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer's disease (AD). We have previously shown that hypoxia in vitro alters Ca2+ homeostasis in astrocytes and promotes increased production of amyloid beta peptides (Abeta) of AD. Indeed, alteration of Ca2+ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O2, 24h) in a manner that is independent of amyloid beta peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid beta peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either beta or gamma secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Abeta production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.

Water-soluble tripeptide Aβ (9−11) forms amyloid-Like fibrils and exhibits neurotoxicity

A water-soluble, hydrophilic tripeptide GYE, having sequence identity with the N-terminal segment of amyloid peptides A�(9-11), upon selfassociation exhibits amyloid-like fibrils and significant neurotoxicity towards the Neuro2A cell line. However, the tripeptides GFE and GWE, in which the centrally located tyrosine residue has been replaced by phenylalanine or tryptophan, fail to show amyloidogenic behavior and exhibit little or no neurotoxicity.

Electrochemical sensing of 2D condensation in amyloid peptides

The interfacial behavior of the model amyloid peptide octamer YYKLVFFC (peptide 1) and two other amyloid peptides YEVHHQKLVFF (peptide 2) and KKLVFFA (peptide 3) at the metal|aqueous solution interface was studied by voltammetric and constant current chronopotentiometric stripping (CPS). All three peptides are adsorbed in a wide potential range and exhibit different interfacial organizations depending on the electrode potential. At the least negative potentials, chemisorption of peptide 1 occurs through the formation of a metal sulfur bond. This bond is broken close to −0.6 V. The peptide undergoes self-association at more negative potentials, leading to the formation of a “pit” characteristic of a 2D condensed film. Under the same conditions the other peptides do not produce such a pit. Formation of the 2D condensed layer in peptide 1 is supported by the time, potential and temperature dependences of the interfacial capacity and it is shown that presence of the 2D layer is reflected by the peptide CPS signals due to the catalytic hydrogen evolution. The ability of peptide 1 to form the potential-dependent 2D condensed layer has been reported neither for any other peptide nor for any protein molecule. This ability might be related to the well-known oligomerization and aggregation of Alzheimer amyloid peptides.

Heme oxygenase-1 protects against Alzheimer’s amyloid-β1-42 induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer‟s disease (AD), catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediate cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentrationdependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of fibroblast

Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2)(-) levels was observed by treatment of fibroblasts with SAA (r = 0.99 and P <= 0.001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0.001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2)(-) by 50%. Also, SAA raised fibroblast proliferation (P < 0.001) and this effect was completely abolished by the addition of anti-oxidants (P < 0.001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.

Albumin-induced circular dichroism in Congo red: applications for studies of amyloid-like fibril aggregates and binding sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)