Modification of amyloid-ß1-42 fibril structure by methionine-35 oxidation.

Oxidative stress and amyloid-ß (Aß) formation are important processes that occur in Alzheimer's disease (AD). Amyloid formation is associated with the aggregation and precipitation of the Aß peptide, while oxidative stress results from an imbalance in pro-oxidant/antioxidant homeostasis that produces harmful reactive oxygen species. The methionine-35 (Met35) residue of the Aß peptide plays an important role in AD oxidative stress events and the associated neurotoxicity. We and other research groups previously demonstrated that in vitro oxidation of the Met35 side-chain to the sulfoxide (Met35red → Met35ox) impedes assembly and aggregation of monomeric Aß peptide into protofibrils, the latter being the immediate precursors of amyloid plaques. Here, we report that Met35 oxidation state affects the stability of preexisting amyloid fibrils and plaques, where the Met35red → Met35ox process leads to changes in the morphology of filaments, protofibrils, mature fibrils, and loss of Congo red birefringence in senile plaques isolated from the brains of AD patients. The most notable differences were in fibril flexibility, as evidenced by changes from straight fibrils to irregularly shaped, rope-like fibrils. These findings suggest that the Met35 oxidation state and amyloid plaque formation may be intimately linked.

Phenolic compounds prevent amyloid ß-protein oligomerization and synaptic dysfunction by site-specific binding.

Cerebral deposition of amyloid ß protein (Aß) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aß aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aß aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aß oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aß. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aß oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aß through different binding.

Antibodies to potato virus Y bind the amyloid beta peptide: immunohistochemical and NMR studies.

Studies in transgenic mice bearing mutated human Alzheimer disease (AD) genes show that active vaccination with the amyloid beta (Abeta) protein or passive immunization with anti-Abeta antibodies has beneficial effects on the development of disease. Although a trial of Abeta vaccination in humans was halted because of autoimmune meningoencephalitis, favorable effects on Abeta deposition in the brain and on behavior were seen. Conflicting results have been observed concerning the relationship of circulating anti-Abeta antibodies and AD. Although these autoantibodies are thought to arise from exposure to Abeta, it is also possible that homologous proteins may induce antibody synthesis. We propose that the long-standing presence of anti-Abeta antibodies or antibodies to immunogens homologous to the Abeta protein may produce protective effects. The amino acid sequence of the potato virus Y (PVY) nuclear inclusion b protein is highly homologous to the immunogenic N-terminal region of Abeta. PVY infects potatoes and related crops worldwide. Here, we show through immunocytochemistry, enzyme-linked immunosorbent assay, and NMR studies that mice inoculated with PVY develop antibodies that bind to Abeta in both neuritic plaques and neurofibrillary tangles, whereas antibodies to material from uninfected potato leaf show only modest levels of background immunoreactivity. NMR data show that the anti-PVY antibody binds to Abeta within the Phe4-Ser8 and His13-Leu17 regions. Immune responses generated from dietary exposure to proteins homologous to Abeta may induce antibodies that could influence the normal physiological processing of the protein and the development or progression of AD.

Antigen-antibody dissociation in Alzheimer disease: a novel approach to diagnosis.

With the ever-increasing population of aged individuals at risk of developing Alzheimer's disease (AD), there is an urgent need for a sensitive, specific, non-invasive, and diagnostic standard. The majority of efforts have focused on auto-antibodies against amyloid-beta (Abeta) protein, both as a potential treatment, and a reliable biomarker of AD pathology. Naturally occurring antibodies against Abeta are found in the CSF and plasma of patients with AD as well as healthy control subjects. To date, differences between diseased and control subjects have been highly variable. However, some of the antibody will be in preformed antigen-antibody complexes and the extent and nature of such complexes may provide a potential explanation for the variable results reported in human studies. Thus, measuring total amounts of antigen or antibody following unmasking is critical. Here, using a technique for dissociating antibody-antigen complexes, we found significant differences in serum antibodies to Abeta between AD and aged-matched control subjects. While the current study demonstrates the relevance of measuring total antibody, bound and unbound, against Abeta in AD, this technique may be applicable to diseases such as acquired immune deficiency syndrome and hepatitis B where determination of antigen and antibody levels are important for disease diagnosis and assessing disease progression.

NMR reveals anomalous copper(II) binding to the amyloid Abeta peptide of Alzheimer's disease.

The Abeta peptide is the major protein component of amyloid deposits in Alzheimer's disease (AD). Age-related microenvironmental changes in the AD brain promote amyloid formation that leads to cell injury and death. Altered levels of metals (such as Cu and Zn) exist in the AD brain, and because Cu and Zn can be bound to the Abeta in the amyloid plaques, it is thought that these binding events in vivo may trigger or prevent Abeta amyloid formation in the AD brain. Although several structural models have been proposed, all of these are undefined due to the lack of definitive structural data. The present NMR studies utilized uniformly 15N-labeled Abeta(1-40) peptide and 1H-15N HSQC experiments and demonstrate for the first time that the Abeta binds Cu and Zn in a distinct manner. The binding promotes NH signal disappearance of E3-V18, which was not due to the paramagnetic effect of Cu2+, as identical NMR studies were seen with Zn2+, which is diamagnetic. NMR titration experiments showed that the amide NH peak intensities of R5-L17 showed the most pronounced intensity reduction, and that the 1H signals for the side chain aromatic signals of the three histidines shift upfield (H6, H13, and H14). We propose that initially Cu2+ is anchored to the Abeta monomer (fast exchange rate) and is followed by deprotonation and/or severe line broadening of the backbone amide NH for E3-V18 (intermediate exchange rate). By contrast, Cu2+ binding to soluble Abeta aggregates leads to rapid aggregation and nonfibrillar amorphous structures, and without metal, the Abeta can undergo the normal time-dependent aggregation, eventually producing more ordered, late-stage parallel beta-sheet structures. These anomalous (rare) binding events may account for some of the unique properties associated with the Abeta, such as its proposed "dual role", where sequestration of metal ions by the monomer is neuroprotective, while that by beta-aggregates generates oxygen radicals and causes neuronal death.

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy.

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.

ABri peptide associated with familial British dementia forms annular and ring-like protofibrillar structures.

Amyloid plaque deposition involves the aggregation of normally soluble proteins into insoluble amyloid fibrils (fibrillization) and proceeds through intermediates with distinct morphologies, including spherical aggregates, protofibrils, and mature fibrils. Recently, a novel annular protofibril-like intermediate with unique pore-like properties was produced by alpha-synuclein, A beta-Arctic and amylin, which are proteins associated with Parkinson's disease, Alzheimer's disease, and type-II diabetes. The observation of annular structures coupled with size selective channel-like activity by these proteins suggests that these structures may be responsible for vesicle permeability by ion-channel formation. Using atomic force spectroscopy, we report here that the ABri peptide associated with familial British dementia produces similar annular and ring-like protofibril structures during the following sequence of events: spherical aggregates (0.4-1.5 nm height)-->chain-like protofibrils (1.5-2.3 nm height)-->ring-like protofibrils and annular protofibrils (1.5-2.3 nm height). This suggests that ABri fibrillization occurs in a similar fashion to other amyloidogenic proteins and that the annular protofibrillar structures may represent a common amyloid intermediate.

Solution NMR studies of the A beta(1-40) and A beta(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation.

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --> Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --> Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia.

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.

Methionine 35 oxidation reduces fibril assembly of the amyloid abeta-(1-42) peptide of Alzheimer's disease.

The major component of amyloid plaques in Alzheimer's disease (AD) is Abeta, a small peptide that has high propensity to assemble as aggregated beta-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35(ox)) significantly hinders the rate of fibril formation for the 42-residue Abeta-(1-42) at physiological pH. Met-35(ox) also alters the characteristic Abeta fibril morphology and prevents formation of the protofibril, which is a key intermediate in beta-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Abeta with oxidative stress in AD are discussed.

Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides.

Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide c[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D- and 2D-(1)H NMR techniques. The peptide backbone has a single predominant conformation. The tryptophan side chain has three chi(1) rotamers: a major chi(1) = -60 degrees rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the chi(1) rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the chi(1) = -60 degrees rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to peptide bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8- and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the chi(1) = 180 degrees rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the epsilon-ammonium does not occur in these peptides.