Cross-seeding of WT amyloid-ß with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease.

Alzheimer's disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-ß (Aß). Inherited early-onset AD can be caused by single point mutations within the Aß sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aß isoform expression. It is therefore important to understand how these mixtures of Aß isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aß42 and Aß40. We confirm that Aß40(WT) does not readily cross-seed Aß42(WT) fibril formation. In contrast, fibril formation of Aß40(Arctic) is hugely accelerated by Aß42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aß40(Arctic) and Aß42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aß42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aß40/42 isoforms, which can impact the rate and structure of fibril assembly.

Imaging Amyloid-ß Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-ß peptides (Aß) is central to the development of Alzheimer's disease. The mechanism by which Aß triggers a cascade of events that leads to dementia is a topic of intense investigation. Aß self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aß can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aß ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aß induced membrane disruption. Understanding the relationship between different Aß structures and membrane permeability will inform therapeutics targeting Aß cytotoxicity.

pH Dependence of Amyloid-ß Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aß) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aß's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pKa of 7.0, close to the pKa of Aß histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aß, in particular histidine protonation, has little impact on this stage of Aß assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aß into toxic oligomers and fibrils.

Amyloid-ß oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy.

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

Copper2+ Binding to α-Synuclein. Histidine50 Can Form a Ternary Complex with Cu2+ at the N-Terminus but Not a Macrochelate.

α-Synuclein (αSyn) forms amyloid fibrils in the neurons of Parkinson's disease (PD) patients'. Despite a role for Cu2+ in accelerating αSyn fibril formation, coupled with reports of copper dis-homeostasis in PD, there remain controversies surrounding the coordination geometry of Cu2+ with αSyn. Here we compare visible circular dichroism (CD) spectra of Cu2+ loaded on to full-length αSyn together with four peptides that model aspects of Cu2+ binding to the N-terminus and histidine50 of αSyn. With glycine as a competitive ligand, the affinity of Cu2+ for full-length αSyn is determined to have a conditional dissociation constant, at pH 7.4, of 0.1 nM. A similar affinity of 0.3 nM is determined for the tripeptide Met-Asp-Val(MDV) that mimics the N-terminus of αSyn, while the incorporation of a putative histidine side chain in the N-terminal complex facilitates the formation of a macrochelate with the histidine, which results in an increase in the affinity for Cu2+ to 0.03 nM at pH 7.4. Comparisons of the visible absorbance and CD spectra over a range of pH values also indicates that the MDV tripeptide closely models Cu2+ binding to full-length αSyn and rules out a role for His50 in the primary Cu2+ binding complex of monomeric αSyn. However, there are reports that suggest His50 does form a macrochelate with the N-terminal Cu2+ complex; we reconcile these conflicting observations by identifying a concentration dependence of the interaction. Only at the higher concentrations can the imidazole nitrogen bind to the N-terminal Cu2+ to form a ternary complex rather than via a macrochelate. This work shows even for this intrinsically disordered protein a large macrochelate with Cu2+ is not favored. Understanding Cu2+ coordination to αSyn gives a more complete picture of its place in amyloid assembly and cytotoxicity.

Ion Channel Formation by Amyloid-ß42 Oligomers but Not Amyloid-ß40 in Cellular Membranes.

A central hallmark of Alzheimer's disease is the presence of extracellular amyloid plaques chiefly consisting of amyloid-ß (Aß) peptides in the brain interstitium. Aß largely exists in two isoforms, 40 and 42 amino acids long, but a large body of evidence points to Aß(1-42) rather than Aß(1-40) as the cytotoxic form. One proposed mechanism by which Aß exerts toxicity is the formation of ion channel pores that disrupt intracellular Ca2+ homeostasis. However, previous studies using membrane mimetics have not identified any notable difference in the channel forming properties between Aß(1-40) and Aß(1-42). Here, we tested whether a more physiological environment, membranes excised from HEK293 cells of neuronal origin, would reveal differences in the relative channel forming ability of monomeric, oligomeric, and fibrillar forms of both Aß(1-40) and Aß(1-42). Aß preparations were characterized with transmission electron microscopy and thioflavin T fluorescence. Aß was then exposed to the extracellular face of excised membranes, and transmembrane currents were monitored using patch clamp. Our data indicated that Aß(1-42) assemblies in oligomeric preparations form voltage-independent, non-selective ion channels. In contrast, Aß(1-40) oligomers, fibers, and monomers did not form channels. Ion channel conductance results suggested that Aß(1-42) oligomers, but not monomers and fibers, formed three distinct pore structures with 1.7-, 2.1-, and 2.4-nm pore diameters. Our findings demonstrate that only Aß(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the differential neurotoxic effect of Aß(1-40) and Aß(1-42) in Alzheimer's disease.

Methionine oxidation reduces lag-times for amyloid-ß(1-40) fiber formation but generates highly fragmented fibers.

Oxidative stress and the formation of amyloid plaques containing amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease. A proportion of methionine (Met) at position 35 within Aß is oxidized to methionine sulphoxide (Met(OX)) within the Alzheimer's plaques. These oxidative processes may be the key to understanding the early stages of Alzheimer's disease. In vitro oxidation of Aß, by the physiological oxidant H2O2, was monitored using (1)H NMR and mass spectrometry. Here we investigate the effect of Aß methionine oxidation on fiber formation kinetics and morphology using the amyloid specific fluorescence dye Thioflavin T (ThT) and Transmission Electron Microscopy (TEM). Methionine oxidation reduces the total amount of fibers generated for both dominant forms of Aß, however there are marked differences in the effect of Met(OX) between Aß(1-40) and Aß(1-42). Surprisingly the presence of Met(OX) reduces lag-times for Aß(1-40) fiber formation but extends lag-times for Aß(1-42). TEM indicates a change in fiber morphology with a pronounced reduction in fiber length for both methionine oxidized Aß(1-40) and Aß(1-42). In contrast, the morphology of preformed amyloid fibers is largely unaffected by the presence of H2O2. Our studies suggest that methionine oxidation promotes highly fragmented fiber assemblies of Aß. Oxidative stress associated with Alzheimer's disease can cause oxidation of methionine within Aß and this in turn will influence the complex assembly of Aß monomer into amyloid fibers, which is likely to impact Aß toxicity.

N-Terminally Truncated Amyloid-ß(11-40/42) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease.

Amyloid-ß peptide (Aß) isoforms of different lengths and aggregation propensities coexist in vivo. These different isoforms are able to nucleate or frustrate the assembly of each other. N-terminally truncated Aß(11-40) and Aß(11-42) make up one fifth of plaque load yet nothing is known about their interaction with full-length Aß(1-40/42) . We show that in contrast to C-terminally truncated isoforms, which do not co-fibrillize, deletions of ten residues from the N terminus of Aß have little impact on its ability to co-fibrillize with the full-length counterpart. As a consequence, N-terminally truncated Aß will accelerate fiber formation and co-assemble into short rod-shaped fibers with its full-length Aß counterpart. This has implications for the assembly kinetics, morphology, and toxicity of all Aß isoforms.

Truncated Amyloid-ß(11-40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly.

Alzheimer disease coincides with the formation of extracellular amyloid plaques composed of the amyloid-ß (Aß) peptide. Aß is typically 40 residues long (Aß(1-40)) but can have variable C and N termini. Naturally occurring N-terminally truncated Aß(11-40/42) is found in the cerebrospinal fluid and has a similar abundance to Aß(1-42), constituting one-fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aß(11-40/42) would have an elevated affinity for Cu(2+). Various spectroscopic techniques, complemented with transmission electron microscopy, were used to determine the properties of the Cu(2+)-Aß(11-40/42) interaction and how Cu(2+) influences amyloid fiber formation. We show that Cu(2+)-Aß(11-40) forms a tetragonal complex with a 34 ± 5 fm dissociation constant at pH 7.4. This affinity is 3 orders of magnitude tighter than Cu(2+) binding to Aß(1-40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu(2+) transport protein. Furthermore, Aß(11-40/42) forms fibers twice as fast as Aß(1-40) with a very different morphology, forming bundles of very short amyloid rods. Substoichiometric Cu(2+) drastically perturbs Aß(11-40/42) assembly, stabilizing much longer fibers. The very tight fm affinity of Cu(2+) for Aß(11-40/42) explains the high levels of Cu(2+) observed in Alzheimer disease plaques.

Cu²âº accentuates distinct misfolding of Aß1₋40 and Aß1₋42 peptides, and potentiates membrane disruption.

Central to Alzheimer's disease is the misfolding of amyloid-beta (Aß) peptide, which generates an assorted population of amorphous aggregates, oligomers and fibres. Metal ion homoeostasis is disrupted in the brains of sufferers of Alzheimer's disease and causes heightened Alzheimer's disease phenotype in animal models. In the present study, we demonstrate that substochiometric Cu²âº affects the misfolding pathway of Aß1₋40, and the more toxic Aß1₋42, in markedly different ways. Cu²âº accelerates Aß1₋40 fibre formation. In contrast, for Aß1₋42, substoichiometric levels of Cu²âº almost exclusively promote the formation of oligomeric and protofibrillar assemblies. Indeed, mature Aß1₋42 fibres are disassembled into oligomers when Cu²âº is added. These Cu²âº stabilized oligomers of Aß1₋42 interact with the lipid bilayer, disrupting the membrane and increasing permeability. Our investigation of Aß1₋40/Aß1₋42 mixtures with Cu²âº revealed that Aß1₋40 neither contributed to nor perturbed formation of Aß1₋42 oligomers, although Cu²âº-Aß1₋42 does frustrate Cu²âº-Aß1₋40 fibre growth. Small amounts of Cu²âº accentuate differences in the propensity of Aß1₋40 and Aß1₋42 to form synaptotoxic oligomers, providing an explanation for the connection between disrupted Cu²âº homoeostasis and elevated Aß1₋42 neurotoxicity in Alzheimer's disease.

A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic Acid).

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-ß (Aß) from Alzheimer's disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aß fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aß and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aß and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution.

Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: the N-terminal region of S2 facilitates self-association.

The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo.

Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2.

The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1 with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation.

Copper(II) sequentially loads onto the N-terminal amino group of the cellular prion protein before the individual octarepeats.

The cellular prion protein (PrPC) binds to Cu2+ ions in vivo, and a misfolded form of PrPC is responsible for a range of transmissible spongiform encephalopathies. Recently, disruption of Cu2+ homeostasis in mice has been shown to impart resistance to scrapie infection. Using full-length PrPC and model peptide fragments, we monitor the sequential loading of Cu2+ ions onto PrPC using visible circular dichroism. We show the N-terminal amino group of PrPC is not the principal binding site for Cu2+; however, surprisingly, it has an affinity for Cu2+ tighter than that of the individual octarepeat binding sites present within PrPC. We re-evaluate what is understood about the sequential loading of Cu2+ onto the full-length protein and show for the first time that Cu2+ loads onto the N-terminal amino group before the single octarepeat binding sites.

The cellular prion protein traps Alzheimer's Aß in an oligomeric form and disassembles amyloid fibers.

There is now strong evidence to show that the presence of the cellular prion protein (PrP(C)) mediates amyloid-ß (Aß) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrP(C) and Aß and discover that substoichiometric amounts of PrP(C), as little as 1/20, relative to Aß will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrP(C). Electron microscopy indicates PrP(C) is able to trap Aß in an oligomeric form. Unlike fibers, this oligomeric Aß contains antiparallel ß sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrP(C), notably residues 95-113, binds to Aß oligomers, but only once Aß misfolds. The ability of PrP(C) to trap and concentrate Aß in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrP(C) might confer Aß toxicity in AD, as oligomers are thought to be the toxic form of Aß. Identification of a specific recognition site on PrP(C) that traps Aß in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.

Impact of Membrane Phospholipids and Exosomes on the Kinetics of Amyloid-ß Fibril Assembly.

Alzheimer's disease (AD) is linked with the self-association of the amyloid-ß peptide (Aß) into oligomers and fibrils. The brain is a lipid rich environment for Aß to assemble, while the brain membrane composition varies in an age dependent manner, we have therefore monitored the influence of lipid bilayer composition on the kinetics of Aß40 fibril assembly. Using global-fitting models of fibril formation kinetics, we show that the microscopic rate constant for primary nucleation is influenced by variations in phospholipid composition. Anionic phospholipids and particularly those with smaller headgroups shorten fibril formation lag-times, while zwitterionic phospholipids tend to extend them. Using a physiological vesicle model, we show cellular derived exosomes accelerate Aß40 and Aß42 fibril formation. Two distinct effects are observed, the presence of even small amounts of any phospholipid will impact the slope of the fibril growth curve. While subsequent additions of phospholipids only affect primary nucleation with the associated change in lag-times. Heightened anionic phospholipids and cholesterol levels are associated with aging and AD respectively, both these membrane components strongly accelerate primary nucleation during Aß assembly, making a link between disrupted lipid metabolism and Alzheimer's disease.

Copper(II) Can Kinetically Trap Arctic and Italian Amyloid-ß40 as Toxic Oligomers, Mimicking Cu(II) Binding to Wild-Type Amyloid-ß42: Implications for Familial Alzheimer's Disease.

The self-association of amyloid-ß (Aß) peptide into neurotoxic oligomers is believed to be central to Alzheimer's disease (AD). Copper is known to impact Aß assembly, while disrupted copper homeostasis impacts phenotype in Alzheimer's models. Here we show the presence of substoichiometric Cu(II) has very different impacts on the assembly of Aß40 and Aß42 isoforms. Globally fitting microscopic rate constants for fibril assembly indicates copper will accelerate fibril formation of Aß40 by increasing primary nucleation, while seeding experiments confirm that elongation and secondary nucleation rates are unaffected by Cu(II). In marked contrast, Cu(II) traps Aß42 as prefibrillar oligomers and curvilinear protofibrils. Remarkably, the Cu(II) addition to preformed Aß42 fibrils causes the disassembly of fibrils back to protofibrils and oligomers. The very different behaviors of the two Aß isoforms are centered around differences in their fibril structures, as highlighted by studies of C-terminally amidated Aß42. Arctic and Italian familiar mutations also support a key role for fibril structure in the interplay of Cu(II) with Aß40/42 isoforms. The Cu(II) dependent switch in behavior between nonpathogenic Aß40 wild-type and Aß40 Arctic or Italian mutants suggests heightened neurotoxicity may be linked to the impact of physiological Cu(II), which traps these familial mutants as oligomers and curvilinear protofibrils, which cause membrane permeability and Ca(II) cellular influx.

Human serum albumin can regulate amyloid-ß peptide fiber growth in the brain interstitium: implications for Alzheimer disease.

Alzheimer disease is a neurodegenerative disorder characterized by extracellular accumulation of amyloid-ß peptide (Aß) in the brain interstitium. Human serum albumin (HSA) binds 95% of Aß in blood plasma and is thought to inhibit plaque formation in peripheral tissue. However, the role of albumin in binding Aß in the cerebrospinal fluid has been largely overlooked. Here we investigate the effect of HSA on both Aß(1-40) and Aß(1-42) fibril growth. We show that at micromolar cerebrospinal fluid levels, HSA inhibits the kinetics of Aß fibrillization, significantly increasing the lag time and decreasing the total amount of fibrils produced. Furthermore, we show that the amount of amyloid fibers generated directly correlates to the proportion of Aß not competitively bound to albumin. Our observations suggest a significant role for HSA regulating Aß fibril growth in the brain interstitium.

Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway.

Oxidative stress and misfolding of the prion protein (PrP(C)) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrP(C). Urea unfolding studies indicate that H(2)O(2) oxidation reduces the thermodynamic stability of PrP(C) by as much as 9 kJ/mol. (1)H-(15)N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrP(C) causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended ß-strand structures that lack a cooperative fold. This transition from the helical molten globule to ß-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrP(C), facilitating the transition to PrP(Sc). This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates.