Rapid α-oligomer formation mediated by the Aβ C terminus initiates an amyloid assembly pathway (original) (raw)

Pathways of Amyloid-β Aggregation Depend on Oligomer Shape

Journal of the American Chemical Society, 2018

One of the main research topics related to Alzheimer's disease is the aggregation of the amyloid-β peptide, which was shown to follow different pathways for the two major alloforms of the peptide, Aβ40 and the more toxic Aβ42. Experimental studies emphasized that oligomers of specific sizes appear in the early aggregation process in different quantities and might be the key toxic agents for each of the two alloforms. We use transition networks derived from all-atom molecular dynamics simulations to show that the oligomers leading to the type of oligomer distributions observed in experiments originate from compact conformations. Extended oligomers, on the other hand, contribute more to the production of larger aggregates thus driving the aggregation process. We further demonstrate that differences in the aggregation pathways of the two Aβ alloforms occur as early as during the dimer stage. The higher solvent-exposure of hydrophobic residues in Aβ42 oligomers contributes to the di...

Structure−Activity Relationships in Peptide Modulators of β-Amyloid Protein Aggregation: Variation in α,α-Disubstitution Results in Altered Aggregate Size and Morphology

ACS Chemical Neuroscience, 2010

Neuronal cytotoxicity observed in Alzheimer's disease (AD) is linked to the aggregation of β-amyloid peptide (Aβ) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aβ fibrils; disruption or inhibition of Aβ self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aβ neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aβ "hydrophobic core" Aβ 17-20 , with R,R-disubstituted amino acids (RRAAs) added into this core as potential disrupting agents of fibril selfassembly. The number, positional distribution, and side-chain functionality of RRAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered RRAA with a diisobutyl side chain in the core sequence, disrupted Aβ 1-40 fibril formation. However, AAMP-6, with a less sterically hindered RRAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aβ 1-40). Remarkably, RRAA-AAMPs caused disassembly of existing Aβ fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures.

Biophysical Analyses of Synthetic Amyloid-β(1−42) Aggregates before and after Covalent Cross-Linking. Implications for Deducing the Structure of Endogenous Amyloid-β Oligomers

Biochemistry, 2009

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40-and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that soluble Aβ aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Aβ aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Aβ oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Aβ have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Aβ aggregates in vitro that resemble the endogenous soluble Aβ aggregates, we previously found that monomeric Aβ(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Aβ(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Aβ cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Aβ(1-42) with three cross-linking agents: transglutaminase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS. † Acknowledgment is made to the donors of ADR, a program of the American Health Assistance Foundation, for support of this research.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Journal of Biological Chemistry, 2012

Background: Oligomers of amyloid-␤ peptides are implicated in the etiology of Alzheimer disease. Results: Specific "off-pathway" oligomers of A␤42 show unique replication properties upon interacting with monomers. Conclusion: The results indicate that oligomers that are formed along pathways outside the fibril formation pathway may undergo replication. Significance: Mechanistic details of A␤ soluble oligomers will enable better understanding of Alzheimer disease pathology. Aggregates of amyloid-␤ (A␤) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of A␤ aggregates, low molecular weight species ranging between ϳ2and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleationdependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of A␤42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein

Nature Chemical Biology, 2011

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β 2 m fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β 2 m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation, and diverting the course of assembly to the formation of spherical aggregates. The results reveal the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers towards alternative assembly pathways. Keywords β 2-microglobulin; amyloid; rifamycin SV; oligomers; NMR; ESI-MS Aberrant aggregation of proteins into amyloid fibrils is a characteristic of over twenty-five human disorders. 1 In each case, the precursor protein is dissimilar in terms of its native fold and primary sequence, yet amyloid fibrils share common structural and tinctorial features, such as a cross-β organization of the polypeptide chain and the ability to bind dyes such as thioflavin-T (ThT) and Congo red. 2 The observation of a common structural architecture for amyloid fibrils has motivated efforts to elucidate the molecular mechanisms of fibril formation in vitro with the aim of revealing possible targets for therapeutic intervention. Such investigations have led to the observation of different species populated en route to amyloid fibrils, including oligomers of different size and morphology, as well as protofibrils, annular aggregates and worm-like assemblies. 3-6 The heterogeneity, dynamic

Slow Amyloid Nucleation via α-Helix-Rich Oligomeric Intermediates in Short Polyglutamine-Containing Huntingtin Fragments

Journal of Molecular Biology, 2012

The 17-amino-acid N-terminal segment (htt NT) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this diseaseassociated aggregation. Although all of these peptides, in addition to htt NT itself, form α-helixrich oligomeric intermediates, only peptides with Q N of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt NT sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and αhelical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt NT Q N peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on-and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.

Diverse Structural Conversion and Aggregation Pathways of Alzheimerʼs Amyloid-β (1–40)

ACS Nano, 2019

Complex amyloid aggregation of amyloid- (1-40) (A 1-40) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of A 1-40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of A 1-40 induced by low solvent polarity accelerated cytotoxic A 1-40 amyloid fibrillation while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of -structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying A 1-40 aggregation with conceptual energy diagrams and propose that aggregationprone partial helical structures are key to inducing amyloidogenesis. We demonstrate that contextdependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.

Amyloid-beta peptide Aβp3-42 affects early aggregation of full-length Aβ1-42

Peptides, 2009

The major amyloid beta (Aβ) peptides found in the brain of familial and late onset Alzheimer's disease include the full-length Aβ1-42 and N-terminally truncated, pyroglutamylated peptides Aβp3-42 and Aβp11-42. The biophysical properties of Aβ1-42 have been extensively studied, yet little is known about the other modified peptides. We investigated the aggregation kinetics of brainspecific Aβ peptides to better understand their potential roles in plaque formation. Synthetic peptides were analyzed individually and in mixtures representing various ratios found in the brain. Spectrofluorometric analyses using Thioflavin-T showed that the aggregation of Aβ1-42 was faster compared to Aβp3-42; however, Aβp11-42 displayed similar kinetics. Surprisingly, mixtures of fulllength Aβ1-42 and Aβp3-42 showed an initial delay in beta-sheet formation from both equimolar and non-equimolar samples. Electron microscopy of peptides individually and in mixtures further supported fluorescence data. These results indicate that Aβ-Aβ peptide interactions involving different forms may play a critical role in senile plaque formation and maintenance of the soluble Aβ pool in the brain.

The Ratio of Monomeric to Aggregated Forms of Aβ40 and Aβ42 Is an Important Determinant of Amyloid-β Aggregation, Fibrillogenesis, and Toxicity

Journal of Biological Chemistry, 2008

Aggregation and fibril formation of amyloid-␤ (A␤) peptides A␤40 and A␤42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of A␤40 to A␤42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of A␤. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of A␤40 to A␤42, we probed the concentration-and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric A␤40 alters the kinetic stability, solubility, and morphological properties of A␤42 aggregates and prevents their conversion into mature fibrils. A␤40, at approximately equimolar ratios (A␤40/A␤42 ϳ 0.5-1), inhibits (>50%) fibril formation by monomeric A␤42, whereas inhibition of protofibrillar A␤42 fibrillogenesis is achieved at lower, substoichiometric ratios (A␤40/A␤42 ϳ 0.1). The inhibitory effect of A␤40 on A␤42 fibrillogenesis is reversed by the introduction of excess A␤42 monomer. Additionally, monomeric A␤42 and A␤40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar A␤ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, A␤42 protofibrils selectively seed the fibrillogenesis of monomeric A␤42 but not monomeric A␤40. Finally, we also show that the amyloidogenic propensities of different individual and mixed A␤ species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of A␤40 to A␤42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.