Probing and trapping a sensitive conformation: amyloid-β fibrils, oligomers, and dimers (original) (raw)
Related papers
Scientific Reports, 2015
The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of welldefined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples. Aβ oligomers, species that form early during Aβ aggregation, are considered the pathogenic molecular form of Aβ in AD 1. Consequently, they have been singled out as a target to treat this disease 2. However, the characterization of Aβ oligomers is challenging because they are heterogeneous-comprising a range of aggregation states-and because they form transiently-evolving as a function of time 3,4. In spite of these difficulties, different approaches and techniques have been developed to characterize them 5-12. One such approach relies on the production of cross-linked oligomers by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, and their subsequent analysis by SDS-PAGE (Fig. 1) 5,13. Aβ 1-40 (Aβ 40) and Aβ 1-42 (Aβ 42) are the two principal forms of Aβ , differing by only two hydrophobic residues at the C-terminus. Analysis of PICUP cross-linked Aβ 40 and Aβ 42 samples by SDS-PAGE revealed that Aβ 40 oligomerizes through dimers up to tetramers while Aβ 42 does so mainly through pentamers and hexamers 5,13. Since Aβ 42 has been shown to have a more prominent role in AD 14,15 , PICUP/SDS-PAGE analysis led to the conclusion that pentamers and hexamers constituted the basic building blocks for Aβ aggregation 5,13. The importance of pentamers and hexamers in Aβ aggregation has become a scientific paradigm in the field. For example, according to the Web of Science, there are six key papers 7-9,16-18 , frequently cited together (more than one thousand times), that constitute the foundational core for the research front entitled "Aβ oligomers, fibrils and AD". Three of these six papers are based directly 16,18 or indirectly 7 on
The elusive nature and diagnostics of misfolded Aβ oligomers
Frontiers in Chemistry, 2015
Amyloid-beta (Aβ) peptide oligomers are believed to be the causative agents of Alzheimer's disease (AD). Though post-mortem examination shows that insoluble fibrils are deposited in the brains of AD patients in the form of intracellular (tangles) and extracellular (plaques) deposits, it has been observed that cognitive impairment is linked to synaptic dysfunction in the stages of the illness well before the appearance of these mature deposits. Increasing evidence suggests that the most toxic forms of Aβ are soluble low-oligomer ligands whose amounts better correlate with the extent of cognitive loss in patients than the amounts of fibrillar insoluble forms. Therefore, these ligands hold the key to a better understanding of AD prompting the search for clearer correlations between their structure and toxicity. The importance of such correlations and their diagnostic value for the early diagnosis of AD is discussed here with a particular emphasis on the transient nature and structural plasticity of misfolded Aβ oligomers.
Molecular basis of β-amyloid oligomer recognition with a conformational antibody fragment
Proceedings of the National Academy of Sciences, 2012
Oligomers are intermediates of the β-amyloid (Aβ) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer’s disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aβ conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aβ oligomers, such as those formed by Aβ (1–40) and Aβ (1–42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aβ residues 18–20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in li...
Angewandte Chemie International Edition, 2014
Small oligomers of the amyloid b (Ab) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solidstate NMR methods. The smallest Ab 40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Ab mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.
Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways
Proceedings of the National Academy of Sciences, 2002
Amyloid β-protein (Aβ) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Aβ in AD is new evidence that oligomeric forms of Aβ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Aβ, Aβ42, with the disease. Detailed knowledge of the structure and assembly dynamics of Aβ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Aβ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Aβ40 and Aβ42. Carefully prepared aggregate-free Aβ40 existed as monomers, dimers, trimers, and tetra...
Nature Chemistry, 2009
In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-b protein isoforms of Ab40 and Ab42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Ab40 and Ab42 self-assemble via different pathways and provide a candidate in the Ab42 dodecamer for the primary toxic species in Alzheimer's disease. M any diseases share the common trait of peptide-protein misfolding that leads to oligomerization and, eventually, formation of plaques of b-sheet structure. Prominent among these are type 2 diabetes 1 , Parkinson's disease 2 and Alzheimer's disease 3,4. Of these, Alzheimer's disease is the leading cause of late-life dementia and is the focus of this paper. An increasing body of evidence links oligomerization of a ubiquitous peptide, the amyloid-b protein, to disease causation 3-6. For this reason, elucidation of pathways of oligomer formation may be critical for the identification of therapeutic targets. Many types of oligomeric amyloid-b assemblies have been described (for a review, see Lazo et al. 7). Recently, Bitan et al. 8-10 used photoinduced cross-linking of unmodified proteins (PICUP) to reveal that the 42-residue form of amyloid-b, Ab42, formed (Ab42) 5 and (Ab42) 6 oligomers ('paranuclei') that could oligomerize to form structures of higher order. Ab40 did not form paranuclei, but instead existed as a mixture of monomers, dimers, trimers and tetramers. Chen and Glabe 11 , in contrast, used fluorescence and gel electrophoresis to determine oligomer states of amyloid-b refolded from denaturing solutions. They observed only Ab42 monomer and trimer bands, and no oligomers of Ab40. Differences such as these may exist because of the diverse experimental systems used to monitor amyloid-b self-association. Also, it has been argued that, in addition to the intrinsic potential of amyloid-b to traverse different assembly pathways, flaws in experimental design may have misled researchers in their quest to elucidate fully the amyloid-b oligomerization process 12. Hence there is significant uncertainty about amyloid-b oligomer states and their position and relevance to amyloid-b aggregation.
Journal of Alzheimer's disease : JAD, 2014
Alzheimer's disease (AD) is associated with the formation of toxic amyloid-β (Aβ)42 oligomers, and recent evidence supports a role for Aβ dimers as building blocks for oligomers. Molecular dynamics simulation studies have identified clans for the dominant conformations of Aβ42 forming dimers; however, it is unclear if a larger spectrum of dimers is involved and which set(s) of dimers might evolve to oligomers verse fibrils. Therefore, for this study we generated multiple structural conformations of Aβ42, using explicit all-atom molecular dynamics, and then clustering the different structures based on key conformational similarities. Those matching a selection threshold were then used to model a process of oligomerization. Remarkably, we showed a greater diversity in Aβ dimers than previously described. Depending on the clan family, different types of Aβ dimers were obtained. While some had the tendency to evolve into oligomeric rings, others formed fibrils of diverse characteris...
Structural conversion of neurotoxic amyloid-β1–42 oligomers to fibrils
Nature Structural & Molecular Biology, 2010
The Aβ42 peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low temperature and low salt can stabilize disc-shaped oligomers (pentamers) that are significantly more toxic to murine cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the β-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C-terminus is protected from solvent exchange and which have a turn conformation placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Aβ42 oligomers to fibrils involves the association of these loosely aggregated strands into β-sheets whose individual β-strands polymerize in a parallel, inregister orientation and are staggered at an inter-monomer contact between Gln15 and Gly37. A major pathological hallmark of Alzheimer's disease (AD) is the formation of neuritic plaques within the gray matter of AD patients 1. These plaques are composed primarily of filamentous aggregates (fibrils) of the 39-42 amino acid long amyloid-β (Aβ) peptide formed from the proteolytic cleavage of the amyloid precursor protein by βand γ-secretases 2-5. The major species of Aβ production are the Aβ40 and Aβ42 peptides, with Aβ42 being predominant in neuritic plaques of AD patients and exhibiting a higher in vitro propensity to Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A Rational Structured Epitope Defines a Distinct Subclass of Toxic Amyloid-beta Oligomers
ACS chemical neuroscience, 2018
Oligomers of amyloid-β (AβO) are deemed key in synaptotoxicity and amyloid seeding of Alzheimer's disease (AD). However, the heterogeneous and dynamic nature of AβO and inadequate markers for AβO subtypes have stymied effective AβO identification and therapeutic targeting in vivo. We identified an AβO-subclass epitope defined by differential solvent orientation of the lysine 28 side chain in a constrained loop of serine-asparagine-lysine (cSNK), rarely displayed in molecular dynamics simulations of monomer and fibril ensembles. A mouse monoclonal antibody targeting AβO recognizes ∼50-60 kDa SDS-resistant soluble Aβ assemblages in AD brain and prolongs the lag phase of Aβ aggregation in vitro. Acute peripheral infusion of a murine IgG1 anti-AβO in two AD mouse models reduced soluble brain Aβ aggregates by 20-30%. Chronic cSNK peptide immunization of APP/PS1 mice engendered an anti-AβO IgG1 response without epitope spreading to Aβ monomers or fibrils and was accompanied by preserv...