Modulation of beta-amyloid aggregation by engineering the sequence connecting beta-strand forming domains (original) (raw)
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Nature communications, 2016
Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aβ aggregation mechanism that uses Aβ-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aβ-polyQ hybrids, and by extension for full-length Aβ peptides, in which a modular Aβ C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a β-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an enginee...
Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering
Alzheimer's & Dementia, 2011
Soluble oligomeric aggregates of the amyloid-β peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Aβ within these aggregates is not known, a β-hairpin conformation is known to be accessible to monomeric Aβ. Here we show that this β-hairpin is a building block of toxic Aβ oligomers by engineering a doublecysteine mutant (called AβCC) in which the β-hairpin is stabilized by an intramolecular disulfide bond. Aβ 40 CC and Aβ 42 CC both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aβ aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in AβCC oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that β-sheet-containing Aβ 42 CC oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Aβ 42 , in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Aβ oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Aβ 42 is more toxic than the shorter Aβ 40 , and why certain inherited mutations are linked to protofibril formation and early-onset AD.
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...
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.
bioRxiv (Cold Spring Harbor Laboratory), 2022
Author contributions: AWS conceived the project, designed and directed the study. CEH and BG planned and carried out LTP studies. AWS performed mass spectrometry and biochemical analysis. JR and TS planned and carried out IF and array tomography studies on human brain tissue. GM and JSD carried out IF studies on Tg mouse brain. DD performed electron microscopy. VS carried out C. elegans experiments and analysis. MPF performed histological examination of human brain tissue. NR and AWS carried out ELISA measurements. BTH and MPF provided biological samples. AWS and CEH wrote the paper and all authors read and commented on the manuscript. Acknowledgments: This work was funded by the "Enable Grant", kindly awarded by Dr. André Catana from the Technology Transfer Office (TTO) as well as the Catalyze4Life Innovation grant by Prof. Bart Deplancke and Dr. Kostas Kaloulis of the Ecole Polytechnique Fédérale de Lausanne (EPFL). We thank Dr. Adam Swetloff (TTO) for the constructive and strategic discussions on the project development as well as the support by the whole Life Sciences Faculty. The project was partially funded using internal funding of the EPFL. Human brain sample preparation and ELISA development was supported by the ADRC grant P30AG062421.We are grateful to the Roland Bailly Foundation (Geneva, Switzerland) for the funding of the MS instrument. We extend our gratitude to Dr. David Hacker (Protein Production and Structure Core Facility (EPFL) for his help with antibody production. Prof. Carmen Sandi, Laboratory of Behavioural Genetics (EPFL) for kindly providing us with antibodies for the study as well as Dr. Jose Sanchez-Mut, Laboratory of Neuroepigenetics (EPFL, Prof. J. Gräff lab), providing us with the reversed sequence of Aβ42-1 peptide and Prof. Hilal Lashuel (EPFL) for the Aβ40-arc mutant peptide. We thank Dr. Pamela Valdés (LEN, EPFL) for her support with IF imaging. TSJ and JR are funded by the UK Dementia
Journal of Biological Chemistry, 2010
The relationship between amyloid deposition and cellular toxicity is still controversial. In addition to fibril-forming oligomers, other soluble Aβ forms (amyloid β-derived diffusible ligands (ADDLs)) were also suggested to form and to present different morphologies and mechanisms of toxicity. One ADDL type, the "globulomer," apparently forms independently of the fibril aggregation pathway. Even though many studies argue that such soluble Aβ oligomers are off fibril formation pathways, they may nonetheless share some structural similarity with protofibrils. NMR data of globulomer intermediates, "preglobulomers," suggested parallel in-register C-terminal β-sheets, with different N-terminal conformations. Based on experimental data, we computationally investigate four classes of Aβ dodecamers: fibril, fibril oligomer, prefibril/preglobulomer cluster, and globulomer models. Our simulations of the solvent protection of double-layered fibril and globulomer models reproduce experimental observations. Using a single layer Aβ fibril oligomer β-sheet model, we found that the C-terminal β-sheet in the fibril oligomer is mostly curved, preventing it from quickly forming a fibril and leading to its breaking into shorter pieces. The simulations also indicate that β-sheets packed orthogonally could be the most stable species for Aβ dodecamers. The major difference between fibril-forming oligomers and ADDL-like oligomers (globulomers) could be the exposure of Met-35 patches. Although the Met-35 patches are necessarily exposed in fibril-forming oligomers to allow their maturation into fibrils, the Met-35 patches in the globulomer are covered by other residues in the orthogonally packed Aβ peptides. Our results call attention to the possible existence of certain "critical intermediates" that can lead to both seeds and other soluble ADDL-like oligomers.
Foldamer-Mediated Structural Rearrangement Attenuates Aβ Oligomerization and Cytotoxicity
Journal of the American Chemical Society, 2017
The conversion of the native random coil amyloid beta (Aβ) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aβ oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aβ conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aβ oligomers. A library of oligoquinolines was screened to identify antagonists of Aβ oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aβ fibrillation. Biophysical assays including amyloid kinetics, Dot Blot, ELISA, and TEM show that 5 effectively inhibits both Aβ oligomerization and fibrillation. The antagonist activity of 5 towards Aβ aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aβ. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aβ. 5 effectively rescues neuroblastoma cells from Aβ-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aβ and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.
A Mini Review on Aβ Oligomers and its Pathogencity
Journal of the Chosun Natural Science, 2014
Amyloid oligomers are believed to play important causal roles in many types of amyloid-related degenerative diseases. Many different laboratories have reported amyloid oligomers that differ in size, morphology, toxicity, and method of preparation or purification, raising the question of the structural relationships among these oligomer preparations. The structural plasticity that has been reported to occur in amyloid formed from the same protein sequence indicates that it is quite possible that different oligomer preparations may represent distinct structural variants. In view of the difficulty in determining the precise structure of amyloids, conformation-and epitope-specific antibodies may provide a facile means of classifying amyloid oligomer structures. Conformation-dependent antibodies that recognize generic epitopes that are specifically associated with distinct aggregation states of many different amyloid-forming sequences indicate that there are at least two fundamentally distinct types of amyloid oligomers: fibrillar and prefibrillar oligomers. Classification of amyloid oligomers according to their underlying structures may be a more useful and rational approach than relying on differences in size and morphology.
Synthetic toxic Aβ1-42 oligomers can assemble in different morphologies
Biochimica et biophysica acta, 2017
Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aβ peptides. Aβ toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aβ1-42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. Different biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used. The oligomers are mostly populated by a 14mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects as...