Binding of Human Proteins to Amyloid-β Protofibrils (original) (raw)
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Scientific Reports
Protofibrils of the 42 amino acids long amyloid-β peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer's disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aβ 42 cc, which is a stable engineered mimic of wild type Aβ 42 protofibrils. Several binders were identified that bind Aβ 42 cc protofibrils with low nanomolar affinities, and which also recognize wild type Aβ 42 protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aβ 42 cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aβ 42 protofibrils. Aggregation of the amyloid-β peptide (Aβ) in the brain is a hallmark of Alzheimer's disease 1. The end-state of the aggregation is amyloid fibrils, which become deposited into senile plaques. However, Aβ aggregation involves a number of intermediate aggregation states, collectively called soluble oligomers, and evidence suggests direct causative links between soluble Aβ oligomers and synapse dysfunction 2, 3. The aggregation path of the 42-residue Aβ 42 peptide to amyloid fibrils is believed to involve the formation of pentameric or hexameric oligomers (paranuclei) that associate into larger protofibrils, which eventually undergo a structural interconversion into amyloid fibrils 4-6. Please note that we refer to protofibrils as a class of rod-like pre-fibrillar aggregates that are distinct from amyloid fibrils 6. However, smaller dimeric or trimeric (low-n) oligomers 7 , and various aggregates that form in the presence of membranes or detergents, have also been described (and reviewed) 6. In fact, much of the details surrounding oligomer formation and interconversion in vivo as well as in vitro remain elusive. New binding agents that specifically recognize intermediate aggregates, and which can be used to detect the presence of such aggregates, would represent valuable tools in the research and such agents might also be used within diagnostic or even therapeutic applications. In this work, we describe the selection of Affibody molecules 8, 9 that selectively recognize protofibrils of Aβ. There are several advantages and potential applications of in vitro selected binders. One apparent strength is that the state of target is under full control during the selection, which is not normally the case with IgG antibodies
Protofibrillar and Fibrillar Amyloid-β Binding Proteins in Cerebrospinal Fluid
Journal of Alzheimer's disease : JAD, 2018
Aggregation and deposition of misfolded amyloid-β (Aβ) peptide in the brain is central to Alzheimer's disease (AD). Oligomeric, protofibrillar, and fibrillar forms of Aβ are believed to be neurotoxic and cause neurodegeneration in AD, but the toxicity mechanisms are not well understood and may involve Aβ-interacting molecular partners. In a previous study, we identified potential Aβ42 protofibrillar-binding proteins in serum and cerebrospinal fluid (CSF) using an engineered version of Aβ42 (Aβ42CC) that forms protofibrils, but not fibrils. Here we studied binding of proteins to Aβ42 fibrils in AD and non-AD CSF and compared these with protofibrillar Aβ42CC-binding partners. Aβ42 fibrils sequestered 2.4-fold more proteins than Aβ42CC protofibrils. Proteins with selective binding to fibrillar aggregates with low nanomolar affinity were identified. We also found that protofibrillar and fibrillar Aβ-binding proteins represent distinct functional categories. Aβ42CC protofibrils trigg...
Biochemistry, 2012
Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/ hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/ internalization for the different Aβ proteins and their aggregation states or fibrillar structure.
The molecular assembly of amyloid aβ controls its neurotoxicity and binding to cellular proteins
PloS one, 2011
Accumulation of b-sheet-rich peptide (Ab) is strongly associated with Alzheimer's disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Ab species are potent neurotoxins, however the molecular mechanism responsible for Ab toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Ab 1-40 and Ab 1-42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Ab toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Ab receptors in N2a cells, triggering a multi factorial toxicity.
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:
Amyloid -Protein Dimers Rapidly Form Stable Synaptotoxic Protofibrils
Journal of Neuroscience, 2010
Non-fibrillar, water-soluble low-molecular weight assemblies of the amyloid β-protein (Aβ) are believed to play an important role in Alzheimer's disease (AD). Aqueous extracts of human brain contain Aβ assemblies which migrate on SDS-PAGE and elute from size exclusion as dimers (~8 kDa) and can block long term potentiation and impair memory consolidation in the rat. Such species are detected specifically and sensitively in extracts of Alzheimer brain suggesting that SDS-stable dimers may be the basic building blocks of AD-associated synaptotoxic assemblies. Consequently, understanding the structure and properties of Aβ dimers is of great interest. In the absence of sufficient brain-derived dimer to facilitate biophysical analysis, we generated synthetic dimers designed to mimic the natural species. For this, Aβ(1-40) containing cysteine in place of serine 26 was used to produce disulphide cross-linked dimer, (AβS26C) 2 . Such dimers had no detectable secondary structure, produced an analytical ultracentrifugation (AUC) profile consistent for an ~8.6 kDa protein, and had no effect on hippocampal long term potentiation (LTP). However, (AβS26C) 2 aggregated more rapidly than either AβS26C or wild type monomers and formed parastable β-sheet rich, thioflavin T positive, protofibril-like assemblies. Whereas wild type Aβ aggregated to form typical amyloid fibrils, the protofibril-like structures formed by (AβS26C) 2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus. These data support the idea that Aβ dimers may stabilize the formation of fibril intermediates by a process distinct from that available to Aβ monomer and that higher molecular weight pre-fibrillar assemblies are the proximate mediators of Aβ toxicity.
Journal of Peptide Science, 2004
The extracellular accumulation of amyloid-beta (Aβ) in neuritic plaques is one of the characteristic hallmarks of Alzheimer's disease (AD), a progressive dementing neurodegenerative disorder of the elderly. By virtue of its structure, Aβ is able to bind to a variety of biomolecules, including lipids, proteins and proteoglycans. The binding of the various forms of Aβ (soluble or fibrillar) to plasma membranes has been studied with regard to the direct toxicity of Aβ to neurons, and the activation of a local inflammation phase involving microglia.The binding of Aβ to membrane lipids facilitates Aβ fibrillation, which in turn disturbs the structure and function of the membranes, such as membrane fluidity or the formation of ion channels.A subset of membrane proteins binds Aβ. The serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind the monomeric form of Aβ. The α7nicotinic acetylcholine receptor (α7nAChR), integrins, RAGE (receptor for advanced glycosylation end-products) and FPRL1 (formyl peptide receptor-like 1) are able to bind the monomeric and fibrillar forms of Aβ. In addition, APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), the P75 neurotrophin receptor (P75NTR), the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, α6β1–integrin and CD47 have been reported to bind the fibrillar form of Aβ.Heparan sulfate proteoglycans have also been described as cell-surface binding sites for Aβ. The various effects of Aβ binding to these membrane molecules are discussed. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.
Brain Research, 2003
The Alzheimer's disease Ab peptide can increase the levels of cell-associated amyloid precursor protein (APP) in vitro. To determine the specificity of this response for Ab and whether it is related to cytotoxicity, we tested a diverse range of fibrillar peptides including amyloid-b (Ab), the fibrillar prion peptides PrP106-126 and PrP178-193 and human islet-cell amylin. All these peptides increased the levels of APP and amyloid precursor-like protein 2 (APLP2) in primary cultures of astrocytes and neurons. Specificity was shown by a c lack of change to amyloid precursor-like protein 1, t-1 and cellular prion protein (PrP ) levels. APP and APLP2 levels were elevated only in cultures exposed to fibrillar peptides as assessed by electron microscopy and not in cultures treated with non-fibrillogenic peptide variants or aggregated lipoprotein. We found that PrP106-126 and the non-toxic but fibril-forming PrP178-193 increased APP levels in c cultures derived from both wild-type and PrP -deficient mice indicating that fibrillar peptides up-regulate APP through a non-cytotoxic mechanism and irrespective of parental protein expression. Fibrillar PrP106-126 and Ab peptides bound recombinant APP and APLP2 suggesting the accumulation of these proteins was mediated by direct binding to the fibrillated peptide. This was supported by decreased APP accumulation following extensive washing of the cultures to remove fibrillar aggregates. Pre-incubation of fibrillar peptide with recombinant APP18-146, the putative fibril binding site, also abrogated the accumulation of APP. These findings show that diverse fibrillogenic peptides can induce accumulation of APP and APLP2 and this mechanism could contribute to pathogenesis in neurodegenerative disorders. Alzheimer's disease (AD) is characterized by progres-APLP2, amyloid precursor like-protein 2; BC, bathocuprione disulphonsive and specific neuronal degeneration with accumulation c ate; CJD, Creutzfeldt-Jakob disease; LDL, low density lipoprotein; PrP , of amyloid deposits and the appearance of neurofibrillary sc normal prion protein; PrP , infectious PrP; t, tau tangle-bearing neurons [53]. A central component of *Corresponding author. Tel.: 161-3-8344-5882; fax: 161-3-8344amyloid plaques is the amyloid-beta (Ab) protein [15,30]. 4004. (R. Cappai).
BMC Neuroscience, 2007
Background Aggregation of the amyloid peptides, Aβ40 and Aβ42, is known to be involved in the pathology of Alzheimer's disease (AD). Here we investigate the relationship between peptide aggregation and cell surface binding of three forms of Aβ (Aβ40, Aβ42, and an Aβ mutant). Results Using confocal microscopy and flow cytometry with fluorescently labelled Aβ, we demonstrate a correlation between the aggregation propensity of the Alzheimer amyloid peptides and their neuronal cell surface association. We find that the highly aggregation prone Aβ42 associates with the surface of neuronal cells within one hour, while the less aggregation prone Aβ40 associates over 24 hours. We show that a double mutation in Aβ42 that reduces its aggregation propensity also reduces its association with the cell surface. Furthermore, we find that a cell line that is resistant to Aβ cytotoxicity, the non-neuronal human lymphoma cell line U937, does not bind either Aβ40 or Aβ42. Conclusion Taken together...
The Journal of biological chemistry, 2017
Self-association of amyloid beta (Aβ) peptides is a hallmark of Alzheimer's disease and serves as a general prototype for amyloid formation. A key endogenous inhibitor of Aβ self-association is Human Serum Albumin (HSA), which binds ~90% of plasma Aβ. However, the exact molecular mechanism by which HSA binds Aβ monomers and protofibrils is not fully understood. Here, using dark-state exchange saturation transfer (DEST) NMR and relaxation experiments, complemented by morphological characterization, we mapped the HSA-Aβ interactions at atomic resolution by examining HSA's effects on Aβ monomers and soluble high-molecular weight oligomeric protofibrils. We found that HSA binds both monomeric and protofibrillar Aβ, but the affinity of HSA for Aβ monomers is lower than for Aβ protofibrils (Kd ~ sub-mM vs. μM), yet physiologically relevant owing to the ~0.6 - 0.7 mM plasma HSA concentration. In both Aβ protofibrils and monomers, HSA targets key Aβ self-recognition sites spanning t...