Stabilization of neurotoxic Alzheimer amyloid- oligomers by protein engineering

Institute of Biomedicine, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 08/2010; 107(35):15595-600. DOI: 10.1073/pnas.1001740107
Source: PubMed

ABSTRACT Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(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 Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(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 Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta 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 Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

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Available from: Anatoly Dubnovitsky, Aug 26, 2015
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    • "However, there is no conclusive measurement of the structure of the monomers and the oligomers under physiological conditions. Hard and coworkers have suggested that forcing the monomer into a hairpin-like structure increases its toxicity (Sandberg et al., 2010). Smith and coworkers have suggested that different oligomeric species of similar size can have dissimilar toxicity (Ladiwala et al., 2012), pointing toward the role played by folding. "
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    ABSTRACT: Amyloid beta (Aβ) is an extracellular 39-43 residue long peptide present in the mammalian cerebrospinal fluid, whose aggregation is associated with Alzheimer's disease (AD). Small oligomers of Aβ are currently thought to be the key to toxicity. However, it is not clear why the monomers of Aβ are non-toxic, and at what stage of aggregation toxicity emerges. Interactions of Aβ with cell membranes is thought to be the initiator of toxicity, but membrane binding studies with different preparations of monomers and oligomers have not settled this issue. We have earlier found that thermodynamically stable Aβ monomers emerge spontaneously from oligomeric mixtures upon long term incubation in physiological solutions (Nag et al., 2011). Here we show that the membrane-affinity of these stable Aβ monomers is much lower than that of a mixture of monomers and small oligomers (containing dimers to decamers), providing a clue to the emergence of toxicity. Fluorescently labeled Aβ40 monomers show negligible binding to cell membranes of a neuronal cell line (RN46A) at physiological concentrations (250 nM), while oligomers at the same concentrations show strong binding within 30 min of incubation. The increased affinity most likely does not require any specific neuronal receptor, since this difference in membrane-affinity was also observed in a somatic cell-line (HEK 293T). Similar results are also obtained for Aβ42 monomers and oligomers. Minimal amount of cell death is observed at these concentrations even after 36 h of incubation. It is likely that membrane binding precedes subsequent slower toxic events induced by Aβ. Our results (a) provide an explanation for the non-toxic nature of Aβ monomers, (b) suggest that Aβ toxicity emerges at the initial oligomeric phase, and (c) provide a quick assay for monitoring the benign-to-toxic transformation of Aβ.
    Frontiers in Physiology 04/2013; 4:84. DOI:10.3389/fphys.2013.00084 · 3.50 Impact Factor
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    • "The structural characterization of these oligomeric aggregation states, associated to their specific neuronal toxicity is of primary importance in the study of AD pathogenesis, even though it is very difficult to achieve specific information due to the fast aggregation processes, the heterogeneous peptide contents and the occurring of transient organization states. Monomeric A␤ assumes a beta-hairpin conformation in water, stabilized by intramolecular H-bonds and antiparallel ␤-strands that lead, via conformational changes, to intermediate oligomeric states of A␤ subunits with intermolecular H-bonds and parallel ␤-strand organization (Campioni et al., 2010; Ono et al., 2009; Roychaudhuri et al., 2009; Sandberg et al., 2010). New atomic force microscopy (AFM) techniques enabled to image a dynamic A␤ oligomer formation consistent with the hairpin conformation hypothesis. "
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    ABSTRACT: Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide.
    The international journal of biochemistry & cell biology 08/2012; 44(11):2085-93. DOI:10.1016/j.biocel.2012.08.010 · 4.24 Impact Factor
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    • "Thus, the protein model HypF-N has allowed oligomeric states, that would normally be metastable, to be trapped and therefore studied, with conclusions probably valid for the toxic properties of many oligomers in disease-related systems (Campioni et al., 2010). Summarizing (Campioni et al., 2010; Sandbergh et al., 2010), to form amyloid-like structures is generic to polypeptide chains, but whether or not these species are pathogenic depends on their structural features, notably the extent to which hydrophobic residues are flexible and exposed on their surfaces within the environment of a living organism. Thus it seems that for therapeutic purposes the toxicity can be substantially reduced if the hydrophobic residues are incorporated to a greater extent within the interior of the oligomeric assemblies, even in the absence of an effective change in morphology: solvent-exposed structurally disorganized hydrophobic residues within small protein oligomers are at the origin of the pathogenesis of important human diseases. "
    Alzheimer’s Disease Pathogenesis-Core Concepts, Shifting Paradigms and Therapeutic Targets, Edited by Ed Suzanne De La Monte, 09/2011: pages 157-172;
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