Self-Templated Nucleation in Peptide and Protein aggregation

Peptides and proteins exhibit a common tendency to assemble into highly ordered fibrillar aggregates, whose formation proceeds in a nucleation-dependent manner that is often preceded by the formation of disordered oligomeric assemblies. This process has received much attention because disordered oligomeric aggregates have been associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Here we describe a self-templated nucleation mechanism that determines the transition between the initial condensation of polypeptide chains into disordered assemblies and their reordering into fibrillar structures. The results that we present show that at the molecular level this transition is due to the ability of polypeptide chains to reorder within oligomers into fibrillar assemblies whose surfaces act as templates that stabilise the disordered assemblies.Comment: 4 pages, 3 figure

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand.

BACKGROUND: Oligomeric and fibrillar aggregates of the amyloid beta-peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Abeta assemblies is essential for the elucidation of the mechanisms of Abeta neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Abeta. The method is based on the coexpression of the affibody protein ZAbeta3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAbeta3 binds to the amyloidogenic central and C-terminal part of Abeta with nanomolar affinity and consequently inhibits aggregation. RESULTS: Coexpression of ZAbeta3 affords the overexpression of both major Abeta isoforms, Abeta(1-40) and Abeta(1-42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Abeta. ZAbeta3 coexpression moreover permits the recombinant production of Abeta(1-42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Abeta(1-42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. CONCLUSION: The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Abeta peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Abeta(1-42) is reported.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Delivery of Native Proteins into C. elegans Using a Transduction Protocol Based on Lipid Vesicles.

The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. Nevertheless, the challenges associated with the administration of native proteins to C. elegans have limited the range of applications of this animal model in protein-based drug discovery programs. Here, we describe a readily usable protocol for the transduction of native proteins in C. elegans, which is based on the encapsulation of the proteins of interest within cationic lipid vesicles, prior to their administration to worms. This procedure limits the degradation of the proteins in the guts of the animals, and promotes their adsorption into body tissues. To illustrate the efficacy of this approach we apply it to deliver an antibody designed to inhibit α-synuclein aggregation, and show that it can lead to the rescue of the disease phenotype in a C. elegans model of Parkinson's disease. As this transduction protocol is fast and inexpensive, we anticipate that it will be readily applicable to protein-based drug discovery studies that utilize C. elegans as a model organism.Alzheimer’s Society, UK (grant number 317, AS-SF-16-003) Centre For Misfolding Disease

Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates.

Molecular chaperones are key components of the arsenal of cellular defence mechanisms active against protein aggregation. In addition to their established role in assisting protein folding, increasing evidence indicates that molecular chaperones are able to protect against a range of potentially damaging aspects of protein behaviour, including misfolding and aggregation events that can result in the generation of aberrant protein assemblies whose formation is implicated in the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The interactions between molecular chaperones and different amyloidogenic protein species are difficult to study owing to the inherent heterogeneity of the aggregation process as well as the dynamic nature of molecular chaperones under physiological conditions. As a consequence, understanding the detailed microscopic mechanisms underlying the nature and means of inhibition of aggregate formation remains challenging yet is a key objective for protein biophysics. In this review, we discuss recent results from biophysical studies on the interactions between molecular chaperones and protein aggregates. In particular, we focus on the insights gained from current experimental techniques into the dynamics of the oligomerisation process of molecular chaperones, and highlight the opportunities that future biophysical approaches have in advancing our understanding of the great variety of biological functions of this important class of proteins.We acknowledge financial support from the Frances and Augustus Newman Foundation (TPJK), the Biological Sciences Research Council (TPJK), the European Research Council (TPJK and MAW), the Wellcome Trust (CMD, TPJK and MV), and the Marie Curie fellowship scheme (PA).This is the final version of the article. It was first available from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5CC03689

Generic Mechanism of Emergence of Amyloid Protofilaments from Disordered Oligomeric aggregates

The presence of oligomeric aggregates, which is often observed during the process of amyloid formation, has recently attracted much attention since it has been associated with neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. We provide a description of a sequence-indepedent mechanism by which polypeptide chains aggregate by forming metastable oligomeric intermediate states prior to converting into fibrillar structures. Our results illustrate how the formation of ordered arrays of hydrogen bonds drives the formation of beta-sheets within the disordered oligomeric aggregates that form early under the effect of hydrophobic forces. Initially individual beta-sheets form with random orientations, which subsequently tend to align into protofilaments as their lengths increases. Our results suggest that amyloid aggregation represents an example of the Ostwald step rule of first order phase transitions by showing that ordered cross-beta structures emerge preferentially from disordered compact dynamical intermediate assemblies.Comment: 14 pages, 4 figure

Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins.

Residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs) have emerged as valuable parameters for defining the structures and dynamics of disordered proteins by nuclear magnetic resonance (NMR) spectroscopy. Procedures for their measurement, however, may lead to conformational perturbations because of the presence of the alignment media necessary for recording RDCs, or of the paramagnetic groups that must be introduced for measuring PREs. We discuss here experimental methods for quantifying these effects by considering the case of the 40-residue isoform of the amyloid β peptide (Aβ40), which is associated with Alzheimer's disease. By conducting RDC measurements over a range of concentrations of certain alignment media, we show that perturbations arising from transient binding of Aβ40 can be characterized, allowing appropriate corrections to be made. In addition, by using NMR experiments sensitive to long-range interactions, we show that it is possible to identify relatively nonperturbing sites for attaching nitroxide radicals for PRE measurements. Thus, minimizing the conformational perturbations introduced by RDC and PRE measurements should facilitate their use for the rigorous determination of the conformational properties of disordered proteins.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.biochem.5b0067

Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson's disease genetically related mutants.

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson's disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.LT has been recipient of a grant PAT Post Doc Outgoing 2009 – 7th Framework Program Marie Curie COFUND actions. NC was funded by a Royal Society Dorothy Hodgkin Research Fellowship and is currently a Ramón y Cajal Research Fellow (Spanish Ministry of Economy and Competitiveness). MHH thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. AJD is funded by the Schiff Foundation.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1669

Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons.

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.We thank Dr Q. Jeng and Dr A. Stephens for technical assistance and Dr J. Skepper for TEM imaging. This work was funded by grants from the U.K. Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer’s Research UK (ARUK-EG2012A-1), U.K. Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z). D.P. wishes to acknowledge support from the Swiss National Science Foundation and the Wellcome Trust through personal fellowships. A.K.B thanks Magdalene College, Cambridge and the Leverhulme Trust for support.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.1516546113

Amyloid-β and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing.

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-β (Aβ) and α-synuclein (αS), associated with Alzheimer's and Parkinson's diseases, respectively, on the Cu(2+)-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu(2+) is bound to Aβ or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aβ or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu(2+) and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.This work was supported by the Villum Foundation (J.T.P., L.H.), the Lundbeck Foundation (J.T.P., K.T.), the Agency for Science, Technology and Research, Singapore (S.W.C.), The Wellcome Trust (C.M.D.) and the Spanish Ministry of Economy and Competitiveness through the Ramon y Cajal ́ program (N.C.).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/jacs.5b1357