Giorgio Favrin

ANS binding reveals common features of cytotoxic amyloid species

Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimers disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

Structural Reorganisation and Potential Toxicity of Oligomeric Species Formed during the Assembly of Amyloid Fibrils

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of β sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

Oligomerization of Amyloid Aβ16–22 Peptides Using Hydrogen Bonds and Hydrophobicity Forces

The 16-22 amino-acid fragment of the beta-amyloid peptide associated with the Alzheimers disease, Abeta, is capable of forming amyloid fibrils. Here we study the aggregation mechanism of Abeta16-22 peptides by unbiased thermodynamic simulations at the atomic level for systems of one, three, and six Abeta16-22 peptides. We find that the isolated Abeta16-22 peptide is mainly a random coil in the sense that both the alpha-helix and beta-strand contents are low, whereas the three- and six-chain systems form aggregated structures with a high beta-sheet content. Furthermore, in agreement with experiments on Abeta16-22 fibrils, we find that large parallel beta-sheets are unlikely to form. For the six-chain system, the aggregated structures can have many different shapes, but certain particularly stable shapes can be identified.

Monte Carlo update for chain molecules: Biased Gaussian steps in torsional space

We develop a new elementary move for simulations of polymer chains in torsion angle space. The method is flexible and easy to implement. Tentative updates are drawn from a (conformation-dependent) Gaussian distribution that favors approximately local deformations of the chain. The degree of bias is controlled by a parameter b. The method is tested on a reduced model protein with 54 amino acids and the Ramachandran torsion angles as its only degrees of freedom, for different b. Without excessive fine tuning, we find that the effective step size can be increased by a factor of 3 compared to the unbiased b=0 case. The method may be useful for kinetic studies, too.

Folding of a small helical protein using hydrogen bonds and hydrophobicity forces

A reduced protein model with five to six atoms per amino acid and five amino acid types is developed and tested on a three‐helix‐bundle protein, a 46‐amino acid fragment from staphylococcal protein A. The model does not rely on the widely used Gō approximation, which ignores non‐native interactions. We find that the collapse transition is considerably more abrupt for the protein A sequence than for random sequences with the same composition. The chain collapse is found to be at least as fast as helix formation. Energy minimization restricted to the thermodynamically favored topology gives a structure that has a root‐mean‐square deviation of 1.8 Å from the native structure. The sequence‐dependent part of our potential is pairwise additive. Our calculations suggest that fine‐tuning this potential by parameter optimization is of limited use. Proteins 2002;47:99–105.

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimers disease-related amyloid beta peptide (Abeta) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Abeta40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Abeta42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Abeta peptide.

Expression in Drosophila of Tandem Amyloid β Peptides Provides Insights into Links between Aggregation and Neurotoxicity

Background: Investigating the kinetics of Aβ peptide aggregation in vivo is vital to understanding Alzheimer disease. Results: Linking two Aβ40 or Aβ42 peptides together increases their aggregation rates in Drosophila, but only increases the neurotoxicity of Aβ42. Conclusion: Increasing the rate of aggregation of Aβ increases amyloid deposition but not necessarily toxicity. Significance: The toxicity of Aβ depends on the mechanism and not just the rate of amyloid formation. The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ40) or 42 residues (Aβ42), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ40 and Aβ42 compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ42 constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ42 peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ40 rather than simply to its higher rate of aggregation.

Expression in Drosophila of Tandem Aβ Peptides Provides Insights Into the Link Between Aggregation and Neurotoxicity

Background: Investigating the kinetics of Aβ peptide aggregation in vivo is vital to understanding Alzheimer disease. Results: Linking two Aβ40 or Aβ42 peptides together increases their aggregation rates in Drosophila, but only increases the neurotoxicity of Aβ42. Conclusion: Increasing the rate of aggregation of Aβ increases amyloid deposition but not necessarily toxicity. Significance: The toxicity of Aβ depends on the mechanism and not just the rate of amyloid formation. The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ40) or 42 residues (Aβ42), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ40 and Aβ42 compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ42 constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ42 peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ40 rather than simply to its higher rate of aggregation.

Two-state folding over a weak free-energy barrier.

We present a Monte Carlo study of a model protein with 54 amino acids that folds directly to its native three-helix-bundle state without forming any well-defined intermediate state. The free-energy barrier separating the native and unfolded states of this protein is found to be weak, even at the folding temperature. Nevertheless, we find that melting curves to a good approximation can be described in terms of a simple two-state system, and that the relaxation behavior is close to single exponential. The motion along individual reaction coordinates is roughly diffusive on timescales beyond the reconfiguration time for a single helix. A simple estimate based on diffusion in a square-well potential predicts the relaxation time within a factor of two.