Pathways and intermediates of amyloid fibril formation

The lack of understanding of amyloid fibril formation at the molecular level is a major obstacle in devising strategies to interfere with the pathologies linked to peptide or protein aggregation. In particular, little is known on the role of intermediates and fibril elongation pathways as well as their dependence on the intrinsic tendency of a polypeptide chain to self-assembly by β-sheet formation (β-aggregation propensity). Here, coarse-grained simulations of an amphipathic polypeptide show that a decrease in the β-aggregation propensity results in a larger heterogeneity of elongation pathways, despite the essentially identical structure of the final fibril. Protofibrillar intermediates that are thinner, shorter and less structured than the final fibril accumulate along some of these pathways. Moreover, the templated formation of an additional protofilament on the lateral surface of a protofibril is sometimes observed as a collective transition. Conversely, for a polypeptide model with a high β-aggregation propensity, elongation proceeds without protofibrillar intermediates. Therefore, changes in intrinsic β-aggregation propensity modulate the relative accessibility of parallel routes of aggregation.     

Measurements of residual dipolar couplings in peptide inhibitors weakly aligned by transient binding to peptide amyloid fibrils**

In this communication, we suggest that transferred residual dipolar couplings (trRDCs) can be employed to restrain the structure of peptide inhibitors transiently binding to beta-amyloid fibrils. The effect is based on the spontaneous alignment of amyloid fibrils with the fibril axis parallel to the magnetic field. This alignment is transferred to the transiently binding peptide inhibitor and is reflected in the size of the trRDCs. We find that the peptide inhibitor adopts a beta-sheet conformation with the backbone N-H and C-H dipolar vectors aligned preferentially parallel and perpendicular, respectively, to the fibril axis.     

Scrapie-associated fibrils (SAF) purification method yields amyloid proteins from systemic and cerebral amyloidosis

We identified fibrils from non-transmissible systemic and cerebral amyloidosis using the purification method of scrapie-associated fibrils (SAF). The fibrils possessed the same nature of congophilia, filamentous structures and molecular weights as amyloid fibrils, and were resistant to Proteinase K digestion. This SAF method makes for a rapid extraction from amyloid-laden tissues. The method, therefore, may purify nontransmissible amyloids alone or together with SAF proteins.     

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

A molecular dynamics approach to the structural characterization of amyloid aggregation

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.     

Effects of chain length on the aggregation of model polyglutamine peptides: molecular dynamics simulations

Aggregation in the brain of polyglutamine-containing proteins is either a cause or an associated symptom of nine hereditary neurodegenerative disorders including Huntington's disease. The molecular level mechanisms by which these proteins aggregate are still unclear. In an effort to shed light on this important phenomenon, we are investigating the aggregation of model polyglutamine peptides using molecular-level computer simulation with a simplified model of polyglutamine that we have developed. This model accounts for the most important types of intra- and inter-molecular interactions-hydrogen bonding and hydrophobic interactions-while allowing the folding process to be simulated in a reasonable time frame. The model is used to examine the folding of isolated polyglutamine peptides 16, 32, and 48 residues long and the folding and aggregation of systems of 24 model polyglutamine peptides 16, 24, 32, 36, 40, and 48 residues long. Although the isolated polyglutamine peptides did form some alpha and beta backbone-backbone hydrogen bonds they did not have as many of these bonds as they would have if they had folded into a complete alpha helix or beta sheet. In one of the simulations on the isolated polyglutamine peptide 48 residues long, we observed a structure that resembles a beta helix. In the multi-chain simulations we observed amorphous aggregates at low temperatures, ordered aggregates with significant beta sheet character at intermediate temperatures, and random coils at high temperatures. We have found that the temperature at which the model peptides undergo the transition from amorphous aggregates to ordered aggregates and the temperature at which the model peptides undergo the transition from ordered aggregates to random coils increase with increasing chain length. Our finding that the stability of the ordered aggregates increases as the peptide chain length increases may help to explain the experimentally observed relation between polyglutamine tract length and aggregation in vitro and disease progression in vivo. We have also observed in our simulations that the optimal temperature for the formation of beta sheets increases with chain length up to 36 glutamine residues but not beyond. Equivalently, at fixed temperature we find a transition from a region dominated by random coils at chain lengths less than 36 to a region dominated by relatively ordered beta sheet structures at chain lengths greater than 36. Our finding of this critical chain length of 36 glutamine residues is interesting because a critical chain length of 37 glutamine residues has been observed experimentally.     

Non-native protein aggregation kinetics

Experimental kinetics of non-native protein aggregation are of practical importance in that they help dictate viable processing, formulation, and storage conditions for biotechnology products, and appear to play a role in determining the onset of a number of diseases. Fundamentally, aggregation kinetics provide insights into the identity of key intermediates in the process, and quantitative tests of available models of aggregation. Although aggregation kinetics often display seemingly disparate behaviors across different proteins and sample conditions, this review illustrates how many of these can be understood within a general framework that treats aggregation as a multi-stage process, and how most available kinetic models of aggregation can be grouped hierarchically in terms of which stage(s) they include. This provides an aid for workers seeking a mechanistic interpretation of in vitro aggregation kinetics, for discriminating among competing models, and in designing experiments to assess in vitro protein stability. Limitations and the utility of purely kinetic approaches to studying aggregation, clarifications of common misperceptions regarding experimental aggregation kinetics, and some outstanding challenges in the field are briefly discussed.     

Different amyloid aggregation of smooth muscles titin in vitro

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300–500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8–10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.     

Study of tetraphenylporphyrins as modifiers of insulin amyloid aggregation

Amyloid fibrils are rigid β‐pleated protein aggregates that are connected with series of harmful diseases and at the same time are promising as base for novel nanomaterials. Thus, design of compounds able to inhibit or redirect those aggregates formation is important both for the biomedical aims and for nanotechnology applications. Here, we studied the effect of tetraphenylporphyrins (metal free, their Cu and Pd complexes, and those functionalized by carboxy and amino groups on periphery) on insulin amyloid self‐assembling. The strongest impact on insulin aggregation was demonstrated by a metal‐free porphyrin bearing four carboxy groups. This compound strongly suppresses insulin aggregation (about 88% reduction in amyloid‐sensitive probe emission) inducing formation of fibrils with the length close to this of free insulin (1.7 ± 0.6 μm as compared with 1.4 ± 0.4 μm, respectively) with an essentially reduced tendency to lateral aggregation. Contrarily, the presence of tetraphenylporphyrin containing four amino groups only slightly affects fibrils' morphology and makes weaker impact on insulin aggregation yield (about 44% reduction). This is explained by the ability of aromatic carboxy groups of 5,10,15,20‐(tetra‐4‐carboxyphenyl)porphyrin to interact with complementary protein‐binding groups and thus stabilize the supramolecular complex. For 5,10,15,20‐(tetra‐4‐aminophenyl)porphyrin, full protonation takes place in acidic medium of protein aggregation reaction; this results in the high positive charge of TPPN4 (equal or close to +6) and hence higher contribution of coulombic repulsion to interaction of TPPN4 with insulin. One more possible mechanism of the lower inhibition effect of TPPN4 as compared with TPPC4 could be the more restricted possibility of the former as compared with the latter to form H bonds with insulin groups. It was also shown that metal‐free, Pd‐containing, and Cu‐containing tetraphenylporphyrins without peripheral substituents make almost the same impact on the protein self‐assembling. We suppose this to be due to coordination saturation of these metal atoms.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The relationship between the aggregational state of the amyloid-β peptides and free radical generation by the peptides

In the present study, we investigated whether or not the amyloid-beta protein (Abeta) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Abeta and Abeta-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of both Abeta40 and Abeta42 with Ntert-butyl-alpha-phenylnitrone (PBN), but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the Abeta/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. The present study also revealed that either Abeta40 or Abeta42 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. Both a thioflavine-T (Th-T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Abeta in PBS to be much higher than that of Abeta in PB. Moreover, Abeta-induced cytotoxicity assays showed Abeta incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Abeta-associated free radical generation is strongly influenced by the aggregational state of the peptides.     

Insights into the disparate action of osmolytes and macromolecular crowders on amyloid formation

It is widely recognized that amyloid formation sensitively responds to conditions set by myriad cellular solutes. These cosolutes include two important classes: macromolecular crowders and compatible osmolytes. We have recently found that addition of macromolecular PEG only slightly affects fibril formation of a model peptide in vitro. Polyol osmolytes, in contrast, lengthen the lag time for aggregation, and lead to larger fibril mass at equilibrium. To further hypothesize on the molecular underpinnings of the disparate effect of the two cosolute classes, we have further analyzed the experiments using an available kinetic mechanism describing fibril aggregation. Model calculations suggest that all cosolutes similarly lengthen the time required for nucleation, possibly due to their excluded volume effect. However, PEGs may in addition promote fibril fragmentation, leading to lag times that are overall almost unvaried. Moreover, polyols effectively slow the monomer-fibril detachment rates, thereby favoring additional fibril formation. Our analysis provides first hints that cosolutes act not only by changing association or dissociation rates, but potentially also by directing the formation of fibrils of varied morphologies with different mechanical properties. Although additional experiments are needed to unambiguously resolve the action of excluded cosolutes on amyloid formation, it is becoming clear that these compounds are important to consider in the search for ways to modulate fibril formation.     

Understanding and controlling amyloid aggregation with chirality

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.     

Molecular dynamics simulation of the SH3 domain aggregation suggests a generic amyloidogenesis mechanism

We use molecular dynamics simulation to study the aggregation of Src SH3 domain proteins. For the case of two proteins, we observe two possible aggregation conformations: the closed form dimer and the open aggregation state. The closed dimer is formed by "domain swapping"-the two proteins exchange their RT-loops. All the hydrophobic residues are buried inside the dimer so proteins cannot further aggregate into elongated amyloid fibrils. We find that the open structure-stabilized by backbone hydrogen bond interactions-packs the RT-loops together by swapping the two strands of the RT-loop. The packed RT-loops form a beta-sheet structure and expose the backbone to promote further aggregation. We also simulate more than two proteins, and find that the aggregate adopts a fibrillar double beta-sheet structure, which is formed by packing the RT-loops from different proteins. Our simulations are consistent with a possible generic amyloidogenesis scenario.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide: Abeta association kinetics at endosomal pH

The deposition of beta-amyloid peptide (Abeta) fibrils around neurons is an invariable feature of Alzheimer's disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Abeta generation is the endosomes, and we have investigated the kinetics of Abeta association at endosomal pH over physiologically relevant time frames. We have identified three distinct Abeta association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 microM Abeta, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Abeta intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain alpha-helical or beta-structure as determined by circular dichroism spectroscopy. However, after two weeks beta-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.     

Identification of β-aggregate sites in protein chains

The problem of amyloidoses is pressing and have recently attracted special attention throughout the world because of epidemics of prion diseases such as mad cow disease and human Creutzfeldt-Jacob disease. These diseases result from the conversion of a native protein or peptide into a highly stable pathological form. Molecules having a pathological conformation aggregate to form amyloid fibrils, capable of unlimited growth. It is important to study the molecular mechanisms of prion diseases and to identify the protein regions responsible for their development. The review considers theoretical and experimental works focusing on the formation of amyloid fibrils.     

The aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Abeta(1-40) peptide. The material is competent to dissolve in water solutions with "random-coil properties," although its conformation and factual oligomerization state are determined by the physico-chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.     

Ultrastructure of perivascular amyloid fibrils in Alzheimer’s disease

Perivascular amyloid fibrils in the brains of patients with Alzheimer's disease have been examined by electron microscopy. The amyloid fibrils showed a hollow rod structure and consisted of globular substances. Each turn appeared to be composed of five globular subunits. These findings coincide with the ultrastructure of amyloid fibrils obtained from replicas made by a rapid freezing method.