Structural transitions and oligomerization along polyalanine fibril formation pathways from computer simulations

The results of a computer simulation study of the aggregation kinetics of a large system of model peptides with particular focus on the formation of intermediates are presented. Discontinuous molecular dynamic simulations were used in combination with our intermediate-resolution protein model, PRIME, to simulate the aggregation of a system of 192 polyalanine (KA(14) K) peptides at a concentration of 5 mM and a reduced temperature of T* = 0.13 starting from a random configuration and ending in the assembly of a fibrillar structure. The population of various structures, including free monomers, beta sheets, amorphous aggregates, hybrid aggregates, and fibrils, and the transitions between the structures were tracked over the course of 30 independent simulations and averaged together. The aggregation pathway for this system starts with the association of free monomers into small amorphous aggregates that then grow to moderate size by incorporating other free monomers or merging with other small amorphous aggregates. These then rearrange into either small beta sheets or hybrid aggregates formed by association between unstructured chains and beta sheets, both of which grow in size by adding free monomer chains or other small aggregates, one at a time. Fibrillar structures are formed initially either by the stacking of beta sheets, rearrangement of hybrid aggregates or association between beta sheets and hybrid aggregates. They grow by the addition of beta sheets, hybrid aggregates, and other small fibrillar structures. The rearrangement of amorphous aggregates into beta sheets is a critical and necessary step in the fibril formation pathway.     

pH-dependent self-assembly of polyalanine peptides

Polyalanine expansions in the nuclear RNA-binding protein PABP2 induce misfolding and aggregation of the protein into insoluble inclusions in muscle tissues and cell nuclei, leading to the disease oculopharyngeal muscular dystrophy (OPMD). We have explored the effect of solvent conditions and alanine repeat number on the propensity of fibril formation in this protein deposition disease. Three peptides mimicking the N-terminal polyalanine segment of PABP2, having the generic sequence Ac-Lys-Met-(Ala)(n)-Gly-Tyr with n = 7, 11, and 17 (referred to as 7-ala, 11-ala, and 17-ala, respectively), were synthesized and their conformational properties studied as a function of pH. In strongly alkaline medium (pH >10), the two longer peptides (11-ala and 17-ala, but not 7-ala) showed remarkable enhancement of beta-sheet content and formed fibrils after incubation for 1-2 weeks at room temperature. Fluorescence studies suggested that tyrosyl radicals produced at high pH cross-linked to form dityrosine, which provided added stabilization for fibril growth. The kinetic progress curves for fibril formation, obtained by ThT fluorescence assay, showed exponential increase with time after an initial quiescent period (lag time) and an eventual saturation phase, all of which are indicative of a nucleation-controlled polymerization mechanism for fibrillation. Hierarchical self-assembly of the peptides led to the formation of striking fractal-shaped growth patterns on substrates, raising the possibility of designing novel materials using these peptides.     

Computer simulation study of amyloid fibril formation by palindromic sequences in prion peptides

We simulate the aggregation of large systems containing palindromic peptides from the Syrian hamster prion protein SHaPrP 113-120 (AGAAAAGA) and the mouse prion protein MoPrP 111-120 (VAGAAAAGAV) and eight sequence variations: GAAAAAAG, (AG)(4) , A8, GAAAGAAA, A10, V10, GAVAAAAVAG, and VAVAAAAVAV The first two peptides are thought to act as the Velcro that holds the parent prion proteins together in amyloid structures and can form fibrils themselves. Kinetic events along the fibrillization pathway influence the types of structures that occur and variations in the sequence affect aggregation kinetics and fibrillar structure. Discontinuous molecular dynamics simulations using the PRIME20 force field are performed on systems containing 48 peptides starting from a random coil configuration. Depending on the sequence, fibrillar structures form spontaneously over a range of temperatures, below which amorphous aggregates form and above which no aggregation occurs. AGAAAAGA forms well organized fibrillar structures whereas VAGAAAAGAV forms less well organized structures that are partially fibrillar and partially amorphous. The degree of order in the fibrillar structure stems in part from the types of kinetic events leading up to its formation, with AGAAAAGA forming less amorphous structures early in the simulation than VAGAAAAGAV. The ability to form fibrils increases as the chain length and the length of the stretch of hydrophobic residues increase. However as the hydrophobicity of the sequence increases, the ability to form well-ordered structures decreases. Thus, longer hydrophobic sequences form slightly disordered aggregates that are partially fibrillar and partially amorphous. Subtle changes in sequence result in slightly different fibril structures.     

Amyloid fibril formation and chaperone-like activity of peptides from alphaA-crystallin

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we found that these peptides, alphaAC(70-88), the peptide corresponding to amino acids 70-88 of alphaAC (KFVIFLDVKHFSPEDLTVK), and alphaAC(71-88), suppressed the amyloid fibril formation of amyloid beta protein (Abeta). On the other hand, while alphaAC(71-88) exhibited chaperone-like activity toward insulin, alphaAC(70-88) and alphaAC(70-88)K70D promoted rapid growth of aggregates consisting of insulin and these peptides in their solution mixtures. Interestingly, we found that alphaAC(71-88) itself can also form amyloid fibrils. It is possible that the chaperone-like activity of the alphaAC peptides is potentially related to their propensity for amyloid fibril formation. Analysis of variants of the alphaAC peptides suggested that F71 is important for amyloid formation, and interestingly, this same residue has previously been found to be essential for chaperone-like activity. Amyloid fibril formation was also observed with the shorter peptide, alphaAC(70-76)K70D, showing that the ability to form amyloid fibrils is maintained even with significant deletion of the C-terminal sequence. The formation of amyloid fibril was suppressed in alphaAC(70-88), suggesting that the K70 in the substrate binding site may play a role in suppressing the amyloid fibril formation of alphaAC, which agreed with recent proposals about the presence of an aggregation suppressor in the region flanking aggregation-prone hydrophobic sequences.     

Hierarchy and the mechanism of fibril formation in ADan peptides

Familial Danish dementia is a neurodegenerative disease which is a consequence of alterations in the BRI gene. The pathological signatures of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. Synthetic Danish dementia (ADan) peptides are capable of forming fibrillar assemblies in vitro at pH 4.8. However, the morphology of the aggregates formed depends greatly on the form of the peptides (oxidized or reduced). In addition to long slender assemblies (2-5 nm in diameter and several micrometers in length) we report ring-like or annular masses (8-9 nm in diameter and 1-2 mm in perimeter) in the case of the oxidized form of the peptides. The reduced forms mainly aggregate to produce granular heaps. The biophysical and kinetic characterization of the process of aggregation was carried out using different spectroscopic and imaging techniques. Neurotoxicity assays performed on both the forms reveal that the toxicity bears proportionality with the aggregate size.     

Amyloid-beta fibril formation is not necessarily required for microglial activation by the peptides

There is increasing evidence that microglial activation has pathogenic influence on Alzheimer's disease. According to in vitro studies, microglia activated by amyloid-beta (Abeta) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, nitric oxide or reactive oxygen species. Although the relationship between the aggregational state of Abeta peptides and their neurotoxic activities has been well investigated, little is known about the relationship between the aggregational state of Abeta peptides and their ability to induce microglial activation. In the present study, we thus performed both structural and biochemical studies to clarify the relationship between the aggregational state of Abeta peptides and their ability to activate microglia. Our results have shown that, in the presence of interferon-gamma, the Abeta25-35(M(35)Nle) peptide had almost the same potency of activating microglia and producing TNF-alpha as the Abeta25-35 peptide on both protein and mRNA levels, in spite of the fact that former peptide represented much less amyloid fibril formation than the latter in a thioflavine-T fluorometric assay. These results suggest that Abeta fibril formation is not necessarily required for microglial activation by the peptides.     

Length-dependent aggregation of uninterrupted polyalanine peptides

Polyalanine (polyA) is the third-most prevalent homopeptide repeat in eukaryotes, behind polyglutamine and polyasparagine. Abnormal expansion of the polyA repeat is linked to at least nine human diseases, and the disease mechanism likely involves enhanced length-dependent aggregation. Because of the simplicity of its side chain, polyA has been a favorite target of computational studies, and because of their tendency to fold into α-helix, peptides containing polyA-rich domains have been a popular experimental subject. However, experimental studies on uninterrupted polyA are very limited. We synthesized polyA peptides containing uninterrupted sequences of 7 to 25 alanines (A7 to A25) and characterized their length-dependent conformation and aggregation properties. The peptides were primarily disordered, with a modest component of α-helix that increased with increasing length. From measurements of mean distance spanned by the polyA segment, we concluded that physiological buffers are neutral solvents for shorter polyA peptides and poor solvents for longer peptides. At moderate concentration and near-physiological temperature, polyA assembled into soluble oligomers, with a sharp transition in oligomer physical properties between A19 and A25. With A19, oligomers were large, contained only a small fraction of the total peptide mass, and slowly grew into loose clusters, while A25 rapidly and completely assembled into small stable oligomers of ~7 nm radius. At high temperatures, A19 assembled into fibrils, but A25 precipitated as dense, micrometer-sized particles. A comparison of these results to those obtained with polyglutamine peptides of similar design sheds light on the role of the side chain in regulating conformation and aggregation.     

Inhibition of amyloid fibril formation of beta-amyloid peptides via the amphiphilic surfactants

Beta-amyloid peptide (A beta) is the major proteinacious constituent of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration process associated with the disease. While the actual size of the aggregated species responsible for A beta neurotoxicity and fibrillogenesis mechanism(s) remain unknown, retardation of A beta aggregation still holds assurance as an effective strategy in reducing A beta-elicited toxicity. The research presented here is aimed at examining the inhibitory effect of two amphiphilic surfactants, di-C6-PC and di-C7-PC, on the in vitro fibrillogenesis process of A beta(1--40) peptides at physiological pH (pH 7.2). Using ThT-induced fluorescence, turbidity, Congo red binding, and circular dichroism spectroscopy studies, our research demonstrated that the inhibition of A beta(1--40) fibril formation was di-C6-PC and di-C7-PC concentration-dependent. The best inhibitory action on fibril formation was observed when A beta was incubated with di-C7-PC at 100 microM over time. We believe that the outcome from this work will aid in the development and/or design of potential inhibitory agents against amyloid formation associated with Alzheimer's and other amyloid diseases.     

Caspase 8 mediated apoptotic cell death induced by beta-sheet forming polyalanine peptides

Expansion of a polyalanine stretch from 10 to 12-17 residues in the N-terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N-terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11-ala) was found to adopt beta-sheet structure while the shorter one (7-ala) formed alpha-helix over a wide range of concentrations ( approximately 20-500 microM). We observed that treatment with 11-ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7-ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11-ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their beta-sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.     

Studying the structural properties of polyalanine and polyglutamine peptides

Poly-(Ala) and poly-(Gln) peptides have important biological effects, and can cause various human illnesses and neurodegenerative diseases. Conformational analysis of these homo-oligopeptides (HOPs) was carried out by simulated annealing in order to identify their structural properties regarding secondary structures and intramolecular H-bonding patterns. Poly-(Ala) and poly-(Gln) peptides composed of 7, 10, 14 or 20 amino acids were modelled in both charged and terminally blocked forms. In the case of conformers derived from simulated annealing calculations, the presence of various secondary structural elements (different types of β-turns, α-helix, 3₁₀-helix, poly-proline II helix, parallel and antiparallel β-strands) was investigated. Moreover, the intramolecular H-bonding patterns formed either between the backbone atoms for both HOPs or between the backbone and side-chain atoms for the poly-(Gln) peptides were examined. Our results showed that different secondary structural elements (type I and type III β-turns, α-helix, 3₁₀-helix, antiparallel β-strand) could be observed in both poly-(Ala) and poly-(Gln) peptides and, according to their presence, characteristic H-bonding patterns formed mainly by i←i+3 and i←i+4 H-bonds could be found.     

Controlling amyloid fibril formation by partial stirring

Many proteins undergoe self‐assembly into fibrillar structures known as amyloid fibrils. During the self‐assembly process, related structures known as spherulites can be formed. Herein we report a facile method where the balance between amyloid fibrils and spherulites can be controlled by stirring of the reaction mixture during the initial stages of the self‐assembly process. Moreover, we report how this methodology can be used to prepare non‐covalently functionalized amyloid fibrils. By stirring the reaction mixture continuously or for a limited time during the lag phase, the fibril length, and hence the propensity to form liquid crystalline phases, can be influenced. This phenomena is utilized in order to prepare films consisting of aligned protein fibrils incorporating the laser dye Nile red. The resulting films display polarized Nile red fluorescence. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 249–259, 2016.     

Amyloid fibril formation by a helical cytochrome

The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c(552) from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly alpha-helical protein can form beta-sheet structures of the type most commonly associated with protein deposition diseases.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Surface-catalyzed amyloid fibril formation

Light chain (or AL) amyloidosis is characterized by the pathological deposition of insoluble fibrils of immunoglobulin light chain fragments in various tissues, walls of blood vessels, and basement membranes. In the present investigation, the in vitro assembly of a recombinant amyloidogenic light chain variable domain, SMA, on various surfaces was monitored using atomic force microscopy. SMA formed fibrils on native mica at pH 5.0, conditions under which predominantly amorphous aggregates form in solution. Fibril formation was accelerated significantly on surfaces compared with solution; for example, fibrils grew on surfaces at significantly faster rates and at much lower concentrations than in solution. No fibrils were observed on hydrophobic or positively charged surfaces or at pH >7.0. Two novel types of fibril growth were observed on the surface: bidirectional linear assembly of oligomeric units, and linear growth from preformed amorphous cores. In addition to catalyzing the rate of fibrillation, the mechanism of fibril formation on the surfaces was significantly different from in solution, but it may be more physiologically relevant because in vivo the deposits are associated with surfaces.     

Amyloid fibril formation by pulmonary surfactant protein C

Lung surfactant protein C (SP-C) is a lipopeptide that contains two fatty acyl (palmitoyl) chains bound via intrinsically labile thioester bonds. SP-C can transform from a monomeric alpha-helix into beta-sheet aggregates, reminiscent of structural changes that are supposed to occur in amyloid fibril formation. SP-C is here shown to form amyloid upon incubation in solution. Furthermore, one patient with pulmonary alveolar proteinosis (PAP, a rare disease where lung surfactant proteins and lipids accumulate in the airspaces) and six healthy controls have been studied regarding presence and composition of amyloid fibrils in the cell-free fraction of bronchoalveolar lavage (BAL) fluid. Abundant amyloid fibrils were found in BAL fluid from the patient with PAP and, in low amounts, in three of the six healthy controls. SDS-insoluble fibrillar material associated with PAP mainly consists of SP-C, in contrast to the fibrils found in controls. Fibrillated SP-C has to a significant extent lost the palmitoyl groups, and removal of the palmitoyl groups in vitro increases the rate of fibril formation.     

Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.