Cross-seeding of wild-type and hereditary variant-type amyloid beta-proteins in the presence of gangliosides

We investigated the molecular mechanism underlying the ganglioside-induced initiation of the assembly of wild and hereditary variant-type amyloid beta-proteins, including Arctic-, Dutch-, and Flemish-type amyloid beta-proteins. We monitored the assembly of amyloid beta-protein by thioflavin-T assay, western blotting and electron microscopy. We also examined how externally added amyloid beta-protein assembles in a cell culture. The assembly of wild-, Arctic-, Dutch-, and Flemish-type amyloid beta-proteins were accelerated in the presence of GM1, GM1, GM3 and GD3 gangliosides. Notably, all of these amyloid beta-proteins accelerated the assembly of different type of amyloid beta-protein, following prior binding to a specific ganglioside. A specific-ganglioside-bound form of variant-type amyloid beta-protein was recognized by the antibody (4396C) specific to the GM1-ganglioside-induced altered conformation of wild-type amyloid beta-protein. Moreover, the assembly of these amyloid beta-proteins in the presence of a specific ganglioside was markedly suppressed by coincubation with 4396C. This study suggests that cross-seeding can occur between wild and hereditary variant-type amyloid beta-proteins despite differences in their amino acid sequences.     

Accelerated Abeta aggregation in the presence of GM1-ganglioside-accumulated synaptosomes of aged apoE4-knock-in mouse brain

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4- than the apoE3-knock-in mouse brain. Furthermore, we show that Abeta aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.     

The Tottori (D7N) and English (H6R) familial Alzheimer disease mutations accelerate Abeta fibril formation without increasing protofibril formation

A subset of Alzheimer disease cases is caused by autosomal dominant mutations in genes encoding the amyloid beta-protein precursor or presenilins. Whereas some amyloid beta-protein precursor mutations alter its metabolism through effects on Abeta production, the pathogenic effects of those that alter amino acid residues within the Abeta sequence are not fully understood. Here we examined the biophysical effects of two recently described intra-Abeta mutations linked to early-onset familial Alzheimer disease, the D7N Tottori-Japanese and H6R English mutations. Although these mutations do not affect Abeta production, synthetic Abeta(1-42) peptides carrying D7N or H6R substitutions show enhanced fibril formation. In vitro analysis using Abeta(1-40)-based mutant peptides reveal that D7N or H6R mutations do not accelerate the nucleation phase but selectively promote the elongation phase of amyloid fibril formation. Notably, the levels of protofibrils generated from D7N or H6R Abeta were markedly inhibited despite enhanced fibril formation. These N-terminal Abeta mutations may accelerate amyloid fibril formation by a unique mechanism causing structural changes of Abeta peptides, specifically promoting the elongation process of amyloid fibrils without increasing metastable intermediates.     

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.     

Pathological significance of ganglioside clusters in Alzheimer's disease

One of the key questions regarding the pathogenesis of Alzheimer's disease (AD) is how amyloid β-protein (Aβ), a proteinaceous component of senile plaques, starts to assemble into amyloid fibrils in the brain. A body of evidence is growing to suggest that Aβ binds to ganglioside on neuronal membranes, and then, is converted to an endogenous seed with an altered conformation (ganglioside-bound Aβ, GAβ) for amyloid fibril formation in the brain. Notably, the risk factors for the development of AD, including aging and apolipoprotein E4, likely facilitate the formation of ganglioside clusters in lipid raft-like membrane microdomains at pre-synaptic terminals, which provide a favorable milieu for the GAβ generation. Furthermore, it has also been suggested that endocytic pathway abnormality of neurons is involved in the formation of the ganglioside clusters. In this review, the nature of the ganglioside clusters and how gangliosides behave in the clusters leading to the GAβ generation are discussed.     

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20 μM). Importantly, when the 5 μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.     

Role of the N and C-terminal strands of beta 2-microglobulin in amyloid formation at neutral pH

Beta 2-microglobulin (beta(2)m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu(2+). Based on these observations, and other structural data, models for fibril formation of beta(2)m have been proposed that involve the fraying of the N and C-terminal beta-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of beta(2)m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven beta-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of beta(2)m.     

Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1-Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formation by acting as a seed. We have previously found using dye-labeled Abeta that Abeta recognizes a GM1 cluster, the formation of which is facilitated by cholesterol [Kakio, A., Nishimoto, S., Yanagisawa, K., Kozutsumi, Y., and Matsuzaki, K. (2001) J. Biol. Chem. 276, 24985-24990]. In this study, we investigated the ganglioside species-specificity in its potency to induce a conformational change of Abeta, by which ganglioside-bound Abeta acts as a seed for Abeta fibrillogenesis, using a major ganglioside occurring in brains (GM1, GD1a, GD1b, and GT1b) in raft-like membranes composed of cholesterol and sphingomyelin. Abeta recognized ganglioside clusters, the density of which increased with the number of sialic acid residues. Interestingly, however, mixing of gangliosides inhibited cluster formation. In contrast, the affinities of the protein for the clusters were similar irrespective of lipid composition and of the order of 10(6) M(-)(1) at 37 degrees C. Abeta underwent a conformational transition from an alpha-helix-rich structure to a beta-sheet-rich structure with the increase in protein density on the membrane. Ganglioside-bound Abeta proteins exhibited seeding abilities for amyloid formation. GM1-Abeta exhibited the strongest seeding potential, especially under beta-sheet-forming conditions. This study suggested that lipid composition including gangliosides and cholesterol strictly controls amyloid formation.     

Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes

The fully developed lesion of Alzheimer's Disease is a dense plaque composed of fibrillar amyloid beta-proteins with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet secondary structure, it is important to understand factors that induce amyloid beta-proteins to adopt this conformation. In this investigation we used a novel form of infrared spectroscopy that can characterize the conformation, orientation, and rate of accumulation of the protein on various lipid membranes to determine whether oxidatively damaged phospholipid membranes induce the formation of beta-sheet secondary structure in a 42-residue amyloid beta-protein. We found that membranes containing oxidatively damaged phospholipids accumulated amyloid beta-protein significantly faster than membranes containing only unoxidized or saturated phospholipids. Accelerated accumulation was also seen when 3 mol % G(M1) ganglioside was incorporated into a saturated phosphatidylcholine membrane. The accumulated protein more completely adopted a beta-sheet conformation on oxidized membranes, and the plane of the beta-sheet was oriented parallel to the plane of the membrane. These results indicate that oxidatively damaged phospholipid membranes promote beta-sheet formation by amyloid beta-proteins, and they suggest a possible role for lipid peroxidation in the pathogenesis of Alzheimer's Disease.     

The GB1 amyloid fibril: recruitment of the peripheral beta-strands of the domain swapped dimer into the polymeric interface

Three-dimensional domain swapping has been evoked as a mechanism for oligomerization of proteins. Here, we show for the immunoglobulin-binding domain B1 of streptococcal protein G (GB1) that fibril formation is observed readily for variants that exist as domain-swapped dimers. No fibril was formed by a revertant that exhibits the stable wild-type GB1 fold or a mutant comprising a highly destabilized, fluctuating ensemble of conformers. Structural features of the GB1 amyloid fibril were characterized by cysteine disulfide cross-linking. Residues in the outer edge beta-strands of the domain-swapped dimer readily form intermolecular disulfide bonds prior to and during fibril formation. On the basis of these data, a structural model for the assembly of domain-swapped dimers into a polymeric structure of the GB1 fibril is proposed.     

Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates.

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (Abeta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Abeta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Abeta fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Abeta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Amyloid fibril formation by human stefins: Structure,mechanism & putative functions

Many questions in the field of protein aggregation to amyloid fibrils remain open. In this review we describe predominantly in vitro studies of oligomerization and amyloid fibril formation by human stefins A and B. In human stefin B amyloidogenesis in vitro we have observed some general and many specific properties of its prefibrillar oligomers and amyloid fibrils. One characteristic feature in common to stefins and cystatins (and possibly some other amyloid proteins) is domain-swapping. In addition to solution structure of the domain-swapped dimer of stefin A, we recently have determined 3D structure of stefin B tetramer, which proved to be composed from two domain-swapped dimers, whose interaction occurs by a proline switch in the loop surrounding the conserved Pro 74. Studying the mechanism of fibril formation by stefin B, we found that the nucleation and fibril elongation reactions have energies of activation (E_a’s) in the range of proline isomerisation, strongly indicating importance of the Pro at site 74 and/or other prolines in the sequence. Correlation between toxicity of the prefibrillar oligomers and their interaction with acidic phospholipids was demonstrated. Stefin B was shown to interact with amyloid-beta peptide of Alzheimer’s disease in an oligomer specific manner, both in vitro and in the cells. It also has been shown that endogenous stefin B (with E at site 31) but especially the EPM1 mutant R68X and Y31-stefin B variant, and to a lesser extent EPM1 mutant G4R, are prone to form aggregates in cells.     

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.     

Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture.

Deposition of aggregated amyloid beta-protein (Abeta), a proteolytic cleavage product of the amyloid precursor protein (Abeta ), is a critical step in the development of Alzheimer's disease(Abeta++). However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo. Here, we report that a seeding Abeta, which catalyzes the fibrillogenesis of soluble Abeta, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.     

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease

The assembly of amyloid β-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid β-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid β-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

The effects of organic solvents on the membrane-induced fibrillation of human islet amyloid polypeptide and on the inhibition of the fibrillation

The organic solvent dimethylsulphoxide (DMSO) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) have been widely used as a pre-treating agent of amyloid peptides and as a vehicle for water-insoluble inhibitors. These solvents are left in many cases as a trace quantity in bulk and membrane environments with treated amyloid peptides or inhibitors. In the present work, we studied the effects of the two organic solvents on the aggregation behaviors of human islet amyloid polypeptide (hIAPP) and the performances of an all-D-amino-acid inhibitor D-NFGAIL in preventing hIAPP fibrillation both in bulk solution and at phospholipid membrane. We showed that the presence of 1% v/v DMSO or HFIP decreases the rate of fibril formation of hIAPP at the lipid membrane rather than accelerates the fibril formation as what happened in bulk solution. We also showed that the presence of 1% v/v DMSO or HFIP impairs the activity of the inhibitor at the lipid membrane surface dramatically, while it affects the efficiency of the inhibitor in bulk solution slightly. We found that the inhibitor inserts into the lipid membrane more deeply or with more proportion in the presence of the organic solvents than it does in the absence of the organic solvents, which may hinder the binding of the inhibitor to hIAPP at the lipid membrane. Our results suggest that the organic solvents should be used with caution in studying membrane-induced fibrillogenesis of amyloid peptides and in testing amyloid inhibitors under membrane environments to avoid incorrect evaluation to the fibrillation process of amyloid peptides and the activity of inhibitors.     

A toy model for predicting the rate of amyloid formation from unfolded protein

We develop a toy model for predicting the rate of amyloid formation from an unfolded polypeptide. The model assumes irreversible amyloid growth, employs a collision encounter scheme and uses a Gaussian chain approximation to describe the polypeptide sequence. A principal feature of the model is its dependence on a number of key sequence residues whose correct placement, geometric arrangement and orientation in relation to their interacting partners define the success, or otherwise, of the amyloid formation reaction. Although not realistic at the molecular level, the model captures some essential features of the system and is therefore useful from a heuristic standpoint. For the case of amyloid formation from an unstructured state, the model suggests that the major determinants of the rate of fibril formation are the length of the sequence separating the critical amino acids promoting amyloid formation and the positional placement of the critical residues within the sequence. Our findings suggest also that the sequence distance between the key interacting amino acid residues may play a role in defining the maximum width of a fibril and that the addition of non-interacting segments of long structure-less polypeptide chain to an amyloidogenic peptide may act to inhibit fibril formation. We discuss these findings with reference to the placement of critical sequence residues within the polypeptide chain, the design of polypeptides with lower amyloid formation propensities and the development of aggregation inhibitors as potential therapeutics for protein depositional disorders.