Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly a-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90–231 and 121–231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non- thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.Key words: misfolding, aggregation, amyloid, prion, conformational conversion, fluorescence     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.     

Mechanisms of protein fibril formation: nucleated polymerization with competing off-pathway aggregation

The formation of protein fibrils, and in particular amyloid fibrils, underlies many human diseases. Understanding fibril formation mechanisms is important for understanding disease pathology, but fibril formation kinetics can be complicated, making the relationship between experimental observables and specific mechanisms unclear. Here we examine one often-proposed fibril formation mechanism, nucleated polymerization with off-pathway aggregation. We use the characteristics of this mechanism to derive three tests that can be performed on experimental data to identify it. We also find that this mechanism has an especially striking feature: although increasing protein concentrations generally cause simple nucleated polymerizations to reach completion faster, they cause nucleated polymerizations with off-pathway aggregation to reach completion more slowly when the protein concentration becomes too high.     

Defining the pathway of worm-like amyloid fibril formation by the mouse prion protein by delineation of the productive and unproductive oligomerization reactions

Aggregation reactions of proteins leading to amyloid fibril formation are often characterized by early transient accumulation of a heterogeneous population of soluble oligomers differing in size and structure. Delineating the kinetic roles of the different oligomeric forms in fibril formation has been a major challenge. The aggregation of the mouse prion protein to form worm-like amyloid fibrils at low pH is known to proceed via a β-rich oligomer ensemble, which is shown here to be comprised of two subpopulations of oligomers that differ in size and internal structure. The relative populations of the two oligomers can be tuned by varying the concentration of NaCl present. By demonstrating that the apparent rate constant for the formation of fibrils is dependent linearly on the concentration of the larger oligomer and is independent of the concentration of the smaller oligomer, we show that the larger oligomer is a productive intermediate that accumulates on the direct pathway of aggregation from monomer to worm-like fibrils. The smaller oligomer is shown to be populated off the pathway of the larger oligomer and, hence, is not directly productive for fibril formation. The relative populations of the two oligomers can also be tuned by single-amino acid residue changes in the sequence of the protein. The different protein variants yield worm-like fibrils of different lengths, and the apparent rate of formation of the fibrils by the mutant variants is also shown to be dependent on the concentration of the larger but not of the smaller oligomer formed.     

Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.     

Dissociation from the oligomeric state is the rate-limiting step in fibril formation by kappa-casein

Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, kappa-casein, to form amyloid fibrils. Using reduced and carboxymethylated kappa-casein (RCMkappa-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and beta-sheet content of the protein. However, the lag phase of RCMkappa-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleation-dependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr(25) to Lys(86) is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of kappa-casein. Based on these data, we propose that fibril formation by RCMkappa-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.     

Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB.

The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unraveling the mysteries of protein folding and misfolding

This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for example, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.     

Interaction of the Molecular Chaperone DNAJB6 with Growing Amyloid-beta 42 (Aβ42) Aggregates Leads to Sub-stoichiometric Inhibition of Amyloid Formation

The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

The aggregation of proteins into amyloid fibrils is a topic that has attracted great interest because the process is associated with the pathology of numerous human diseases. Despite considerable progress in the elucidation of the structure of amyloid fibrils and the kinetic mechanism of their formation, knowledge on the thermodynamic aspects underlying the formation and stability of amyloid fibrils is limited. In this review, we summarize recent calorimetric studies of amyloid fibril formation, with the goal of obtaining a better understanding of the causal factors that thermally induce proteins to aggregate into amyloid fibrils. Calorimetric data show that differential scanning calorimetry is a useful technique to study the causative factors that thermally trigger the conversion to the amyloid structure and highlight the physics related to the thermal fluctuation of proteins during this conversion.     

Kinetically controlled thermal response of beta2-microglobulin amyloid fibrils

Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 degrees C for characterizing the thermal response of beta2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity-temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid beta peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern.     

Biophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein B.

The peptide CspB-1, which represents residues 1-22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water [M. Gross et al. (1999) Protein Science 8, 1350-1357] We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed. Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils. A CD spectrum indicative of beta structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases. The characteristic wavelength shift of the amyloid-binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform-infrared spectrum indicative of beta structure. Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well-defined amyloid fibrils are established on a timescale of hours. NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process. A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease-related systems. These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins.     

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization

The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.     

Template-assisted lateral growth of amyloid-β42 fibrils studied by differential labeling with gold nanoparticles

Amyloid-β protein (Aβ) aggregation into amyloid fibrils is central to the origin and development of Alzheimer's disease (AD), yet this highly complex process is poorly understood at the molecular level. Extensive studies have shown that Aβ fibril growth occurs through fibril elongation, whereby soluble molecules add to the fibril ends. Nevertheless, fibril morphology strongly depends on aggregation conditions. For example, at high ionic strength, Aβ fibrils laterally associate into bundles. To further study the mechanisms leading to fibril growth, we developed a single-fibril growth assay based on differential labeling of two Aβ42 variants with gold nanoparticles. We used this assay to study Aβ42 fibril growth under different conditions and observed that bundle formation is preceded by lateral interaction of soluble Aβ42 molecules with pre-existing fibrils. Based on this data, we propose template-assisted lateral fibril growth as an additional mechanism to elongation for Aβ42 fibril growth.     

Nucleation of Polymorphic Amyloid Fibrils

One and the same protein can self-assemble into amyloid fibrils with different morphologies. The phenomenon of fibril polymorphism is relevant biologically because different fibril polymorphs can have different toxicity, but there is no tool for predicting which polymorph forms and under what conditions. Here, we consider the nucleation of polymorphic amyloid fibrils occurring by direct polymerization of monomeric proteins into fibrils. We treat this process within the framework of our newly developed nonstandard nucleation theory, which allows prediction of the concentration dependence of the nucleation rate for different fibril polymorphs. The results highlight that the concentration dependence of the nucleation rate is closely linked with the protein solubility and a threshold monomer concentration below which fibril formation becomes biologically irrelevant. The relation between the nucleation rate, the fibril solubility, the threshold concentration, and the binding energies of the fibril building blocks within fibrils might prove a valuable tool for designing new experiments to control the formation of particular fibril polymorphs.     

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.