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The β-sheet-rich amyloid core is the defining feature of protein aggregates associated with neurodegenerative disorders. Recent investigations have revealed that there exist multiple examples of the same protein, with the same sequence, forming a variety of amyloid cores with distinct structural characteristics. These structural variants, termed as polymorphs, are hypothesized to influence the pathological profile and the progression of different neurodegenerative diseases, giving rise to unique phenotypic differences. Thus, identifying the origin and properties of these structural variants remain a focus of studies, as a preliminary step in the development of therapeutic strategies. Here, we review the potential role of the flanking regions of amyloid cores in inducing polymorphism. These regions, adjacent to the amyloid cores, show a preponderance for being structurally disordered, imbuing them with functional promiscuity. The dynamic nature of the flanking regions can then manifest in the form of conformational polymorphism of the aggregates. We take a closer look at the sequences flanking the amyloid cores, followed by a review of the polymorphic aggregates of the well-characterized proteins amyloid-β, α-synuclein, Tau, and TDP-43. We also consider different factors that can potentially influence aggregate structure and how these regions can be viewed as novel targets for therapeutic strategies by utilizing their unique structural properties., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro , in vivo , and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

The culmination of many years of increasing research into the toxicity of tau aggregation in neurodegenerative disease has led to the consensus that soluble, oligomeric forms of tau are likely the most toxic entities in disease. While tauopathies overlap in the presence of tau pathology, each disease has a unique combination of symptoms and pathological features; however, most study into tau has grouped tau oligomers and studied them as a homogenous population. Established evidence from the prion field combined with the most recent tau and amyloidogenic protein research suggests that tau is a prion-like protein, capable of seeding the spread of pathology throughout the brain. Thus, it is likely that tau may also form prion-like strains or diverse conformational structures that may differ by disease and underlie some of the differences in symptoms and pathology in neurodegenerative tauopathies. The development of techniques and new technology for the detection of tau oligomeric strains may, therefore, lead to more efficacious diagnostic and treatment strategies for neurodegenerative disease. [Formula: see text]., Competing Interests: Declaration of Interest Statement Rakez Kayed has patent applications on the compositions and methods related to tau oligomers and antibodies. No competing financial interests exist for other authors.

The tumor suppressor p53 plays an important role in genome integrity. It is frequently mutated in all types of human cancers, making p53 a key factor in cancer progression. Two phenotypic consequences of these alterations are dominant; a loss of function and a gain of function of p53, which, in several cases, accumulates in intracellular aggregates. Although the nature of such aggregates is still unclear, recent evidence indicates that p53 can undergo conformational transitions leading to amyloid formation. Amyloid diseases, such as, Alzheimer's disease, are characterized by the accumulation of insoluble aggregates displaying the fibrillar conformation. We decided to investigate the propensity of wild type p53 to aggregate and its consequent assembly into different amyloid species, such as oligomers and fibrils; and to determine if these changes in conformation lead to a loss of function of p53. Furthermore, we analyzed cases of Basal Cell Carcinoma (BCC), for the presence of p53 amyloids. Here, we show that p53 forms amyloid oligomers and fibrils, which coincide with p53 inability of binding to DNA consensus sequences. Both p53 amyloid oligomers and fibrils were detected in BCC cancer samples. Additionally, we demonstrate that p53 oligomers are the most cytotoxic to human cell cultures. Our study reveals p53 amyloid formation and demonstrates its dual role in the pathogenesis of cancer by producing a loss of protein function and a gain of toxic function, extensively described in several amyloidogenic diseases. Our results suggest that under certain circumstances, cancer could be considered a protein-conformation disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Amyloid oligomers have emerged as the most toxic species of amyloid-β (Aβ). This hypothesis might explain the lack of correlation between amyloid plaques and memory impairment or cellular dysfunction. However, despite the numerous published research articles supporting the critical role Aβ oligomers in synaptic dysfunction and cell death, the exact definition and mechanism of amyloid oligomers formation and toxicity still elusive. Here we review the evidence supporting the many molecular mechanisms proposed for amyloid oligomers toxicity and suggest that the complexity and dynamic nature of amyloid oligomers may be responsible for the discrepancy among these mechanisms and the proposed cellular targets for amyloid oligomers.

Background: Primary cutaneous nodular amyloidosis (PCNA) is thought to be a plasma cell dyscrasia. The amyloid deposits are found in the dermis and subcutis, and they contain clonal immunoglobulin light chains, produced by a local proliferation of plasma cells. New insights into amyloid diseases have revealed that the pathology is due more to the presence of small, misfolded protein species termed oligomers than to the deposition of fibrillar material., Objectives: To demonstrate the presence of amyloid oligomers in PCNA and to provide evidence that cutaneous amyloid diseases share a common pathogenic pathway similar to other amyloid diseases., Methods: Immunohistochemical staining with conformation-specific and sequence-specific antibodies was used to localize different amyloid species of light chain immunoglobulins in a case of PCNA. Additionally, in vitro characterization of immunoglobulin oligomers and fibrils was performed to determine, through toxicity studies in a human keratinocyte cell line, which amyloidogenic form of the immunoglobulin is toxic in PCNA., Results: Amyloid oligomers were identified in PCNA. Oligomers were mainly formed by lambda light chain immunoglobulins, and kappa light chain oligomers were detected in lesser amounts. Amyloid species were detected intra- and extracellularly. In addition, amyloid oligomers and fibrils, derived from unknown protein sources, were detected. This finding suggests that immunoglobulin amyloids can act as seeds capable of inducing the aggregation of heterogeneous proteins in the skin. Furthermore, cytotoxicity studies demonstrated that immunoglobulin oligomers, but not monomers or fibrils, are toxic to human keratinocytes., Conclusions: These data indicate that PCNA has common pathways with other amyloid diseases with respect to protein misfolding and pathogenesis. Immunoglobulin oligomers may prove to be targets for the treatment of PCNA., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.)

Annular protofibrils (APFs) represent a newly described and distinct class of amyloid structures formed by disease-associated proteins. In vitro, these pore-like structures have been implicated in membrane permeabilization and ion homeostasis via pore formation. Still, their formation and relevance in vivo are poorly understood. Herein, we report that APFs are in human Alzheimer's disease brain samples and that amyloid-β APFs were associated with activated astrocytes. Moreover, we show that amyloid-β APFs in astrocytes adopt a conformation in which the N-terminal region is buried inside the wall of the pore. Our results together with previous studies suggest that the formation of amyloid-β APFs in astrocytes could be a relevant event in the pathogenesis of Alzheimer's disease and validate this amyloidogenic structure as a target for the prevention of the disease., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In the skin, amyloidosis can be found with or without systemic disease. Primary cutaneous amyloidosis defines those amyloidoses restricted to the skin without involvement of other systems. Here, we used conformation-specific antibodies to characterise both fibrillar and oligomeric amyloid aggregates in the skin from patients with cutaneous amyloidosis. Localised cutaneous amyloidosis with different morphology was reproduced in mice by intra-dermal (i.d.) and subdermal administration of amyloid-enhancing factor. Moreover, we demonstrated that conformational antibodies were effective in clearing amyloid deposits caused by localised intra-lesional injections without the necessity of an immune response. Given the accessibility and amyloid localization in this disease, direct i.d. injections of conformational antibodies could be a convenient and direct method for treatment., (© 2010 John Wiley & Sons A/S.)

According to the amyloid hypothesis, the accumulation of oligomerized amyloid-β (Aβ) is a primary event in the pathogenesis of Alzheimer's disease (AD). The trigger of the amyloid cascade and of Aβ oligomerization in sporadic AD, the most prevalent form of the disease, remains elusive. Here, we examined the hypothesis that apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for AD, triggers the accumulation of intraneuronal oligomerized Aβ following activation of the amyloid cascade. We investigated the intracellular organelles that are targeted by these processes and govern their pathological consequences. This revealed that activation of the amyloid cascade in vivo by inhibition of the Aβ degrading enzyme neprilysin specifically results in accumulation of Aβ and oligomerized Aβ and of ApoE4 in the CA1 neurons of ApoE4 mice. This was accompanied by lysosomal and mitochondrial pathology and the co-localization of Aβ, oligomerized Aβ, and ApoE4 with enlarged lysosomes and of Aβ and oligomerized Aβ with mitochondria. The time course of the lysosomal effects paralleled that of the loss of CA1 neurons, whereas the mitochondrial effects reached an earlier plateau. These findings suggest that ApoE4 potentiates the pathological effects of Aβ and the amyloid cascade by triggering the oligomerization of Aβ, which in turn, impairs intraneuronal mitochondria and lysosomes and drives neurodegeneration.

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (alphaAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in alphaAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric alpha-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a beta barrel structural motif.

Background: The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon--prostate tissue remodelling in middle-aged and elderly men., Methodology/principal Findings: By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions., Conclusions/significance: These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.

Objective: Islets in type 2 diabetes are characterized by a deficit in beta-cells, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). The toxic form of amyloidogenic protein oligomers are distinct and smaller than amyloid fibrils and act by disrupting membranes. Using antibodies that bind to toxic IAPP oligomers (but not IAPP monomers or fibrils) and a vaccination-based approach, we sought to establish whether IAPP toxic oligomers form intra- or extracellularly and whether vaccination to induce anti-toxic oligomer antibodies prevents IAPP-induced apoptosis in human IAPP (h-IAPP) transgenic mice., Research Design and Methods: Pancreas was sampled from two h-IAPP transgenic mouse models and examined by immunohistochemistry for toxic oligomers. The same murine models were vaccinated with toxic oligomers of Alzheimer beta protein (AbetaP(1-40)) and anti-oligomer titers, and blood glucose and islet pathology were monitored., Results: Toxic oligomers were detected intracellularly in approximately 20-40% of h-IAPP transgenic beta-cells. Vaccine induced high titers of anti-h-IAPP toxic oligomers in both transgenic models, but beta-cell apoptosis was, if anything, further increased in vaccinated mice, so that neither loss of beta-cell mass nor diabetes onset was delayed., Conclusions: IAPP toxic oligomers form in h-IAPP transgenic mouse models, and anti-toxic oligomer antibodies do not prevent h-IAPP-induced beta-cell apoptosis. These data suggest that prevention of h-IAPP oligomer formation may be more useful than a vaccination-based approach in the prevention of type 2 diabetes.

Alzheimer disease is characterized by the abnormal aggregation of amyloid beta peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid beta oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid beta aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid beta monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.

The R120G mutation in the small heat shock-like protein alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM), which is characterized by the formation of desmin- and CryAB-containing aggregates within muscle fibers. Mice with cardiac-specific overexpression of CryAB(R120G) develop cardiomyopathy at 3 months and die at 6-7 months from heart failure (HF). Previous studies showed that overexpression of CryAB(R120G) results in accumulation of preamyloid oligomer (PAO). PAO is considered to be the cytotoxic entity in many of the protein misfolding-based neurodegenerative diseases. On the basis of data from mouse models of neurodegenerative diseases showing that exercise or environmental enrichment reduces the amyloid oligomer level and improves cognitive ability, we hypothesized that CryAB(R120G)-induced DRM would also respond favorably to prolonged voluntary exercise, reducing HF symptoms and rescuing the mice from premature death. Six months of voluntary exercise in CryAB(R120G) animals resulted in 100% survival at a time when all unexercised mice had died. After 22 weeks of exercise, PAO levels were decreased by 47% compared with the unexercised CryAB(R120G) control mice (P = 0.00001). Although CryAB(R120G) expression led to decreased levels of the metallomembrane endopeptidase neprilysin, normal levels were maintained in the exercised CryAB(R120G) mice, and in vitro loss-of-function and gain-of-function experiments using adenovirus-infected cardiomyocytes confirmed the importance of neprilysin in ameliorating PAO accumulation. The data demonstrate that voluntary exercise slows the progression to HF in the CryAB(R120G) DRM model and that PAO accumulation is mediated, at least in part, by decreased neprilysin activity.

Protein misfolding and aggregation are thought to underlie the pathogenesis of many amyloid diseases, such as Alzheimer and Parkinson diseases, whereby a stepwise protein misfolding process begins with the conversion of soluble protein monomers to prefibrillar oligomers and progresses to the formation of insoluble amyloid fibrils. Drusen are extracellular deposits found in aging eyes and in eyes afflicted with age-related macular degeneration (AMD). Recent characterizations of drusen have revealed protein components that are shared with amyloid deposits. However, characteristic amyloid fibrils have thus far not been identified in drusen. In this study, we tested the hypothesis that nonfibrillar oligomers may be a common link in amyloid diseases. Oligomers consisting of distinct amyloidogenic proteins and peptides can be detected by a recently developed antibody that is thought to recognize a common structure. Notably, oligomers exhibit cellular toxicity, which suggests that they play a role in the pathogenesis of neurodegenerative diseases. Through use of the anti-oligomer antibody, we came to observe the presence of nonfibrillar, toxic oligomers in drusen. Conversely, no reactivity was observed in age-matched control eyes without drusen. These results suggest that amyloid oligomers may be involved in drusen biogenesis and that similar protein misfolding processes may occur in AMD and amyloid diseases.

Recent findings indicate that soluble amyloid oligomers may represent the primary pathologic species in degenerative diseases. These amyloid oligomers share common structural features and the ability to permeabilize membranes, suggesting that they also share a common primary mechanism of pathogenesis. Membrane permeabilization by amyloid oligomers may initiate a common group of downstream pathologic processes, including intracellular calcium dyshomeostasis, production of reactive oxygen species, altered signaling pathways, and mitochondrial dysfunction that represent key effectors of cellular dysfunction and cell death in amyloid-associated degenerative disease, such as sporadic inclusion-body myositis.

Although abundant evidence suggests that amyloid accumulation plays a significant role in the pathogenesis of degenerative disease, the mechanism of amyloid formation and toxicity remains elusive. Early hypotheses for disease pathogenesis proposed that large amyloid deposits, which are composed primarily of 6-10-nm mature amyloid fibrils, were the primary causative agent in pathogenesis, but this hypothesis required modification to consider the central role of oligomers or aggregation intermediates, because the accumulation of these large aggregates does not correlate well with pathogenesis. Recent evidence supports the hypothesis that small soluble aggregates representing intermediates in the fibril assembly process may represent the primary culprits in a variety of amyloid-related degenerative diseases. Investigating the role of soluble amyloid oligomers in pathogenesis presents a problem for distinguishing these aggregates from the mature fibrils, soluble monomer, and natively folded precursor proteins, especially in vivo and in complex mixtures. Recently, we generated a conformation-specific antibody that recognizes soluble oligomers from many types of amyloid proteins, regardless of sequence. These results indicate that soluble oligomers have a common, generic structure that is distinct from both fibrils and low-molecular-weight soluble monomer/dimer. Conformation-dependent, oligomer-specific antibodies represent powerful tools for understanding the role of oligomers in pathogenesis. The purpose of this chapter is to review the methods for the production, characterization, and application of this antibody to understanding the contribution of amyloid oligomers to the disease process.

Amyloid oligomers, similar to the toxic entities found in Alzheimer's disease patients and in other amyloid-based diseases, are present in cardiomyocytes derived from human heart-failure patients and in animal models of desmin-related cardiomyopathy (DRM). The R120G mutation in alpha-B-crystallin (CryAB) causes DRM and is characterized by aggresomes containing CryAB(R120G) and amyloid oligomer. In this study, we show that aggresome levels do not correlate with disease. Blocking aggresome formation results in increased levels of toxic amyloid oligomer and decreased cardiomyocyte viability. We confirmed the primary toxicity of intrasarcoplasmic amyloid accumulation in the cardiomyocytes by ectopic expression of the amyloidogenic peptide PQ81, which consists of multiple repeats of a polyglutamine tract. We then addressed the issue of disease reversibility by placing CryAB(R120G) under inducible cardiomyocyte-specific expression in transgenic mice. The mice developed aggresomes and contained high concentrations of amyloid oligomer in the heart, resulting in cardiac disease. Cessation of CryAB(R120G) expression in symptomatic mice improved cardiac function and rescued all of the animals from premature death. Rescue was accompanied by significant decreases in amyloid oligomer without a significant reduction in aggresomes. Blocking cardiac amyloid oligomer formation, even after cardiac dysfunction presents, may be a therapeutic strategy in DRM as well as in other types of cardiac disease in which significant amyloid accumulation occurs.

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Abeta, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6-6 microg ml-1) rapidly (approximately 5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Abeta42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 microm). Instead, observations that Abeta42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid beta (Abeta) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, Abeta40 and Abeta42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.

Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer's disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into beta-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 microM peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 degrees C. The state at 45 degrees C was also found to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of beta-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases., (Copyright 1999 Academic Press.)