Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

Shear Pulses Nucleate Fibril Aggregation

We have studied the effect of shear flow on the formation of amyloid fibrils of the whey protein β-lactoglobulin. β-Lactoglobulin aggregates into long, thin, and semiflexible fibrils upon heating at low pH and low ionic strength. Solutions with a protein concentration of 0.5% (w/w) were used, and the formation of fibrils was quantified with flow-induced birefringence, a proportional measure of the length concentration of the fibrils. From the decay of the birefringence after cessation of the flow, a length distribution could be fitted. Pulsed and continuous shear treatment of the samples resulted in a comparable enhancement of the fibrillar growth as compared to the fibrillar growth under quiescent conditions. This indicates that the onset of shear flow is the key parameter for the enhancement of fibrillar growth and not the continuous shear flow itself. This behavior is comparable to a nucleation-like process, during which preaggregates of the fibrils are induced during the onset of the flow and orthokinetic coagulation is absent. However, a difference was present in the length distribution between the pulsed and continuously sheared samples, which can be explained by the homogenizing effect of shear flow.     

Hydrophobic tail length plays a pivotal role in amyloid beta (25–35) fibril–surfactant interactions

The amyloid β‐peptide fragment comprising residues 25–35 (Aβ_25‐35) is known to be the most toxic fragment of the full length Aβ peptide which undergoes fibrillation very rapidly. In the present work, we have investigated the effects of the micellar environment (cationic, anionic, and nonionic) on preformed Aβ_25‐35 fibrils. The amyloid fibrils have been prepared and characterized by several biophysical and microscopic techniques. Effects of cationic dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB), anionic sodium dodecyl sulfate (SDS), and nonionic polyoxyethyleneoctyl phenyl ether (Triton X‐100 or TX) on fibrils have been studied by Thioflavin T fluorescence, UV–vis spectroscopy based turbidity assay and microscopic analyses. Interestingly, DTAB and SDS micelles were observed to disintegrate prepared fibrils to some extent irrespective of their charges. CTAB micelles were found to break down the fibrillar assembly to a greater extent. On the other hand, the nonionic surfactant TX was found to trigger the fibrillation process. The presence of a longer hydrophobic tail in case of CTAB is assumed to be a reason for its higher fibril disaggregating efficacy, the premise of their formation being largely attributed to hydrophobic interactions. Proteins 2016; 84:1213–1223. © 2016 Wiley Periodicals, Inc.     

Chiral bias of amyloid fibrils revealed by the twisted conformation of Thioflavin T: an induced circular dichroism/DFT study

Since it was implicated in a number of neurodegenerative conditions, such as Alzheimer disease, formation of beta-sheet-rich protein fibrils (amyloids) has been drawing a lot of attention. One of elusive aspects of amyloidogenesis concerns the mechanisms of specific binding of molecules such as Congo red, or Thioflavin T by amyloid fibrils. A comprehensive understanding of these docking interactions is needed, however, for the sake of furthering biochemical studies and developing molecular, pharmacological strategies preventing proliferation of amyloids in vivo. Through the application of circular dichroism, here we show that upon binding to insulin fibrils, a twisted conformation is enforced in molecules of Thioflavin T, manifested in a strong negative Cotton effect around 450 nm, which is supported by density functional theory-based calculations. This finding may lead to circular dichroism of Thioflavin T becoming a new diagnostic technique for protein fibrils, complementary to fluorescence spectroscopy.     

The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.     

Cyclin-dependent Kinase 5 Phosphorylation of Familial Prion Protein Mutants Exacerbates Conversion into Amyloid Structure

Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion.     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

Amyloid fibril formation by macrophage migration inhibitory factor

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

Antiaggregation Potential of Padina gymnospora against the Toxic Alzheimer’s Beta-Amyloid Peptide 25–35 and Cholinesterase Inhibitory Property of Its Bioactive Compounds

Inhibition of β-amyloid (Aβ) aggregation in the cerebral cortex of the brain is a promising therapeutic and defensive strategy in identification of disease modifying agents for Alzheimer’s disease (AD). Since natural products are considered as the current alternative trend for the discovery of AD drugs, the present study aims at the evaluation of anti-amyloidogenic potential of the marine seaweed Padina gymnospora. Prevention of aggregation and disaggregation of the mature fibril formation of Aβ _25–35 by acetone extracts of P. gymnospora (ACTPG) was evaluated in two phases by Thioflavin T assay. The results were further confirmed by confocal laser scanning microscopy (CLSM) analysis and Fourier transform infrared (FTIR) spectroscopic analysis. The results of antiaggregation and disaggregation assay showed that the increase in fluorescence intensity of aggregated Aβ and the co-treatment of ACTPG (250 μg/ml) with Aβ _25–35, an extensive decrease in the fluorescence intensity was observed in both phases, which suggests that ACTPG prevents the oligomers formation and disaggregation of mature fibrils. In addition, ACTPG was subjected to column chromatography and the bioactivity was screened based on the cholinesterase inhibitory activity. Finally, the active fraction was subjected to LC-MS/MS analysis for the identification of bioactive compounds. Overall, the results suggest that the bioactive compound alpha bisabolol present in the alga might be responsible for the observed cholinesterase inhibition with the IC50 value < 10 μg/ml for both AChE and BuChE when compared to standard drug donepezil (IC50 value < 6 μg/ml) and support its use for the treatment of neurological disorders.     

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed.     

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington''s disease, in vitro. Huntington''s disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer''s and Parkinson''s disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.     

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.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

The Critical Aggregation Concentration of β-Lactoglobulin-Based Fibril Formation

The critical aggregation concentration (CAC) for fibril formation of β-lactoglobulin (β-lg) at pH 2 was determined at 343, 353, 358, 363, and 383 K using a Thioflavin T assay and was approximately 0.16 wt%. The accuracy of the CAC was increased by measuring the conversion into fibrils at different stirring speeds. The corresponding binding energy per mol, as determined from the CAC, was 13 RT (∼40 kJ mol⁻¹) for the measured temperature range. The fact that the CAC was independent of temperature within the experimental error indicates that the fibril formation of β-lg at pH 2 and the measured temperature range is an entropy-driven process.     

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.     

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.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.     

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

Amyloid formation by salmon calcitonin

It is demonstrated using three independent methods that salmon calcitonin can form amyloid fibrils in vitro. Large aggregates are shown to exhibit a blue-green birefringence in cross polarised light after staining with congo red. Individual fibrils were observed using electron microscopy. These fibrils are approx. 50–60 Å in diameter and up to 20 000 Å in length and are similar in appearance to those observed in Alzheimer's disease. Finally, X-ray diffraction studies of the large aggregates reveal the cross-β conformation characteristic of the monomers in the fibre.