No abstract

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 ␤2-microgobulin (␤2-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 ␤2-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 ␤-peptide.There is an increasing body of evidence showing that many proteins including the Alzheimer's amyloid ␤-peptide (A␤), 1 prion protein, transthyretin, and ␤2-microgobulin (␤2-m) tend to misfold and aggregate into amyloid fibrils (1-3). Moreover, several proteins not known to be involved in disease and various polyamino acids have been shown to form amyloid fibrils in vitro under carefully selected conditions (4, 5). Although no sequence or structural similarity between the amyloid precursor proteins has been found, amyloid fibrils share several common structural and spectroscopic properties (6). Irrespective of the protein species, electron microscopy and x-ray fiber diffraction indicate that the amyloid fibrils have relatively rigid structures with diameters of 10 -15 nm consisting of cross-␤-strands. Making use of an NMR technique in combination with hydrogen/deuterium exchange of amide protons and dissolution of amyloid fibrils by dimethyl sulfoxide, we have shown that the amyloid fibrils of ␤2-m are stabilized by a hydrogenbond network which is more extensive than that in the native state (7).Amyloid fibril formation is considered to be a nucleation-dependent process in which non-native precursor proteins slowly associate to form the nuclei (8). This process is followed by an extension reaction, where the nucleus grows by sequential incorporation of more precursor protein molecules. This model has been validated by the observation that fibril extension kinetics is accelerated by the addition of preformed fibrils, i.e. by a seeding effect. However, the mechanism of fibril formation by individual polypeptide chains is not completely understood, and there are several variations of the nucleation-dependent model (9, 10). To address the mechanism of amyloid fibril formation, it is important to observe the process at the singlefibril level. Recently, epifluorescence with a newly introduced fluorescent dye (11) and atomic force microscopy (AFM) (12, 13) have been utilized for the d...

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.

The conformational change in amyloid ␤ (A␤) peptide from its monomeric form to aggregates is crucial in the pathogenesis of Alzheimer's disease (AD). In the healthy brain, some unidentified chaperones appear to prevent the aggregation of A␤. aggregation ͉ Alzheimer's disease ͉ mouse ͉ surface plasmon resonance ͉ thioflavin T

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AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease.

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.