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Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

ABSTRACT: We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers-exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the ?-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a ?-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.

SUBMITTER: Pease LF

PROVIDER: S-EPMC3000482 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

Pease Leonard F LF Sorci Mirco M Guha Suvajyoti S Tsai De-Hao DH Zachariah Michael R MR Tarlov Michael J MJ Belfort Georges G

Biophysical journal 20101201 12

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomer ...[more]

PMID: 21156140

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Project description:Cerebral amyloid angiopathy (CAA) induces various forms of cerebral infarcts and hemorrhages from vascular amyloid-? accumulation, resulting in acceleration of cognitive impairment, which is currently untreatable. Soluble amyloid-? protein likely impairs cerebrovascular integrity as well as cognitive function in early stage Alzheimer's disease. Taxifolin, a flavonol with strong anti-oxidative and anti-glycation activities, has been reported to disassemble amyloid-? in vitro but the in vivo relevance remains unknown. Here, we investigated whether taxifolin has therapeutic potential in attenuating CAA, hypothesizing that inhibiting amyloid-? assembly may facilitate its clearance through several elimination pathways. Vehicle- or taxifolin-treated Tg-SwDI mice (commonly used to model CAA) were used in this investigation. Cognitive and cerebrovascular function, as well as the solubility and oligomerization of brain amyloid-? proteins, were investigated. Spatial reference memory was assessed by water maze test. Cerebral blood flow was measured with laser speckle flowmetry and cerebrovascular reactivity evaluated by monitoring cerebral blood flow changes in response to hypercapnia. Significantly reduced cerebrovascular pan-amyloid-? and amyloid-?1-40 accumulation was found in taxifolin-treated Tg-SwDI mice compared to vehicle-treated counterparts (n?=?5). Spatial reference memory was severely impaired in vehicle-treated Tg-SwDI mice but normalized after taxifolin treatment, with scoring similar to wild type mice (n?=?10-17). Furthermore, taxifolin completely restored decreased cerebral blood flow and cerebrovascular reactivity in Tg-SwDI mice (n?=?4-6). An in vitro thioflavin-T assay showed taxifolin treatment resulted in efficient inhibition of amyloid-?1-40 assembly. In addition, a filter trap assay and ELISA showed Tg-SwDI mouse brain homogenates exhibited significantly reduced levels of amyloid-? oligomers in vivo after taxifolin treatment (n?=?4-5), suggesting the effects of taxifolin on CAA are attributable to the inhibition of amyloid-? oligomer formation. In conclusion, taxifolin prevents amyloid-? oligomer assembly and fully sustains cognitive and cerebrovascular function in a CAA model mice. Taxifolin thus appears a promising therapeutic approach for CAA.

Project description:The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

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Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

Publications

Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

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