Novel method for quantitative determination of amyloid fibrils of α-synuclein and amyloid β/A4 protein by using resveratrol

Ahn, Jung Sun; Lee, Jung-Ho; Kim, Je-Hoon; Paik, Seung R.

doi:10.1016/j.ab.2007.05.023

Cited by 49 publications

(36 citation statements)

References 26 publications

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“…Under these conditions, wild type IAPP began to aggregate, and hence bind ThT, within minutes of incubation. The decrease in ThT fluorescence after reaching its maximum is commonly witnessed in these assays and is consistent with previously reported amyloid aggregation reactions (25, 26). …”

Section: Resultssupporting

confidence: 92%

Selection for Nonamyloidogenic Mutants of Islet Amyloid Polypeptide (IAPP) Identifies an Extended Region for Amyloidogenicity

et al. 2010

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The aggregation of the 37-residue polypeptide IAPP, as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type II diabetes. While IAPP has been known to be the primary component of type II diabetes amyloid, the molecular interactions responsible for this aggregation have not been identified. To identify the aggregation-prone region(s), we constructed a library of randomly generated point mutants of IAPP. This mutant IAPP library was expressed in E. coli as genetic fusions to the reporter protein enhanced green fluorescent protein (EGFP). Because IAPP aggregates rapidly, both independently and when fused to EGFP, the fusion protein does not yield a functional, fluorescent EGFP. However, mutations of IAPP that result in non-amyloidogenic sequences remain soluble and allow EGFP to fold and fluoresce. Using this screen, we identified 22 single mutations, 4 double mutations and 2 triple mutations of IAPP that appear to be less amyloidogenic than wild type human IAPP. A comparison of these sequences suggests residues 13 and 15-17 comprise an additional aggregation-prone region outside of the main amyloidogenic region of IAPP.The aggregation of misfolded proteins into toxic oligomers and fibers has been linked to a variety of diseases such as type II diabetes, Alzheimer's disease and Parkinson's disease. In type II diabetes the amyloid-forming peptide is islet amyloid polypeptide (IAPP, amylin). This 37 amino acid polypeptide misfolds and forms aggregates within the pancreas. This misfolding into toxic aggregates, such as small soluble oligomers or large fibers, is believed to contribute to the loss of pancreatic β-cells. While the exact role of IAPP in type II diabetes is unclear, it is known that IAPP is found as extracellular deposits of amyloid in approximately 95% of patients afflicted with type II diabetes (1-3). IAPP has also been shown to be a toxic agent in vitro when added to human islet β-cells (4).Many of the therapeutic strategies for preventing or slowing the progression of amyloid diseases such as type II diabetes, involve slowing or preventing the aggregation of the amyloidogenic proteins. To this end, a great deal of effort has been made in identifying the amino acids responsible for the aggregation-prone nature of amyloidogenic peptides such as †

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Section: Resultssupporting

confidence: 92%

Selection for Nonamyloidogenic Mutants of Islet Amyloid Polypeptide (IAPP) Identifies an Extended Region for Amyloidogenicity

et al. 2010

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“…At the highest concentration of myricetin tested, where myricetin was at a 10-fold molar excess over IAPP, the ThT-binding of IAPP was approximately the same as ThT in solution in the absence of IAPP. The decrease in ThT fluorescence after reaching its maximum is commonly witnessed in these assays and is consistent with previously reported amyloid aggregation reactions [12, 13]. …”

supporting

confidence: 92%

Myricetin Inhibits Islet Amyloid Polypeptide (IAPP) Aggregation and Rescues Living Mammalian Cells from IAPP Toxicity

Zelus¹,

Fox²,

Calciano³

et al. 2012

Open Biochem J

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The aggregation of the amyloidogenic polypeptide IAPP (Islet Amyloid Polypeptide, amylin) is believed to play a direct role in the death of pancreatic β-islet cells in type II diabetes. Preventing the initial aggregation event of IAPP is one strategy for slowing, and possibly preventing, the progression of this disease. Here, we investigate myricetin’s potential as an inhibitor of IAPP aggregation. We show that myricetin prevented thioflavin T binding in a concentration dependent manner. Atomic force microscopy revealed that myricetin prevented fiber formation under rigorous conditions conducive to forming IAPP aggregates. Using an IAPP-EGFP (Enhanced Green Fluorescent Protein) protein construct, we find that high concentrations of myricetin slowed the in vivo aggregation of IAPP-EGFP. Myricetin was also found to rescue living mammalian cells from the toxic effects of IAPP. These results indicate that myricetin is a strong inhibitor of IAPP amyloid aggregation and a potential lead molecule for the development of an amyloid inhibiting therapeutic.

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“…To avoid artifacts, the results were confirmed by SDS-PAGE before and after PK digestion followed by Coomassie Blue staining. This revealed that also resveratrol (which has fluorescence emission in the presence of β-sheet structures that overlaps with the emission of ThT (25)) was inhibiting α-synuclein aggregation during PMCA (Fig. 3 B ).…”

Section: Resultsmentioning

confidence: 85%

“…Other previously studied drugs, non-steroidal anti-inflammatory drugs such as Ibuprofen (21), acetaminophen (22), and aspirin (23) were used to evaluate the specificity of the assay. The remaining drugs tested included lacmoid and resveratrol, with reported binding to α-synuclein (24, 25); tannic acid and (−)epigallocatechin gallate (EGCG), previously described as potential inhibitors of α-synuclein aggregation (26–29). …”

Section: Resultsmentioning

confidence: 99%

Anti-amyloid Compounds Inhibit α-Synuclein Aggregation Induced by Protein Misfolding Cyclic Amplification (PMCA)

Herva¹,

Zibaee

Fraser

et al. 2014

Journal of Biological Chemistry

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Background: α-Synuclein filaments can be formed in vitro, but the process is slow and unreliable, unsuitable for high throughput screening.Results: Protein misfolding cyclic amplification (PMCA) rapidly assembles α-synuclein into filaments that form aggregates in cell culture and whose formation is inhibited by anti-amyloid compounds.Conclusion: Anti-amyloid compounds affect PMCA cell-transmissible α-synuclein fibril formation.Significance: α-Synuclein PMCA is useful for the screening of anti-aggregation compounds as treatments for α-synucleinopathies.

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Novel method for quantitative determination of amyloid fibrils of α-synuclein and amyloid β/A4 protein by using resveratrol

Cited by 49 publications

References 26 publications

Selection for Nonamyloidogenic Mutants of Islet Amyloid Polypeptide (IAPP) Identifies an Extended Region for Amyloidogenicity

Selection for Nonamyloidogenic Mutants of Islet Amyloid Polypeptide (IAPP) Identifies an Extended Region for Amyloidogenicity

Myricetin Inhibits Islet Amyloid Polypeptide (IAPP) Aggregation and Rescues Living Mammalian Cells from IAPP Toxicity

Anti-amyloid Compounds Inhibit α-Synuclein Aggregation Induced by Protein Misfolding Cyclic Amplification (PMCA)

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