[19] Quantifying amyloid by congo red spectral shift assay

Klunk, William E.; Rf, Jacob; Mason, Ronald P.

doi:10.1016/s0076-6879(99)09021-7

Cited by 331 publications

(279 citation statements)

References 25 publications

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“…Binding of Congo red to the TFE aggregates gives rise to a small shoulder centered at Ϸ565 nm (Fig. 3B); this change is also smaller and different from the large increase in absorbance at 541 nm typically seen for binding to amyloid (26). These observations suggest that the TFE aggregates are distinct from amyloid and have similar tinctorial properties to in vivo ALS aggregates (27).…”

Section: Figmentioning

confidence: 82%

Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Stathopulos

Rumfeldt²,

Scholz³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

246

273

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Mutations in Cu͞Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo. Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: (i) an increase in the equilibrium population of unfolded or of partially unfolded states, (ii) an increase in the rate of unfolding, and (iii) a decrease in the rate of folding. Our data support the hypothesis that the gain of toxic function for many different familial ALS-associated mutant SODs is a consequence of protein destabilization, which leads to an increase in the formation of cytotoxic protein aggregates.

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Section: Figmentioning

confidence: 82%

Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Stathopulos

Rumfeldt²,

Scholz³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

246

273

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“…In the presence of amyloid fibrils, a spectral shift of the max of the bound dye from 488 to 540 nm is observed (38). The dye spectra in the presence of the 25-, 12-, 8-, and 6-amino acid peptides are shown in Fig.…”

Section: Table Imentioning

confidence: 94%

Amyloid Fibril Formation from Sequences of a Natural β-Structured Fibrous Protein, the Adenovirus Fiber

Papanikolopoulou

Schoehn

Forge

et al. 2005

Journal of Biological Chemistry

112

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Amyloid fibrils are fibrous ␤-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, ␤-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, ␤-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, ␤-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last ␤-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of ␤-strand repeat units is an underlying common feature of amyloid formation.

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“…CR Binding Assay-CR is a dye routinely used for detecting amyloid formation in protein/peptide aggregation studies (44). In order to confirm the amyloid nature of BSA aggregates, CR binding studies were performed.…”

Section: Methodsmentioning

confidence: 99%

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

Jacob¹,

George²,

Singh

et al. 2016

Journal of Biological Chemistry

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Amyloids are highly ordered, cross-␤-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.

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[19] Quantifying amyloid by congo red spectral shift assay

Cited by 331 publications

References 25 publications

Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Amyloid Fibril Formation from Sequences of a Natural β-Structured Fibrous Protein, the Adenovirus Fiber

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

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