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Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

Ramshini H, Parrini C, Relini A, Zampagni M, Mannini B, Pesce A, Saboury AA, Nemat-Gorgani M, Chiti F - PLoS ONE (2011)

Bottom Line: The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger.In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding.Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy.

ABSTRACT
The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

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TFE-induced unfolding of YHKB monitored by [θ]222 in the absence (▪) and/or presence (□) of 1 mM α-cyclodextrin.
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pone-0016075-g006: TFE-induced unfolding of YHKB monitored by [θ]222 in the absence (▪) and/or presence (□) of 1 mM α-cyclodextrin.

Mentions: Solution conditions containing moderate concentrations of TFE have also been widely used to form amyloid-like fibrils by both disease-related and disease-unrelated proteins [10], [12], [19], [21]–[23]. Similarly to the study performed at low pH, we first acquired far-UV CD and fluorescence spectra in the presence of various concentrations of TFE, ranging from 0 to 45% (v/v) at pH 5.5. The intrinsic fluorescence of YHKB was found to decay rapidly with TFE concentration, due to the ability of TFE to quench dramatically tryptophan and tyrosine fluorescence. By contrast, the far-UV CD spectra were found to be similar from 0 to 15% (v/v) TFE, decrease in intensity at 20–26% (v/v) TFE and then increase dramatically to reach the appearance typical of an α-helical-enriched conformational state at TFE concentrations higher than 40%. The plot of [θ]222 versus TFE concentration allowed this behavior to be clearly observed (Fig. 6). The first transition causing a decrease of the CD signal at 20–26% (v/v) TFE is due to aggregation (see below), whereas the second transition is likely to arise from the conversion of the protein into a monomeric α-helical conformational state.


Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

Ramshini H, Parrini C, Relini A, Zampagni M, Mannini B, Pesce A, Saboury AA, Nemat-Gorgani M, Chiti F - PLoS ONE (2011)

TFE-induced unfolding of YHKB monitored by [θ]222 in the absence (▪) and/or presence (□) of 1 mM α-cyclodextrin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020945&req=5

pone-0016075-g006: TFE-induced unfolding of YHKB monitored by [θ]222 in the absence (▪) and/or presence (□) of 1 mM α-cyclodextrin.
Mentions: Solution conditions containing moderate concentrations of TFE have also been widely used to form amyloid-like fibrils by both disease-related and disease-unrelated proteins [10], [12], [19], [21]–[23]. Similarly to the study performed at low pH, we first acquired far-UV CD and fluorescence spectra in the presence of various concentrations of TFE, ranging from 0 to 45% (v/v) at pH 5.5. The intrinsic fluorescence of YHKB was found to decay rapidly with TFE concentration, due to the ability of TFE to quench dramatically tryptophan and tyrosine fluorescence. By contrast, the far-UV CD spectra were found to be similar from 0 to 15% (v/v) TFE, decrease in intensity at 20–26% (v/v) TFE and then increase dramatically to reach the appearance typical of an α-helical-enriched conformational state at TFE concentrations higher than 40%. The plot of [θ]222 versus TFE concentration allowed this behavior to be clearly observed (Fig. 6). The first transition causing a decrease of the CD signal at 20–26% (v/v) TFE is due to aggregation (see below), whereas the second transition is likely to arise from the conversion of the protein into a monomeric α-helical conformational state.

Bottom Line: The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger.In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding.Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy.

ABSTRACT
The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

Show MeSH
Related in: MedlinePlus