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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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H-induced unfolding of YHKB.(A) pH-induced unfolding of YHKB monitored by the fluorescence emission intensity at the maximum wavelength (▪) and [θ]222 (□). (B) Spectral diagram of pH-induced equilibrium unfolding of YHKB, reporting on F340 versus [θ]222.
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pone-0016075-g002: H-induced unfolding of YHKB.(A) pH-induced unfolding of YHKB monitored by the fluorescence emission intensity at the maximum wavelength (▪) and [θ]222 (□). (B) Spectral diagram of pH-induced equilibrium unfolding of YHKB, reporting on F340 versus [θ]222.

Mentions: Amyloid fibril formation by globular proteins generally requires the presence of a partially unfolded state [8], [29]. Intrinsic fluorescence and far-UV CD spectroscopy were used to monitor possible conformational changes of the protein as the pH is decreased from neutral to acid values in the absence of salts. Both spectroscopic probes monitored substantial changes as the pH is reduced from 4 to 3, with a single sharp transition (Fig. 2A). The pH dependence of ANS fluorescence also showed a similar transition within the same pH range (data not shown). The spectral diagram reporting intrinsic fluorescence at 340 nm (F340) versus the mean residue ellipticity at 222 nm ([θ]222) possesses only one linear part (Fig. 2B). This indicates the existence of one transition in the pH titration of the enzyme and suggests that a two-state model is a good approximation to describe the pH unfolding of the enzyme under the investigated pH range.


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)

H-induced unfolding of YHKB.(A) pH-induced unfolding of YHKB monitored by the fluorescence emission intensity at the maximum wavelength (▪) and [θ]222 (□). (B) Spectral diagram of pH-induced equilibrium unfolding of YHKB, reporting on F340 versus [θ]222.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016075-g002: H-induced unfolding of YHKB.(A) pH-induced unfolding of YHKB monitored by the fluorescence emission intensity at the maximum wavelength (▪) and [θ]222 (□). (B) Spectral diagram of pH-induced equilibrium unfolding of YHKB, reporting on F340 versus [θ]222.
Mentions: Amyloid fibril formation by globular proteins generally requires the presence of a partially unfolded state [8], [29]. Intrinsic fluorescence and far-UV CD spectroscopy were used to monitor possible conformational changes of the protein as the pH is decreased from neutral to acid values in the absence of salts. Both spectroscopic probes monitored substantial changes as the pH is reduced from 4 to 3, with a single sharp transition (Fig. 2A). The pH dependence of ANS fluorescence also showed a similar transition within the same pH range (data not shown). The spectral diagram reporting intrinsic fluorescence at 340 nm (F340) versus the mean residue ellipticity at 222 nm ([θ]222) possesses only one linear part (Fig. 2B). This indicates the existence of one transition in the pH titration of the enzyme and suggests that a two-state model is a good approximation to describe the pH unfolding of the enzyme under the investigated pH range.

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