Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.
ABSTRACT As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.
SourceAvailable from: Eva Zerovnik[Show abstract] [Hide abstract]
ABSTRACT: EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P and they similarly showed numerous aggregates upon over expression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 06/2014; 1843(9). DOI:10.1016/j.bbamcr.2014.05.018 · 5.30 Impact Factor
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ABSTRACT: Conformational alterations and aggregates of chickpea cystatin (CPC) were investigated upon sequential addition of trifluoroethanol (TFE) over a range of 0-70% v/v. CPC on 30% and 40% v/v TFE addition exhibited non-native β-sheet, altered intrinsic fluorescence, increased thioflavin T fluorescence, prominent red shifted shoulder peak in Congo Red absorbance, and enhanced turbidity as well as Rayleigh scattering, suggesting the aggregate formation. TEM results confirmed the formation of prefibrills at 30% v/v and fibrillar aggregates at 40% v/v TFE. On increasing concentration of TFE to 70% v/v, CPC showed retention of native-like secondary structure, increased intrinsic and ANS fluorescence. Thus our results show that favourable condition for fibrillation of CPC is in the range of 30-40% TFE. Moreover, anti-aggergational effects of polyphenols, epicatechin (EC) and tannic acid (TA) were analysed using ThT binding assay and other biophysical assays. EC and TA produced a concentration dependent decline in ThT fluorescence suggesting inhibition of the fibril formation. Furthermore, TA in comparison to EC, served as a more effective inhibitor against amyloid fibril formation of CPC. This work supports the universality of the amyloid-like aggregation not restricted to some special categories of protein and the fact that this aggregation can be prevented.Archives of Biochemistry and Biophysics 08/2014; 562. DOI:10.1016/j.abb.2014.08.015 · 3.04 Impact Factor
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ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (140 K)Download as PowerPoint slideBiochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2014; · 5.30 Impact Factor