In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amy... more In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1—EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.
Human stefin B is a protease inhibitor from the family of cystatins. It was reported that it form... more Human stefin B is a protease inhibitor from the family of cystatins. It was reported that it forms oligomers in cells. We have shown that it has a role in cell’s response to misfolded proteins. We also have shown that its oligomers bind amyloid-beta (Aβ). Here, we discuss ways, how stefin B could reduce build-up of protein aggregates by other proteins and consequently reduces ROS and, how this might be connected to autophagy. When overexpressed, stefin B forms protein aggregates itself and these protein aggregates induce autophagy. Similarly, cystatin C was shown to bind Aβ and to induce autophagy. It is also suggested how more knowledge about the role of stefin B in a cell’s response to misfolded proteins could be used to modulate progressive myoclonus epilepsy of type 1 EPM1 disease.
To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuro... more To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.
Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, al... more Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-α-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.
Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Seve... more Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing. The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility. Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions. From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results. Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms. It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate. Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation.
The mechanism by which proteins form amyloid fibrils is of high interest to the scientific commun... more The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.
In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amy... more In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1—EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.
Human stefin B is a protease inhibitor from the family of cystatins. It was reported that it form... more Human stefin B is a protease inhibitor from the family of cystatins. It was reported that it forms oligomers in cells. We have shown that it has a role in cell’s response to misfolded proteins. We also have shown that its oligomers bind amyloid-beta (Aβ). Here, we discuss ways, how stefin B could reduce build-up of protein aggregates by other proteins and consequently reduces ROS and, how this might be connected to autophagy. When overexpressed, stefin B forms protein aggregates itself and these protein aggregates induce autophagy. Similarly, cystatin C was shown to bind Aβ and to induce autophagy. It is also suggested how more knowledge about the role of stefin B in a cell’s response to misfolded proteins could be used to modulate progressive myoclonus epilepsy of type 1 EPM1 disease.
To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuro... more To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.
Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, al... more Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-α-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.
Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Seve... more Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing. The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility. Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions. From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results. Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms. It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate. Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation.
The mechanism by which proteins form amyloid fibrils is of high interest to the scientific commun... more The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.
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Papers by Eva Zerovnik