The lattice of stefin B crystals (obtained at pH 10) is composed of five tetramer layers: four well ordered layers which are followed by an additional layer of alternatively placed tetramers.
In this paper we have shown that cells with certain EPM1 mutants aggregates (such as G50E, G4R and Q71P) increase cell death. A correlation exists between the aggregates ability to increase ROS and cell death. 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 (soluble) or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Of interest, wt stefin B at over expression also forms scattered aggregates and increases oxidative stress. However, cell viability is not affected that much, which shows that a compensatory mechanism is taking place with stefin B exerting a protective function against cellular protein misfolding stress – next paper in PLOS One.
discovered for stefin B. Astrocytes isolated from stefin B knockout (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. This in turn contributes to accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated, solubilized and analyzed by mass spectrometry.
In this paper we improved the model for the mechanism of fibrillation of stefin B. We simulated the kinetic data obtained by CD and ThT fluorescence as a function of protein concentration and temperature. We observe a typical polymerization that starts with a lag phase, where oligomers accumulate till a critical mass (cca 60-mers) and a conformational change (involving nearly a complete unforlding, Ea= 55 kcal/mol). Later protofibrils and fibrils grow, ending in a plateau phase (possibly leading by a proline isomerization, Ea= 25 kcal/mol).
We suggest that impaired autophagy could be in common to neurodegenerative diseases – NDs and progressive myoclonus epilepsies – PMEs. In some of the PMEs, as for example Lafora disease, evidence exists for such an impairment. On the other side some authors have shown that autophagy is impaired in AD, on account of protein aggregates, which have to be cleared from the cell. Such new data imply new therapies based on autophagy enhancement.