Gain in toxic function of stefin B EPM1 mutants aggregates: Correlation between cell death, aggregate number/size and oxidative stress

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 overexpression. 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.

Characterization of a rare Unverricht–Lundborg disease mutation

“…Cstb present in the nucleus has been described to bind to histones and indirectly regulate the cell cycle through inhibition of cathepsin L [12] , and this increase in the expression of Cstb in the nucleus delays caspase activation and apoptosis, although not preventing cell death [19] . Recently Cstb protein mutants have been described as having a tendency to aggregate in cells [14] . Nevertheless, the cause of the degree of disease progression and neurodegeneration remains unclear.…”

“…Nevertheless, the cause of the degree of disease progression and neurodegeneration remains unclear. It is not known whether it may be due to altered/missing protein function or to protein abnormal misfolding and aggregation or a combination of both [14] . Recently, Cstb protein mutants have been described as having a tendency to aggregate in cells [14] .…”

“…To date only 13 mutations have been described in this gene. The most common mutation, in all populations, accounts for about 90% of the alleles and is an unstable dodecamer repeat expansion in the promoter region of CSTB (13) which leads to reduced mRNA levels and decreased protein levels [14] . Mostly due to the difficult diagnosis of ULD, the disease is believed to be underdiagnosed [15] .…”

Pore-Forming Proteins as Mediators of Novel Epigenetic Mechanism of Epilepsy

“…Several missense mutations in stefin B are linked to epilepsy (50), whereas rare missense mutations in α-synuclein gene (Figure 2B) are associated with Parkinson’s disease (51–53). …”

“…Although the function of stefin B in the pathogenesis of epilepsy is not completely understood, experiments in vitro show that two missense mutants G50E and Q71P aggregate to a much greater extent than the wild-type (WT) protein (50). …”