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Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and
Mira Polajnar1, Tina Zavašnik-Bergant2, Nataša Kopitar-Jerala2
1Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.
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.
Insights
Protein aggregation and oxidative stress in EPM1 disease are linked to cell death. The study found that the form of stefin B aggregates, not their size, influences toxicity, impacting neuronal function.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Epilepsy, progressive myoclonus type 1 (EPM1) is a rare neurological disorder characterized by cerebellar apoptosis.
- Mutations in the stefin B (cystatin B) gene are the primary cause of EPM1, leading to protein aggregation.
- The interplay between protein aggregation, oxidative stress, and cell death in EPM1 pathogenesis requires further elucidation.
Purpose of the Study:
- To investigate the relationship between stefin B aggregation, oxidative stress, and cell death in EPM1.
- To compare the toxicity of different stefin B mutant forms and aggregate structures.
- To understand the functional consequences of stefin B mutations on protease inhibition.
Main Methods:
- Overexpression of wild-type and mutant stefin B (G4R, R68X, G50E, Q71P) in cellular models.
- Analysis of protein aggregation, oxidative stress levels, and cell death.
- Transmission electron microscopy for characterizing aggregate morphology.
- Assessment of stefin B's cysteine protease inhibitory activity.
Main Results:
- Overexpression of EPM1-associated stefin B mutants (G50E, Q71P) induced significant protein aggregation, oxidative stress, and cell death.
- No direct correlation was found between aggregate size/number and cell death.
- Aggregate toxicity appeared dependent on their form (oligomeric/protofibrillar vs. fibrillar) and the mutant's 3D structure, with unfolded proteins showing less toxicity.
- All studied EPM1 mutants exhibited loss of cysteine protease inhibitory function.
Conclusions:
- The form and structure of stefin B aggregates, rather than their quantity, are critical determinants of toxicity in EPM1.
- Loss of stefin B's protease inhibitory function and gain of toxic function through aggregation contribute to EPM1 pathogenesis.
- These findings offer insights into the molecular mechanisms underlying EPM1 and potential therapeutic targets.
