title: 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.
authors.affiliation: Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Mira
authors.initials: M
authors.lastname: Polajnar
authors.affiliation: Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Tina
authors.initials: T
authors.lastname: Zavašnik-Bergant
authors.affiliation: Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Nataša
authors.initials: N
authors.lastname: Kopitar-Jerala
authors.affiliation: National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Magda
authors.initials: M
authors.lastname: Tušek-Žnidarič
authors.affiliation: Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia. Electronic address: eva.zerovnik@ijs.si.
authors.forename: Eva
authors.initials: E
authors.lastname: Zerovnik
mesh_terms: D000682:Amyloid / Q000378:metabolism; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D052160:Benzothiazoles; D016466:CHO Cells; D002452:Cell Count; D016923:Cell Death; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D055313:Cystatin B / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000648:ultrastructure; D018384:Oxidative Stress; D011419:Propidium / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000378:metabolism; D014162:Transfection; D020194:Unverricht-Lundborg Syndrome / Q000235:genetics* / Q000473:pathology*
chemical_list: D000682:Amyloid; D017304:Annexin A5; D052160:Benzothiazoles; D050505:Mutant Proteins; D013844:Thiazoles; C009462:thioflavin T; D011419:Propidium; D055313:Cystatin B
keywords: Amyloid; Cell death; Cystatin B; Oxidative stress; Protein aggregate; Toxic oligomer