title: Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo.
abstract: Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.
authors.affiliation: Department of Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Cipollini
authors.forename: Massimo
authors.initials: M
authors.lastname: Riccio
authors.forename: Rossella
authors.initials: R
authors.lastname: Di Giaimo
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Dal Piaz
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pulice
authors.forename: Sandra
authors.initials: S
authors.lastname: Catania
authors.forename: Ilaria
authors.initials: I
authors.lastname: Caldarelli
authors.forename: Maja
authors.initials: M
authors.lastname: Dembic
authors.forename: Spartaco
authors.initials: S
authors.lastname: Santi
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Melli
mesh_terms: D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D020191:Myoclonic Epilepsies, Progressive / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: C524994:CSTB protein, human; D015891:Cystatins; D050505:Mutant Proteins; D016877:Oxidants; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D055313:Cystatin B; D003545:Cysteine