Insights in progressive myoclonus epilepsy: HSP70 promotes cystatin B polymerization.

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu(2+) for chaperone-dependent polymerization. Cu(2+) interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu(2+) modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu(2+)/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s).

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo.

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.

Cell-specific expression of the epm1 (cystatin B) gene in developing rat cerebellum.

Cystatin B (cystB) is an anti-protease implicated in EPM1, a degenerative disease of the central nervous system. This work analyzes the pattern of expression of cystB in developing and adult cerebellum, identifying the cystB positive cells by double immune-fluorescence microscopy using specific cell markers. In primary glial cells, cystB is found in progenitor and differentiated oligodendrocytes as well as in astrocytes. In the cerebellum, only oligodendrocyte progenitors express cystB. In myelin-producing cells, cystB synthesis is strongly down-regulated and the protein is not detectable. Astrocytes and Bergmann glia express cystB at all the developmental stages analyzed both in the cell body and in the fibers. Most neurons of developing and adult rat cerebellum do not express detectable amounts of cystB, with the exception of the Purkinje cells and of some cells of the differentiated molecular layer. In human cerebellum, cystB is present in Purkinje cells and Bergmann glial fibers only. cystB is also found in the cortical neurons of the dentate gyrus of the hippocampus. In rat cerebellum, cystB forms a complex with a number of proteins, two of which are specific to the nervous system. The cellular co-localization of cystB and its partners in developing and adult cerebellum is also shown.

New insights into the molecular basis of progressive myoclonus epilepsy: a multiprotein complex with cystatin B.

Cystatin B is an anti-proteolytic polypeptide implicated in progressive myoclonus epilepsy (EPM1), a degenerative disease of the central nervous system. The knock-out mouse model of the disease shows apoptosis of the cerebellar granule cells. We have identified five recombinant proteins interacting with cystatin B and none of them is a protease. We show that three of these proteins (RACK-1, beta-spectrin and NF-L) co-immunoprecipitate with cystatin B in rat cerebellum. Confocal immunofluorescence analysis shows that the same proteins are present in the granule cells of developing cerebellum, as well as in Purkinje cells of adult rat cerebellum. We propose that a cystatin B multiprotein complex has a specific cerebellar function and that the loss of this function might contribute to the disease in EPM1 patients.