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).
Department of Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.
Ada
A
Rispoli
Elena
E
Cipollini
Sandra
S
Catania
Rossella
R
Di Giaimo
Giuseppe
G
Pulice
Stineke
S
van Houte
Francesca
F
Sparla
Fabrizio
F
Dal Piaz
Davide
D
Roncarati
Paolo
P
Trost
Marialuisa
M
Melli
D000818:Animals; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D055332:Cystatin M / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020191:Myoclonic Epilepsies, Progressive / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D051381:Rats
C075076:Cst6 protein, rat; D055332:Cystatin M; D018840:HSP70 Heat-Shock Proteins; D003300:Copper
CSTB polymer; Cystatin B mutant; Cysteine as a redox sensitive sensor; EPM1; Native and denatured CSTB polymer