Effect of proteasome inhibition by MG-132 on HSP27 oligomerization, phosphorylation, and aggresome formation in the OLN-93 oligodendroglia cell line.

Small heat shock proteins (sHsps) are characteristically observed in glial cell inclusion bodies in a variety of neurodegenerative diseases. The small Hsp27 (HspB1) is stress inducible and participates in the defence against misfolded proteins. Under normal conditions, it forms oligomeric structures with molecular masses ranging from 50 to 800 kDa. These structures are highly dynamic and their dynamic organization is regulated by phosphorylation, which has been suggested to be a crucial factor in modulating the activity of the protein. To investigate the responses of Hsp27 to stress induced by proteasomal inhibition, and its state of phosphorylation and oligomerization during aggregate formation, three oligodendroglial cell lines stably expressing Hsp27 were established: OLN27 wild type, OLN27DDD expressing triple mutant Hsp27 in its phosphorylation mimicking form, and OLN27AAA, expressing non-phosphorylatable Hsp27. Our data show that the proteasomal inhibitor MG-132 promotes Hsp27 phosphorylation by activation of p38 mitogen activated protein kinase and leads to the formation of small oligomers. However, Hsp27 is recruited to aggresomes independently of its state of phosphorylation. After 24 h of treatment, all three cell lines had formed protein accumulations in the vicinity of the centrosome, but in cells over-expressing Hsp27, aggresome formation was delayed in comparison with OLN-93 oligodendroglial cells not expressing Hsp27.