LARGE UNPHOSPHORYLATED AGGREGATES AS THE ACTIVE FORM OF HSP27 WHICH CONTROLS INTRACELLULAR REACTIVE OXYGEN SPECIES AND GLUTATHIONE LEVELS AND GENERATES A PROTECTION AGAINST TNF-ALPHA IN NIH-3T3-RAS CELLS

The mammalian small stress protein hsp27 is an oligomeric phosphoprote in which interferes with the cell death induced by several stimuli. In that sense, we and others have recently shown that human hsp27 expres sion induced cellular protection against tumor necrosis factor (TNF al pha), a protection which depends on the ability of hsp27 to decrease t he level of reactive oxygen species and increase that of glutathione. Here, we have analyzed unphosphorylatable mutants of human hsp27 in wh ich serines 15, 78, and 82 were replaced by alanines, glycines, or asp artic acids. Depending on the amino acid which was used to substitute the serine sites, a different pattern of hsp27 structural organization was observed. Alanine substitution generated large hsp27 aggregates w hile glycine and aspartic acid did the reverse. Hence, these phosphory latable serine residues can be considered as key elements affecting hs p27 structural organization. Only the large aggregates of hsp27 were a ble to modulate reactive oxygen species and glutathione and generated cellular protection against TNF alpha. Moreover, using drugs that modu late the intracellular level of glutathione, we show that an increase in glutathione by itself was sufficient to generate large hsp27 struct ures while the reverse was observed in the case of glutathione depriva tion. (C) 1997 Academic Press.