The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27C137A) and comparing it to wild-type protein (Hsp27WT). Both proteins were multi-subunit oligomers, but subunits of Hsp27WT were disulfide-linked unlike those of Hsp27C137A, which were monomeric. Hsp27C137A was indistinguishable from Hsp27WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27WT. The anti-apoptotic function of Hsp27C137A and Hsp27WT was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27C137A and Hsp27WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H2O2 in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress. J. Cell. Biochem. 110: 408–419, 2010. © 2010 Wiley-Liss, Inc.
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