INTRODUCTION M AMMALIAN SMALL STRESS PROTEINS [small heat shock proteins (sHsps)] are oligomeric polypeptides related to -crystallin (17). Expression of several of these proteins was found to confer thermotolerance (22), resistance to cytotoxic drugs (38), or oxidative stress (3, 6, 16, 26, 28, 30, 40, 41, 43, 44, 49–51). Some sHsps can also act as actin capping/decap- ping enzymes (23, 37). sHsps also counteract apoptosis in- duced by agents that do not trigger an oxidative stress (1, 2, 5, 42, 45, 47) or that takes place during early differentiation (33, 34). As a consequence, the antiapoptotic activity of human heat shock protein 27 (Hsp27) expression can promote tu- morigenesis (14). Hsp27 belongs to the so-called family of “survival proteins” (18), which includes several antiapoptotic proteins (i.e., Hsp70, Bcl-2, IAP, and survivin) whose expres- sion is often up-regulated in cancer cells and results in ag- gressively growing and therapy-resistant tumors. Evidence have been obtained that sHsps act as ATP- independent chaperones (15, 20) that bind misfolded poly- peptides. This creates reservoirs of folding intermediates (10, 12, 24) counteracting the formation of deleterious protein ag- 436 Laboratoire Stress Oxydant, Chaperons et Apoptose, Centre de Génétique Moléculaire et Cellulaire, CNRS UMR 5534, Université Claude Bernard, Lyon-1, Villeurbanne, France. Substitution of the Unique Cysteine Residue of Murine Hsp25 Interferes with the Protective Activity of This Stress Protein Through Inhibition of Dimer Formation CHANTAL DIAZ-LATOUD, EMILIE BUACHE, ETIENNE JAVOUHEY, and ANDRÉ-PATRICK ARRIGO ABSTRACT Murine small stress protein [heat shock protein 25 (Hsp25)] expression confers thermotolerance and protec- tion against oxidative stress. Hsp25 is an oligomeric ATP-independent phospho-chaperone that can generate a glutathione-dependent pro-reducing state in cells that are normally devoid of small stress protein constitutive expression. Hsp25 contains only one cysteine residue (position 141) that is highly susceptible to oxidation. We have explored the significance of this reactive residue by generating a mutant in which cysteine-141 was sub- stituted by an alanine residue (C141A mutant). We report here that the C141A mutant did not form dimers when expressed in either murine L929 or human HeLa cells, hence, demonstrating that cysteine-141 regulates Hsp25 dimer formation. The C141A mutant also interfered with the dimerization of human Hsp27, a constitu- tively expressed small stress protein in HeLa cells. The mutated polypeptide showed a decreased ability to multimerize, but its expression was still able to induce cellular protection against oxidative stress. The C141A mutant was, however, less efficient than the wild-type protein in counteracting staurosporine-induced apopto- sis, and it showed no in vivo chaperone activity. Hence, the cellular protection mediated against different stres- sors may require specific structural organizations of Hsp25 that are differently altered by the mutation. Of interest, when expressed concomitantly with wild-type Hsp25, the C141A polypeptide induced a dominant- negative effect, a phenomenon that may result from the ability of small stress proteins to interact and multi- merize with each other. Antioxid. Redox Signal. 7, 436–445. Forum Original Research Communication ANTIOXIDANTS & REDOX SIGNALING Volume 7, Numbers 3 & 4, 2005 © Mary Ann Liebert, Inc. LS____ LE____ LL____