Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2

• Published in: Free radical biology & medicine 2006-02, Vol.40 (4), p.651-659
• Main Authors: Choi, Hoon-In, Lee, Sang Pil, Kim, Kyung Soon, Hwang, Chae Young, Lee, Yu-Ran, Chae, Suhn-Kee, Kim, Yeon-Soo, Chae, Ho Zoon, Kwon, Ki-Sun
• Format: Article
• Language: English
• Subjects: Chaperone; Cysteine - chemistry; Cysteine - metabolism; Free radical; HSC70 Heat-Shock Proteins - metabolism; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - pharmacology; Humans; Hydrogen Peroxide - pharmacology; Luciferases - metabolism; Molecular Chaperones; Oxidants - pharmacology; Oxidation-Reduction; Oxidative stress; Protein folding; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Thioredoxin; Thioredoxins - metabolism; Two-Hybrid System Techniques; Zinc - metabolism; Zinc finger; Zinc Fingers
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2005.09.018

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H 2O 2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H 2O 2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.