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(Circulation Research. 2007;100:213.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Glutathiolation Regulates Tumor Necrosis Factor-–Induced Caspase-3 Cleavage and Apoptosis

Key Role for Glutaredoxin in the Death Pathway

Shi Pan, Bradford C. Berk

From the Cardiovascular Research Institute and Department of Medicine, University of Rochester, NY.

Correspondence to Shi Pan, PhD, University of Rochester, Box 679, Cardiovascular Research Institute, 601 Elmwood Ave, Rochester, NY 14642. E-mail shi_pan{at}urmc.rochester.edu

Caspase-3 cleavage and activation are known to play central roles in apoptosis. However, the mechanisms that regulate caspase-3 cleavage remain elusive. Glutaredoxin (Grx) is a ubiquitous redox molecule that is unique in its ability to regulate S-glutathiolation (glutathiolation) of proteins. Here we show the essential role of Grx in caspase-3 cleavage via regulation of caspase-3 glutathiolation. Grx activity was significantly upregulated by tumor necrosis factor- in endothelial cells. Small interference RNA knock down of Grx significantly inhibited tumor necrosis factor-–induced endothelial cell death because of attenuated caspase-3 cleavage concomitant with increased caspase-3 glutathiolation. Enhanced caspase-3 cleavage by wild-type Grx overexpression was reversed by catalytically inactive Grx (C22S), demonstrating a requirement for thioltransferase activity. Cysteine-to-serine mutations (C163S, C184S, and C220S) of caspase-3 that were predicted to prevent glutathiolation showed increased cleavage compared with wild-type caspase-3. This inverse correlation between caspase-3 glutathiolation and cleavage was further confirmed by the observation that in vitro glutathiolation of caspase-3 inhibited its cleavage with recombinant caspase-8. Furthermore, Grx association with caspase-3 was decreased by tumor necrosis factor-. These findings demonstrate a novel mechanism of caspase-3 regulation by Grx in tumor necrosis factor-–induced apoptosis.

Key Words: tumor necrosis factor • glutaredoxin • endothelial cell • caspase-3

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