Inhibition of caspase-3 activity and activation by protein glutathionylation

Protein glutathionylation is a post-translational modification that may account for a broad mechanism of redox signaling. The caspase family of cysteine proteases represents a potential target for regulation by glutathionylation. To examine this, caspase proteins, derived from HL-60 cells after acti...

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Bibliographic Details
Published in:Biochemical pharmacology 2008-06, Vol.75 (11), p.2234-2244
Main Authors: Huang, Zhishan, Pinto, John T., Deng, Haiteng, Richie, John P.
Format: Article
Language:English
Subjects:
Apoptosis
Biological and medical sciences
Caspase
Caspase 3 - metabolism
Caspase 9 - metabolism
Caspase Inhibitors
Cell-Free System
Dose-Response Relationship, Drug
Enzyme Activation - drug effects
Enzyme Activation - physiology
Glutathione
Glutathione - metabolism
Glutathione Disulfide - pharmacology
Glutathionylation
HL-60 Cells
Humans
Liver - enzymology
Medical sciences
Oxidative stress
Pharmacology. Drug treatments
Thionylation
Time Factors
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Summary:Protein glutathionylation is a post-translational modification that may account for a broad mechanism of redox signaling. The caspase family of cysteine proteases represents a potential target for regulation by glutathionylation. To examine this, caspase proteins, derived from HL-60 cells after activation with actinomycin D, were incubated with GSSG. Total protein glutathionylation was enhanced and caspase-3 activity was inhibited in a dose- and time-dependent manner by GSSG. Caspase inhibition was reversible by thiol-specific reducing reagents. Proteolytic activation of caspases was also affected, as the activation of procaspase-3 and procaspase-9 in HL-60 cell extracts induced by cytochrome c and dATP was inhibited by pre-incubation with GSSG. When biotin-labeled GSSG was incubated with recombinant caspase-3, biotin label was found associated with both p12 and p17 subunits of active caspase-3 by non-reducing SDS-PAGE. Caspase-3 glutathionylation was confirmed by matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis of GSSG-treated recombinant caspase-3. Specific sites of glutathionylation were identified as Cys135 of the p17 protein (the active site) and Cys45 of the p12 protein. These results indicate that glutathionylation of caspase can occur at physiologically relevant concentrations of GSSG and results in the inhibition of caspase activation and activity.
ISSN:0006-2952
1873-2968
DOI:10.1016/j.bcp.2008.02.026