Inhibition of caspase-3 activity and activation by protein glutathionylation

Zhishan Huang, John T. Pinto, Haiteng Deng, John P. Richie

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2234-2244
Number of pages11
JournalBiochemical Pharmacology
Volume75
Issue number11
DOIs
StatePublished - Jun 1 2008

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Glutathione Disulfide
Caspases
Caspase 3
Chemical activation
Proteins
HL-60 Cells
Biotin
Cysteine Proteases
Caspase 9
Dactinomycin
Post Translational Protein Processing
Cytochromes c
Cell Extracts
Sulfhydryl Compounds
Oxidation-Reduction
Ionization
Labels
Polyacrylamide Gel Electrophoresis
Desorption
Catalytic Domain

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

Cite this

Huang, Zhishan ; Pinto, John T. ; Deng, Haiteng ; Richie, John P. / Inhibition of caspase-3 activity and activation by protein glutathionylation. In: Biochemical Pharmacology. 2008 ; Vol. 75, No. 11. pp. 2234-2244.
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Inhibition of caspase-3 activity and activation by protein glutathionylation. / Huang, Zhishan; Pinto, John T.; Deng, Haiteng; Richie, John P.

In: Biochemical Pharmacology, Vol. 75, No. 11, 01.06.2008, p. 2234-2244.

Research output: Contribution to journalArticle

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