Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST

doi:10.1073/pnas.0400181101

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST

Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104

Communicated by Mildred Cohn, University of Pennsylvania School of Medicine, Philadelphia, PA, January 9, 2004 (received for review May 20, 2003)

Abstract

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of πGST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated πGST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated πGST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express πGST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of πGST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with πGST followed by its GSH-mediated reduction and enzyme activation.

Footnotes

↵ * To whom correspondence should be addressed at: Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. E-mail: abf{at}mail.med.upenn.edu.
This work was presented in part at Experimental Biology 2003, April 11-15, 2003, San Diego, CA [Manevich, Y., Feinstein, S. I. & Fisher, A. B. (2003) FASEB J. 17, A156 (abstr.)].
Abbreviations: 1-cysPrx, 1-cys peroxiredoxin; GSH, glutathione; PLPCOOH, 1-palmitoyl-2-linolenoyl hydroperoxide-sn-glycero-3-phosphocholine; sulfo-SBED, sulfosuccinimidyl[2,6-(biotinamido)-2-(p-azidobenzamido)-hexanamido]ethyl-1,3-dithiopropionate.
↵ † This protein also has been called antioxidant protein 2 (AOP2), nonselenium GSH peroxidase (NSPGPx), acidic Ca²⁺-independent phospholipase A₂ (aiPLA₂), and p67^phox-binding protein. The corresponding cDNA has been called ORF6.
↵ ‡ It is not clear whether the active form of 1-cysPrx is the monomer or the homodimer.