Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase Plays a Major Role in Preventing Oxidative Injury to Cells

Abstract

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is synthesized as a long form (L-form; 23 kDa) and a short form (S-form; 20 kDa). The L-form contains a leader sequence that is required for transport to mitochondria, whereas the S-form lacks the leader sequence. A construct encoding the leader sequence of PHGPx tagged with green fluorescent protein was used to transfect RBL-2H3 cells, and the fusion protein was transported to mitochondria. The L-form of PHGPx was identified as the mitochondrial form of PHGPx and the S-form as the non-mitochondrial form of PHGPx since preferential enrichment of mitochondria for PHGPx was detected in M15 cells that overexpressed the L-form of PHGPx, whereas no similar enrichment was detected in L9 cells that overexpressed the S-form. Cell death caused by mitochondrial injury due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was considerably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytotoxic effects of KCN. Flow cytometric analysis showed that mitochondrial PHGPx suppressed the generation of hydroperoxide, the loss of mitochondrial membrane potential, and the loss of plasma membrane integrity that are induced by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functions and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cyanide m-chlorophenylhydrazone, which directly reduces membrane potential without the generation of hydroperoxides. M15 cells were more resistant than L9 cells to cell death caused by direct damage to mitochondria and to extracellular oxidative stress. L9 cells were more resistant to tert-butylhydroperoxide than S1 cells, whereas resistance to t-butylhydroperoxide was even more pronounced in M15 cells than in L9 cells. These results suggest that mitochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx, might play a primary role in protecting cells from oxidative stress.

Oxidative stress and regulation of glutathione in lung inflammation

“…CHEN et al [220] have recently demonstrated that depletion of mitochondrial GSH in human umbilical vein endothelial cells (HUVECs) increased TNF-a-induced adhesion molecule (VCAM-1) expression but not ICAM-1 expression and mononuclear leukocyte adhesion in HUVECs, suggesting that mitochondrial GSH is involved in endothelial cell function [220]. Recent studies have shown that gene delivery of glutathione reductase to mitochondria and overexpression of GPx in various cell lines provided protection against oxidative stress [224,225]. This finding demonstrates the importance of mitochondrial GSH homeostasis in the regulation of cell function.…”

Depletion of Selenoprotein GPx4 in Spermatocytes Causes Male Infertility in Mice

“…The most abundant GPx4 of testis is associated with mitochondria (16). Overexpression of mitochondrial GPx4 in RBL2h3 cells suppresses mitochondrial-derived reactive oxygen species produced by the respiratory chain inhibitors (17) and suppresses apoptosis induced by 2-deoxyglucose, staurosporine, etoposide, and UV (18,19). In spermatozoa, GPx4 is also mainly localized in the mitochondria (13,16).…”

Resistance of Mitochondrial DNA-depleted Cells against Cell Death

“…Next we studied the expression of H 2 O 2 metabolizing en- (25). RT-PCR analysis showed that the expression of 2 types of GPx (cGPx and PHGPx) that are expressed in mitochondria (25) was increased in SK-Hep1 0 cells compared with parental cells (Fig.…”