Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells

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 lea der sequence of PHGPx tagged with green fluorescent protein was used to tra nsfect RBL-2H3 cells, and the fusion protein was transported to mitochondri a. The L-form of PHGPx was identified as the mitochondrial form of PHGPx an d the S-form as the non-mitochondrial form of PHGPx since preferential enri chment of mitochondria for PHGPx was detected in M15 cells that overexpress ed the L-form of PHGPx, whereas no similar enrichment was detected in L9 ce lls that overexpressed the S-form. Cell death caused by mitochondrial injur y due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was conside rably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytot oxic effects of KCN, Flow cytometric analysis showed that mitochondrial PHG Px suppressed the generation of hydroperoxide, the loss of mitochondrial me mbrane potential, and the loss of plasma membrane integrity that are induce d by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functi ons and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cya nide m-chlorophenylhydrazone, which directly reduces membrane potential wit hout the generation of hydroperoxides. M15 cells were more resistant than L 9 cells to cell death caused by direct damage to mitochondria and to extrac ellular oxidative stress. L9 cells were more resistant to tert-butylhydrope roxide than S1 cells, whereas resistance to t-butylhydroperoxide was even m ore pronounced in M15 cells than in L9 cells. These results suggest that mi tochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHG Px, might play a primary role in protecting cells from oxidative stress.