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.
Highlights
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 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
Chimeric proteins that included green fluorescent protein (GFP) were expressed in RBL-2H3 cells in order to determine whether the leader sequence of the L-form could serve to target GFP to the mitochondria of living cells
Summary
Reagents—Antibodies against PHGPx and cGPx were prepared as described previously [17]. Cytochrome c oxidase was distributed in nuclear (7.9%), mitochondrial (75.6%), microsomal (3.5%), and cytosolic (12.9%) fractions of M15 cells. NADPH-cytochrome c reductase activities were found in nuclear (5.9%), mitochondrial (16.7%), microsomal (68.3%), and cytosolic (9.1%) fractions of M15 cells. Activities of PHGPx and cGPx were measured after the fractionation of cytosol and mitochondria from each cell line (1.5 ϫ 108 cells). The supernatants obtained from mitochondrial fraction and cytosolic fraction were used for assays of PHGPx and cGPx activities. GFP and mitochondria were simultaneously detected in the same cells by the double staining with GFP fluorescence and a monoclonal antibody of Cy3-conjugated anti-cytochrome c oxidase subunit IV that was a specific probe for the mitochondrial staining [27].
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