Pig heart peroxisomal carbonyl reductase (PerCR) belongs to the short-chain dehydrogenase/reductase family, and its sequence comprises a C-terminal SRL tripeptide, which is a variant of the type 1 peroxisomal targeting signal (PTS1) Ser-Lys-Leu. PerCR is imported into peroxisomes of HeLa cells when the cells are transfected with vectors expressing the enzyme. However, PerCR does not show specific targeting when introduced into the cells with a protein transfection reagent. To understand the structural basis for peroxisomal localization of PerCR, we determined the crystal structure of PerCR. Our data revealed that the C-terminal PTS1 of each subunit of PerCR was involved in intersubunit interactions and was buried in the interior of the tetrameric molecule. These findings indicate that the PTS1 receptor Pex5p in the cytosol recognizes the monomeric form of PerCR whose C-terminal PTS1 is exposed, and that this PerCR is targeted into the peroxisome, thereby forming a tetramer.
We have recently purified a tetrameric carbonyl reductase from the cytosolic fraction of pig heart (pig heart carbonyl reductase, PHCR), using 4-benzoylpyridine (4-BP) as the substrate. PHCR has the ability to catalyze efficiently the reduction of 9,10-phenanthrenequinone (PQ) contained in diesel exhaust particles (DEPs). Thus, the present study was attempted to characterize the inhibitory effect of PQ on the reduction of 4-BP and all-trans-retinal in pig heart cytosol. Of the DEP components examined, PQ was the most potent inhibitor for the reduction of 4-BP and all-trans-retinal in pig heart cytosol. PQ also inhibited competitively the 4-BP reduction. These results indicate that PQ inhibits the reduction of 4-BP and all-trans-retinal by acting PHCR present in pig heart cytosol. Furthermore, whether PQ induces the formation of superoxide anion radical was examined in pig heart cytosol. The absorbance of cytochrome c at 550 nm was increased with the time by adding PQ, and the increased absorbance was decreased in the presence of superoxide dismutase. A similar result was observed in the reaction system of recombinant PHCR. On the basis of these results, it is concluded that PQ not only inhibits the reduction of 4-BP and all-trans-retinal catalyzed by PHCR but also mediates superoxide formation through its redox cycling involved in PHCR. We propose the possibility that PQ disturbs the homeostasis of retinoid metabolism and induces oxidative stress in pig heart.
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