Abstract
Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans.
Highlights
Heme oxygenase (HO) is the only known enzyme degrading pro-oxidant heme to the antioxidant biliverdin, iron and carbon monoxide [1]
When homogenates of Sf9 cells were used instead of cytosolic fractions both HO-1 and HO-2 were catalytically active regardless of exogenous cytochrome P450 reductase (CPR) (Figure 1B). This indicates that endogenous CPR activity in Sf9 cells can support HO activity, but the functional interaction is lost for HO-1 upon the preparation of cytosol
In the present study we show for the first time that CPR prevents nuclear translocation of HO-1 under hypoxic conditions and promotes oligomerization of HO-1 into higher ordered complexes
Summary
Heme oxygenase (HO) is the only known enzyme degrading pro-oxidant heme to the antioxidant biliverdin, iron and carbon monoxide [1]. This reaction requires three mol of oxygen and seven electrons supplied by NADPH cytochrome P450 reductase (CPR) [2]. The constitutive HO-2 (36 kDa) is mainly found in brain and testis [5]. Both HO-isoforms as well as the CPR are anchored with a stretch of hydrophobic amino acids to the outer membrane of the endoplasmic reticulum [6,7]. Regulated intramembrane proteolysis of the carboxy-terminus of HO-1 under conditions of hypoxia will weaken the functional interaction with CPR leading to a loss in enzyme activity [10]
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