Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/ oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266 -285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the selfassembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS ␣-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO) 3 catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common pheno...
Heme oxygenase‐1 (HO‐1) catalyzes the oxidative degradation of heme to biliverdin. Previous studies suggest that HO‐1 exists as a monomer in ER. In the present study, we performed chemical cross‐linking and fluorescence resonance energy transfer (FRET) experiments to show that HO‐1 forms dimers in ER. However, the dimerization was not observed with a truncated HO‐1 which lacks the C‐terminal transmembrane segment (TMS) (aa 266‐285) and exhibits cytosolic and nuclear localization, indicating that the TMS is essential for the self‐assembly of HO‐1 in native membrane. To identify the interface of TMS‐TMS interaction, W270 predicted by molecular modeling with stronger hydrophobic force in the interface was mutated and the effect on protein subcellular localization and dimerization was assessed. The results showed that the W270A mutant was present exclusively in ER and formed dimers as the wild type HO‐1. Interestingly, W270N mutant was localized not only in ER but also in cytosol and nucleus as well, suggesting that it is susceptible to proteolytic cleavage. Moreover, W270N mutation appeared to interfere with dimeric state as revealed by the lower FRET efficiency. Collectively, these data suggest that the dimerization driven by the hydrophobic interaction in the TMS is implicated in the stabilization of HO‐1 in ER. This work was supported by a grant from National Science Council of Taiwan (NSC‐95‐2320‐B‐001‐008).
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