Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

Liu Liu,Arti B Dumbrepatil,Angela S Fleischhacker,E Neil G Marsh,Stephen W Ragsdale

doi:10.1074/jbc.ra120.014919

Liu Liu, Arti B Dumbrepatil + Show 3 more

Open Access

https://doi.org/10.1074/jbc.ra120.014919

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Abstract

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

Highlights

Heme is a commonly used prosthetic group in a myriad of biological processes
Given that the most common role of heme regulatory motif 3-MA (HRM) is to promote protein degradation upon binding heme, we first asked whether the two HRMs in Heme oxygenase-2 (HO2) control protein stability and degradation
We showed that substituting Cys-265 or Cys282 with Ala abolishes heme binding to HRM1 or HRM2, 17228 J

Summary

Introduction

Heme is a commonly used prosthetic group in a myriad of biological processes. High levels (.1 mM) of free heme are seen in many pathological processes and diseases, such as cardiovascular diseases [1]. In contrast to what has been observed with ALAS1 or Bach1, in which a loss-of-function mutation in the HRM leads to a higher cellular level of protein [19, 21], all three HRM variants of HO2 maintained a steady-state expression level very similar to that of WT protein (Fig. 1, B and C).

Results

Conclusion