Journal of Biological Chemistry
Volume 295, Issue 50, 11 December 2020, Pages 17227-17240
Journal home page for Journal of Biological Chemistry

Protein Synthesis and Degradation
Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

https://doi.org/10.1074/jbc.RA120.014919Get rights and content
Under a Creative Commons license
open access

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.

heme oxygenase
heme homeostasis
heme regulatory motif
enzyme degradation
autophagy
chaperone-mediated autophagy
metabolic regulation
membrane proteins
protein degradation
post-transcriptional regulation
metal homeostasis
membrane enzyme

Cited by (0)

This article contains supporting information.

Author contributions—L. L., A. B. D., and S. W. R. conceptualization; L. L., A. B. D., A. S. F., and S. W. R. formal analysis; L. L. validation; L. L., A. B. D., A. S. F., and S. W. R. investigation; L. L. and S. W. R. visualization; L. L. and A. B. D. methodology; L. L. and S. W. R. writing-original draft; L. L., E. N. G. M., and S. W. R. project administration; L. L., A. B. D., A. S. F., E. N. G. M., and S. W. R. writing-review and editing; E. N. G. M. and S. W. R. supervision; S. W. R. funding acquisition.

Funding and additional information—This work was supported by National Institutes of Health Grants R01-GM123513 (to S. W. R.) and R01-GM093088 (to E. N. G. M.); and by American Heart Association Predoctoral Fellowship 19PRE34380029 (to L. L.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    6-AN

    6-aminonicotinamide

    ALAS1

    d-aminolevulinic acid synthase 1

    BAF

    bafilomycin

    CHX

    cycloheximide

    CMA

    chaperone-mediated autophagy

    ERAD

    endoplasmic reticulum-associated degradation

    HD

    heme-depleted

    HDX-MS

    hydrogen-deuterium exchange mass spectrometry

    HO

    heme oxygenase

    HRM

    heme regulatory motif

    3-MA

    3-methyladenine

    SA

    succinylacetone

    ER

    endoplasmic reticulum

    DAPI

    4′,6-diamidino-2-phenylindole

    TBST

    Tris-buffered saline with Tween

    PBST

    PBS containing 0.1% Tween 20.