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

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 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, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type 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.

Unravelling the mechanisms controlling heme supply and demand

“…S4 ), the sensor will not perturb the cellular biochemistry by depleting the availability of heme (forming a complex AH) as long as the total concentration of heme exceeds or equals 3 μM (the value reported in ref. 46 from denatured cell lysates). In this model, the total concentration of heme will include a minute fraction of free heme (estimated to be 5 nM; as in Fig.…”

“…The concentration of the holo form of the heme sensor, [AH], is shown in light red), where the total concentration of the expressed sensor, [A] 0 = [A] + [AH] is given on the horizontal axis ([A] = concentration of the apo form of the sensor). The model has been created by assuming that the total concentration of heme, [H] 0 = [H] + [BH] + [AH], is 3 μM ( 46 ); the concentration of free heme, [H], in the absence of a sensor or acceptor protein is 5 nM (as observed in Fig. 2 , Lower Left ); and the K d for heme dissociation from the sensor is 22 nM ( Fig.…”

Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

“…HO-2 binds heme at its catalytic site but also bears two additional heme binding sites termed heme regulatory motifs (HRMs), which contain Cys-Pro motifs [ 67 ]. In recent studies, mutation in the HRMs of HO-2 was found to accelerate HO-2 turnover via chaperone-mediated autophagy, suggesting that heme bioavailability regulates post-translational stability of HO-2 [ 68 ]. Ferric heme bound to HRMs may be reversibly transferred to the HO-1 catalytic site for degradation [ 69 ].…”

The Chemical Basis of Intracerebral Hemorrhage and Cell Toxicity With Contributions From Eryptosis and Ferroptosis

“…Heme oxygenase 2 (HO-2) is expressed continuously in neurons and is not inducible (Chang et al, 2003 ). The regulation mechanism of HO-2 is not clear, however recent work by Liu et al ( 2020 ) demonstrated that HO-2 is stabilized by hemin. HO-2 carries three docking sites for heme but only one is catalytic, the remaining two are heme regulatory motifs that bind heme; unlike other heme-regulated proteins, HO-2 is only destabilized by a loss of heme binding to the catalytic site, not the regulatory sites (Liu et al, 2020 ).…”

“…The regulation mechanism of HO-2 is not clear, however recent work by Liu et al ( 2020 ) demonstrated that HO-2 is stabilized by hemin. HO-2 carries three docking sites for heme but only one is catalytic, the remaining two are heme regulatory motifs that bind heme; unlike other heme-regulated proteins, HO-2 is only destabilized by a loss of heme binding to the catalytic site, not the regulatory sites (Liu et al, 2020 ). Heme oxygenase 1 (HO-1) is not expressed in the brain under normal circumstances but is expressed in macrophages (Naito et al, 2014 ; Vijayan et al, 2018b ), microglia (Schallner et al, 2015 ), and astrocytes localized to the peripheral tissue surrounding the hematoma from ICH (Chang et al, 2003 ; Yu et al, 2016 ).…”