The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Fleischhacker, Angela S.; Gunawan, Amanda L.; Kochert, Brent A.; Liu, Liu; Wales, Thomas E.; Borowy, Maelyn C.; Engen, John R.; Ragsdale, Stephen W.

doi:10.1074/jbc.ra120.012803

Cited by 18 publications

(23 citation statements)

References 49 publications

(78 reference statements)

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“…To explore the role of heme binding to the catalytic site in regulating HO2 stability, we generated a catalytic site loss-of-function mutant, H45W/G159W, for expression in HEK293 cells. In vitro experiments have confirmed that in H45W/G159W variant, there is no detectible heme binding at the catalytic site and that the overall structure and heme binding to the HRMs are unperturbed (32). As expected, no changes were observed in steady-state expression levels of the H45W/G159W mutant upon cellular heme level variation (Fig.…”

Section: Resultssupporting

confidence: 70%

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

Liu

Dumbrepatil

Fleischhacker

et al. 2020

Journal of Biological Chemistry

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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.

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Section: Resultssupporting

confidence: 70%

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

Liu

Dumbrepatil

Fleischhacker

et al. 2020

Journal of Biological Chemistry

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“…To explore the role of heme binding to the catalytic site in regulating HO2 stability, we generated a catalytic site loss-of-function mutant, H45W/G159W, for expression in HEK293 cells. In vitro experiments have confirmed that in H45W/G159W variant, there is no detectible heme binding at the catalytic site and that the overall structure and heme binding to the HRMs are unperturbed (30). As expected, no changes were observed in steady-state expression levels of the H45W/G159W mutant upon cellular heme level variation ( Fig.…”

Section: Resultssupporting

confidence: 70%

“…The marked effect of heme occupancy at the catalytic site on HO2 stability also led us to explore whether increasing heme affinity of the catalytic site would further stabilize HO2 protein, leading to a higher steady-state HO2 level than wild-type. Therefore, the G163H variant, also catalytically inactive (Table S1), however with a 100-fold higher heme affinity of the catalytic site (30), was tested. Surprisingly, both the G163D and G163H variants showed similar steady-state protein levels as wild type, and the G163H variant has a similar half-life as wild-type HO2 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although these properties contrast with those of other HRM-containing proteins, they are consistent with HO2's role as a heme detoxification enzymeit would be counterproductive for heme to reduce HO2 level and hence its heme metabolic activity. In light of HRM's function in HO2, we recently proposed a heme transfer model in which HO2 readily transfers HRM-bound Fe 3+ -heme to the catalytic site for degradation to facilitate turnover and equilibrates heme between the HRMs and the catalytic core to maintain heme homeostasis through heme-dependent conformational changes in the protein (30). This model defines a novel function for the HRM, while maintaining consistency with a conformational change model proposed for another HRM-containing protein, Bach1, in which heme induces local conformational changes that alter interactions with its binding partners (39).…”

Section: Discussionmentioning

confidence: 99%

“…Our recent hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies demonstrated that in H45W/G159W variant, the tryptophan mutations themselves led to minimal to no structural perturbations and that the only observed differences in HDX-MS related to heme binding to the catalytic core were specific to the distal and proximal helices, which form the heme binding pocket (45). Furthermore, the circular dichroism spectra of wild-type HO2 and the H45W/G159W variant were nearly indistinguishable (30). In a search for potential signals for HO2 degradation in the absence of heme, we identified a KFERQ-like motif, which has been classified as a recognition motif by CMA.…”

Section: Discussionmentioning

confidence: 99%

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Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

Liu

Dumbrepatil

Fleischhacker

et al. 2020

Preprint

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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. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; 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. However, cellular heme overload does not affect HO2 stability. 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.

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A Model Peptide Reveals Insights into the Interaction of Human Hemopexin with Heme

Hopp

George²,

Ramoji

et al. 2022

Int J Pept Res Ther

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Under hemolytic conditions, toxic heme is scavenged by hemopexin. Recently, the heme-binding properties of hemopexin have been reassessed, which revealed a KD of ~ 0.32 nM as well as a stoichiometry of one to two heme molecules binding to hemopexin. A 66mer hemopexin-derived peptide that spans over three heme-binding motifs was used to verify the earlier suggested heme-recruiting mechanism. Herein, we employed spectroscopic and computational methods to substantiate the hypothesis of more than one heme molecule binding to hemopexin and to analyze the heme-binding mode. Both, hemopexin and the 66mer peptide, were found to bind heme in mixed penta- and hexacoordinated states, which strongly indicates that heme binding follows distinct criteria and increases rigidity of the peptide-heme complex. Additional in silico molecular dynamics simulations support these experimental findings and, thus, contribute to our understanding of the molecular basis of the heme-hemopexin interaction. This analysis provides further details for consideration of hemopexin in biomedical applications.

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The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Cited by 18 publications

References 49 publications

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

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

Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

A Model Peptide Reveals Insights into the Interaction of Human Hemopexin with Heme

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