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. 2026 Sep 16;11(38):57354–57361. doi: 10.1021/acsomega.6c05685

Bottom-Up Model Suggesting Membrane Localization of Heme Oxygenase‑1 Contributes to Heme Selection for Degradation

Ryusei Matsutani †, Yuka Kodama †, Tomoichiro Kusumoto †, Masakazu Sugishima ‡, Hiroshi Sakamoto †, Junichi Taira †,*
PMCID: PMC13629402  PMID: 42824789

Abstract

Heme functions as a prosthetic group in numerous hemoproteins, whereas a fraction of intracellular heme is not tightly bound to proteins or membranes and can readily dissociate; this labile heme participates in the regulation of diverse cellular processes. Heme has pro-oxidant properties and is cytotoxic at elevated levels. Accordingly, heme oxygenase-1 (HO-1), an enzyme localized to the endoplasmic reticulum membrane, degrades excess intracellular heme. However, how HO-1 gains access to the excess intracellular heme for degradation remains unclear. In this study, we investigated how the membrane association of HO-1 influences heme capture using a bottom-up approach. A rat HO-1-based fluorescent probe retaining the membrane-anchoring region and capable of detecting heme binding via fluorescence quenching was reconstituted into liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). An HO-1 variant lacking the membrane-anchoring region competitively inhibited heme binding to the probe in the presence of POPC liposomes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein proposed to function as a heme chaperone. In contrast, membrane-associated HO-1 showed enhanced fluorescence quenching associated with heme binding in the presence of liposomes compared with aqueous conditions, although competition with GAPDH was still observed. These findings suggest that membrane environments may facilitate the access of membrane-associated HO-1 to chaperone-unbound heme under the reconstituted conditions. Our results suggest a model in which membrane localization of HO-1 contributes to the spatial regulation of intracellular heme processing and heme selection for degradation.

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1. Introduction

Heme, a porphyrin–iron complex, constitutes a major form of cellular iron and plays essential roles as a prosthetic group in oxygen transport, catalysis, and electron transfer across organisms from bacteria to humans. In addition, the regulatory roles of labile heme, defined as heme that does not function as a prosthetic group of heme proteins, have been extensively studied. For example, 5-aminolevulinic acid synthase, the rate-limiting enzyme in heme biosynthesis, is feedback-inhibited by labile heme. , Labile heme binding to iron regulatory proteins modulates intracellular iron homeostasis through translational control of the transferrin receptor and ferritin. Labile heme has also been implicated in the regulation of circadian rhythms via binding to the nuclear receptor Rev-erbα Furthermore, the induction of heme oxygenase-1 (HO-1) is triggered by labile heme binding to the transcriptional repressor Bach1, a key regulator of cellular stress responses. Although a cytosolic labile heme pool has been debated, accumulating evidence suggests that labile heme is unlikely to be present at significant levels in cells. Instead, intracellular heme trafficking is now proposed to occur through regulated, transient interactions with putative heme chaperone proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ,

While labile heme participates in diverse cellular regulatory processes, it can also promote the generation of reactive oxygen species via the Fenton reaction, thereby exerting strong cytotoxic effects. Excess intracellular labile heme can act as a pro-oxidant and activate innate immune pathways, thereby amplifying cytokine production. , Labile heme accumulation under pathological conditions has been implicated in promoting a vicious cycle of oxidative stress and inflammation in diseases including diabetes and cancer. , To prevent heme-induced oxidative injury, surplus heme is oxidatively degraded into biliverdin, free iron, and carbon monoxide (CO) by heme oxygenase (HO) on the endoplasmic reticulum (ER) membrane, in concert with cytochrome P450 reductase (CPR) (Figure ). Among the HO isoforms, HO-1 is an inducible enzyme that is robustly upregulated in response to oxidative stress, including excess heme. HO-1 has been considered to exert cytoprotective effects against oxidative stress by catalyzing the conversion of pro-oxidant heme into biliverdin, the precursor of the potent antioxidant bilirubin. − In addition to its antioxidant function, HO-1 plays a critical physiological role in intracellular iron recycling. Recombinant HO-1 retaining the membrane-anchoring region is difficult to maintain in a soluble form; consequently, many mechanistic and structural studies have relied on truncated variants lacking the C-terminal membrane-anchoring region. Although membrane localization of HO-1 is required for its interaction with CPR and catalytic activity, − the functional significance of this membrane association in intracellular heme metabolism remains poorly characterized.

1.

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Membrane-dependent heme selection and processing model and HO-1 based heme probe. (A) Proposed model for membrane-dependent processing of intracellular labile heme. Labile heme that fails to associate with heme chaperones accumulates on cellular membranes, where it is captured and degraded by membrane-localized HO-1. (B) Design of membrane-bound HO-1-based heme probes. Upper panel: structural features of mbHO-1 and sHO-1. Structurally defined regions are indicated in gray. Alexa Fluor 555 was site-specifically introduced at Cys18. Lower panel: Schematic representation of a liposome-associated mbHO-1 probe, illustrating fluorescence quenching upon heme binding via energy transfer from fluorescent dye to heme.

HO-1 shares several properties with ER-resident monooxygenases such as cytochrome P450 (CYP) enzymes, including electron transfer from CPR and degradation of hydrophobic substrates. Because membrane localization of CYP enzymes is essential for binding of hydrophobic substrates, − we hypothesized that heme in excess of the buffering capacity of heme chaperones partitions into the ER membrane, where it is captured by HO-1 (Figure A). Accumulation and intracellular trafficking of heme at membranes have been reported; , however, the physiological role of HO-1 membrane localization and whether it facilitates substrate access remain unclear. We therefore reconstructed a minimal system and analyzed competition between membrane-associated HO-1 and cytosolic heme chaperones for heme binding. This bottom-up approach suggests that membrane association of HO-1 enhances access to degradative heme.

2. Materials and Methods

2.1. Preparation of Rat HO-1-Based Fluorescent Probes

The cDNAs for recombinant rat HO-1 (rHO-1; UniProtKB: P06762) variants were constructed based on our previously reported rHO-1­(K18C/D140H). The cDNA was subcloned into the NdeI–XhoI sites of pET15b, and an additional R254K substitution was introduced, yielding rHO-1(1–266)­K18C/D140H/R254K. To generate the full-length variant, the transmembrane region (residues 267–289) was reconstructed by insertion of a codon-optimized synthetic polynucleotide, resulting in rHO-1(1–289)­K18C/D140H/R254K (Figure S1). The plasmids were transformed into Escherichia coli BL21­(DE3) and the transformants were grown in Luria–Bertani broth containing 100 μg/mL ampicillin, and protein expression was induced with 0.1 mM isopropyl β-D-thiogalactopyranoside for 6 h at 30 °C with aeration. Cells were harvested by centrifugation (4 °C, 5,000 × g, 20 min) and resuspended in extraction buffer containing 50 mM Tris-HCl (pH 8.0), 0.2 mg/mL lysozyme, 2 mM ethylenediaminetetraacetic acid, 200 μM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1% Triton X-100, and cOmplete ULTRA Tablets, Mini EDTA-free protease inhibitors (Roche Diagnostics). Following a 1 h incubation, cells were lysed by sonication, and insoluble material was removed by centrifugation (4 °C, 8,000 × g, 30 min). His6-tagged rHO-1 mutants were purified from the supernatant using Ni Sepharose 6 Fast Flow resin (Cytiva). The resin was equilibrated with 20 mM Tris-HCl (pH 8.0), 25 mM imidazole, and 150 mM KCl, and bound proteins were eluted stepwise with 20 mM Tris-HCl (pH 7.4) containing 50, 100, 150, or 200 mM imidazole. Purified rHO-1(1–289)­K18C/D140H/R254K and rHO-1(1–266)­K18C/D140H/R254K were labeled at Cys18 with Alexa Fluor 555 C2-maleimide (Thermo Fisher Scientific). In brief, 3-fold molar excess of the dye was added, and the reaction mixture was incubated for 2 h at room temperature. After labeling, unreacted maleimide groups were quenched by the addition of β-mercaptoethanol. Excess dye and salts were removed by gel-filtration chromatography on a Sephadex G-25 column equilibrated with buffer. The labeled rHO-1 fraction was collected and concentrated using Amicon Ultra-15 (Cytiva). Protein concentration was determined by UV absorbance and the bicinchoninic acid assay, and the samples were used immediately for subsequent experiments. Protein purity was assessed by SDS–PAGE (Figure S2).

2.2. Expression and Purification of hGAPDH

Human GAPDH (hGAPDH; UniProtKB: P04406) cDNA (FlexiORF Clone FXC02698, Promega) was subcloned into the BamHI–SalI sites of pGEX-6P-3 to express glutathione S-transferase (GST)-tagged hGAPDH in E. coli BL21­(DE3). Protein expression and cell lysis were performed under the same conditions described for rHO-1. GST-hGAPDH was purified using Glutathione Sepharose 4B resin (Cytiva). The resin was equilibrated with phosphate-buffered saline, and bound GST-hGAPDH was eluted with 50 mM Tris-HCl (pH 8.0) containing 3 mg/mL reduced glutathione. The GST tag was removed by incubation with 10 unit of Precision Protease (Cytiva) for 2 h at 4 °C, and hGAPDH was recovered as the flow-through fraction after passage through the Glutathione Sepharose 4B resin. Purified hGAPDH was concentrated and desalted by ultrafiltration, and protein purity and concentration were assessed as above.

2.3. Liposome-Binding Assay

A lipid film of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; FUJIFILM Wako Pure Chemical) was hydrated with 2 mL of 20 mM MOPS buffer (pH 7.0) containing 0.1 M sucrose to obtain a final POPC concentration of 1.6 mM. The mixture was incubated at 37 °C for 3 h without agitation to prepare a POPC liposome suspension. The liposome suspension was diluted sixfold by adding 10 mL of 20 mM MOPS buffer containing 0.1 M glucose. To assess hemin adsorption onto liposomes, BODIPY 493/503 (42 nM) and hemin (2 nM) were added to the liposome suspension. To evaluate the adsorption of recombinant rHO-1 onto liposomes, either rHO-1(1–266)­K18C/D140H/R254K or rHO-1(1–289)­K18C/D140H/R254K was incubated with liposomes. The sample was centrifuged at 15,000 × g for 10 min to separate the supernatant from sedimented liposomes. After centrifugation, five sequential 2 mL supernatant fractions were carefully collected from the top of the tube (fractions 1–5). The remaining 2 mL containing the sedimented liposomes was then gently resuspended and collected as fraction 6, resulting in six fractions of identical volume (2 mL each). Liposome sedimentation was assessed by BODIPY 493/503 fluorescence excited at 470 nm. Heme levels in each fraction were quantified using the Hemin Assay Kit (Sigma-Aldrich). Accumulation of recombinant HO-1 on the liposome was assessed by immune blotting visualized with horseradish peroxidase (HRP)-conjugated Anti-His-tag monoclonal antibody (MBL).

2.4. Fluorescence Imaging

The fluorescence–labeled recombinant rHO-1 proteins were mixed with BODIPY 493/503-labeled POPC liposomes and incubated for approximately 1 h on ice in the dark. The samples were then placed on MAS-GP type A amine-coated, anti-peeling microscope slides (Matsunami Glass) and imaged using an FV1200 confocal laser scanning microscope (Olympus). Microscope operation and image acquisition were performed using the FV10-ASW software package, and the resulting images were analyzed using Fiji.

2.5. Hemin Titration

The Alexa Fluor 555-labeled rHO-1 variants (50 nM) were titrated with hemin added incrementally at 0.1-equivalent steps. Fluorescence spectra were recorded on a JASCO FP-8550 spectrofluorometer using an excitation wavelength of 545 nm and monitoring emission from 560 to 650 nm. For measurements in the presence of POPC liposomes (16 mM), a POPC lipid film was hydrated with 20 mM potassium phosphate buffer (pH 7.4) to obtain vesicles, which were subsequently sonicated to prepare small unilamellar vesicles (SUVs). The SUVs were incubated with the recombinant HO-1 for 1 h at 4 °C, then subjected to the hemin titration assay under the same fluorescence measurement conditions as for recombinant HO-1 without liposomes. The apparent dissociation constant (K d) for heme–HO-1 binding was obtained by nonlinear curve fitting as previously reported (Figure S3).

2.6. Fluorescence Measurements by Microplates

Changes in fluorescence intensity of Alexa Fluor 555-labeled rHO-1 variants (50 nM) were measured in the presence of hemin (25 nM, 0.5 molar equivalent), 16 mM POPC SUVs, and 250 nM hGAPDH. Proteins and liposomes were preincubated at 4 °C for 1 h prior to measurement. Fluorescence experiments were performed in a total volume of 100 μL in black 96-well microplates (Nunc). Fluorescence was measured at 25 °C using a TECAN Infinite M200 PRO microplate reader with excitation at 545 nm and emission at 590 nm. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was evaluated using Student’s t-test (two-tailed), with p < 0.05 considered statistically significant.

3. Results

3.1. Heme Capturing Model of rHO-1 on Lipid Membranes

To examine whether membrane association of HO contributes to cellular discrimination of cytotoxic heme, we evaluated heme capture by HO-1 on lipid membranes using a bottom-up approach. We employed an HO-1-based fluorescent probe previously developed for heme detection via fluorescence quenching through intramolecular energy transfer. Alexa Fluor 555-labeled rHO-1(1–289)­K18C/D140H/R254K, which retains the membrane-anchoring region (residues 267–289), is referred to as membrane-bound HO-1 (mbHO-1). The K18C substitution allows site-specific fluorescent labeling, D140H renders the enzyme catalytically inactive, and R254K prevents proteolytic degradation. The truncated variant which lacks the membrane-anchoring region is referred to as soluble HO-1 (sHO-1) (Figure B upper panel). Because mbHO-1 exhibits a strong tendency to aggregate, all handling steps following preparation were performed rapidly without freeze–thaw cycles. A schematic illustration of the liposome-complexed probe, in which mbHO-1 is reconstituted on liposomes, is shown in lower panel of Figure B. Liposomes composed of the zwitterionic lipid POPC were chosen as a model of the highly fluid ER membrane. In the liposome-complexed probe, heme binding to membrane-associated HO-1 was monitored by fluorescence quenching.

3.2. Interaction of Heme and Recombinant rHO-1 Proteins with POPC Liposomes

We examined the interaction of heme with lipid membranes. POPC liposomes encapsulating sucrose were incubated with hemin and subsequently separated into pellet and supernatant fractions by centrifugation. The amounts of heme in the resuspended liposome pellet and in the supernatant were quantified (Figure ). Sedimentation of liposomes by centrifugation was confirmed by BODIPY labeling (Figure S4). A significantly higher heme concentration was detected in the liposome pellet fraction, indicating preferential partitioning of heme into the lipid membrane.

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Interaction of heme with POPC liposomes. Left panel: Schematic representation of the preparation of fractions. Liposomes were incubated with heme prior to sedimentation analysis. Fractions 1–5 represent five sequential 2 mL supernatant fractions obtained from the top of the tube after centrifugation. The remaining 2 mL containing sedimented liposomes was resuspended and collected as fraction 6. No sedimentation of heme alone was observed under the centrifugation conditions. Right panel: Relative hemin concentrations in each fraction were determined using a hemin assay kit. Data are presented as mean ± SD (n = 3) from at least three independent experiments. Statistical significance was evaluated using two-tailed Student’s t-test.

Next, formation of the liposome-complexed probe was evaluated by fluorescence colocalization analysis using BODIPY-labeled liposomes and Alexa Fluor 555 (A555)-labeled mbHO-1. Liposomes large enough to be observed by light microscopy were prepared without sonication, incubated with mbHO-1, and analyzed by confocal fluorescence microscopy (Figure A). Fluorescence derived from A555 conjugated to HO-1 was clearly detected along the liposome membrane, suggesting membrane association of mbHO-1. Quantitative analysis of Alexa Fluor 555 fluorescence revealed a membrane-to-background fluorescence ratio of 3.6 for mbHO-1, indicating enrichment of mbHO-1 on liposomes. In contrast, under the same conditions, no membrane localization was observed for sHO-1 lacking the membrane-anchoring region. To further confirm protein–liposome interactions, mbHO-1 or sHO-1 were incubated with liposomes and separated into pellet and supernatant fractions by centrifugation. The distribution of each protein was analyzed by immunoblotting (Figure B). As expected, mbHO-1 was predominantly detected in the liposome pellet fraction. sHO-1 was also detected in the pellet fraction, although the signal intensity was lower than that of mbHO-1. Densitometric analysis showed that mbHO-1 exhibited a higher pellet-to-supernatant (P/S) ratio than sHO-1 (23.0 vs 8.9). These results indicate that the membrane-anchoring region enhances membrane association but does not completely abolish interactions between the soluble portion of HO-1 and lipid membranes upon its removal.

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Accumulation of an mbHO-1-based heme probe on POPC liposomes. (A) Confocal microscopy of POPC liposomes co-incubated with mbHO-1 or sHO-1. Fluorescence images of BODIPY 493/503 (upper panels) and Alexa Fluor 555 (lower panels) are shown. White arrowheads represent accumulated mbHO-1 on the liposomes. The scale bars indicate 15 μm. Quantitative analysis of membrane-associated Alexa Fluor 555 fluorescence was performed by calculating the membrane-to-background fluorescence ratio. (B) Immunoblot analysis of recombinant proteins accumulated on sedimented liposomes. Upper panel, schematic representation of preparation of subjected samples. No sedimentation of recombinant proteins was observed under the centrifugation conditions. Lower panel, Recombinant proteins visualized with HRP-conjugated anti-His-tag antibody. The pellet-to-supernatant (P/S) ratios were calculated by densitometric analysis of the immunoblot signals.

3.3. Heme Detection Properties of the Liposome-Complexed Probe

Fluorescence spectral changes upon titration of hemin into mbHO-1 or sHO-1 were examined by incremental addition of hemin from 0.1 to 1.5 equiv, as shown in Figure A. For both proteins, fluorescence quenching increased in a heme-dependent manner and reached a plateau at approximately one equivalent of hemin, indicating saturation of heme-binding sites. Figure B shows relationships between fluorescence intensity at 565 nm and hemin concentration. mbHO-1 exhibited a slightly nonlinear quenching profile, whereas sHO-1 showed a linear response in the absence of POPC SUVs (open circles). This nonlinear binding behavior of mbHO-1 is likely attributable to partial self-association mediated by its hydrophobic membrane-anchoring region in aqueous solution, which may reduce accessibility to the heme-binding site.

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Hemin titration experiment. (A) Changes in the fluorescence spectra of mbHO-1 and sHO-1 (50 nM) during titration with hemin. (B) Heme-binding curve showing changes in fluorescence intensity at 565 nm. Open and closed circles represent the absence and presence of POPC SUVs, respectively. The apparent K d values were calculated according to nonlinear curve fitting, and errors represent the standard errors of the fit. Arrows indicate the changes in fluorescence intensity with increasing hemin concentration upon liposome addition.

Heme titration profiles were compared after incubating sHO-1 and mbHO-1 with POPC liposomes at a lipid-to-protein molar ratio of 320. In the presence of liposomes, fluorescence quenching reflecting heme binding to sHO-1 was moderately attenuated, and a higher amount of heme was required to reach stoichiometric saturation. In contrast, mbHO-1 exhibited more efficient fluorescence quenching in the presence of liposomes than in aqueous solution; notably, addition of 0.5 equiv of heme resulted in approximately 10% greater quenching in the presence of liposomes. Consistent with these observations, the apparent K d for heme binding to sHO-1 increased from 1.5 ± 0.5 nM to 13.6 ± 6.5 nM upon addition of liposomes, whereas the apparent K d value for mbHO-1 decreased from 14.5 ± 3.2 nM to 3.8 ± 0.8 nM. These results suggest that the presence of liposomes alters the accessibility of heme to mbHO-1 under these experimental conditions. This effect may involve redistribution of heme between the aqueous and membrane phases in addition to changes in the interaction of mbHO-1 with heme.

3.4. Competitive Effects of Lipid Membranes and hGAPDH on Heme Binding to HO-1

Although further studies will be required to determine whether this mechanism operates in a cellular context, the observed results suggest that membrane structures may facilitate the access of HO-1 to heme under the experimental conditions used, particularly for heme not solubilized by cytosolic heme chaperones. We therefore evaluated the competitive effects of liposomes and hGAPDH, used here as a model heme chaperone, on heme binding to sHO-1 and mbHO-1 by monitoring fluorescence quenching upon heme binding (Figure ). hGAPDH was added at 250 nM, corresponding to a 5-fold molar excess over HO-1, to evaluate its competitive effect on heme binding, as GAPDH has been proposed to function as a heme chaperone that transiently binds and transfers heme. For sHO-1, which lacks a membrane-anchoring region, fluorescence quenching associated with heme binding was reduced by the addition of both liposomes and hGAPDH, indicating that sHO-1 binds heme in competition with lipid membranes and hGAPDH. In contrast, the enhancement of fluorescence quenching observed for mbHO-1 upon liposome addition is consistent with the results of the hemin titration experiments. Notably, even in the presence of hGAPDH, fluorescence quenching of mbHO-1 was increased by liposome addition. These observations suggest that membrane-associated mbHO-1 retains access to heme even in the presence of the cytosolic chaperone model protein hGAPDH under the present experimental conditions. Collectively, these results suggest that membrane-associated mbHO-1 has preferential access to membrane-associated heme under the present experimental conditions, even in the presence of the cytosolic chaperone model protein hGAPDH, although direct measurement of heme distribution was beyond the scope of this study.

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Comparison of fluorescence quenching of sHO-1 and mbHO-1 (50 nM) with half equivalent of hemin (25 nM) in the presence of 16 mM POPC SUVs and/or 250 nM hGAPDH. Data are presented as mean ± SD (n = 3) from at least three independent experiments. Statistical significance was evaluated using Student’s t-test.

4. Discussion

Genetically encoded heme sensors have primarily been used to investigate cytosolic heme trafficking, − though the contribution of lipid membranes to intracellular heme dynamics has remained underexplored. Since the late 2010s, it has been proposed that intracellular labile heme exists in association with proteins that bind heme weakly and function as heme chaperones, thereby conferring solubility to heme in the cytosol. , However, the mechanisms that sort intracellular heme, particularly those directing excess or unnecessary labile heme toward degradation pathways, remain poorly understood. In this study, we investigated whether membrane environments influence the accessibility of heme to HO using a bottom-up reconstitution strategy.

Consistent with the fluorescence-based analysis of heme-binding properties, mbHO-1 exhibited enhanced fluorescence quenching associated with heme binding in the presence of liposomes, whereas sHO-1 showed competitive behavior with liposomes. Comparison of mbHO-1 and sHO-1 indicates that the C-terminal membrane-anchoring region enhances membrane association and promotes heme binding under membrane-associated conditions. Furthermore, inhibition of heme binding to mbHO-1 by the heme chaperone model protein hGAPDH was reduced in the presence of liposomes, in contrast to the behavior observed for sHO-1. These observations suggest that heme chaperones contribute to maintaining heme solubility in the cytosol. In contrast, heme not efficiently solubilized under the experimental conditions may associate with membrane compartments, where it could be accessible to HO-1 (Figure A). Because the present experiments did not directly quantify the distribution of heme among the aqueous phase, lipid membranes, and proteins, the observed fluorescence changes may reflect both redistribution of heme between the aqueous and membrane phases and altered accessibility of membrane-localized HO-1 to membrane-associated heme under the experimental conditions used. However, as shown in Figure B, sHO-1 was also detected in the liposome pellet fraction, which appears inconsistent with the confocal microscopy observations (Figure A). Neither sHO-1 nor mbHO-1 was detected in the pellet fraction when centrifuged in the absence of liposomes under the same conditions, indicating that the recovery of HO-1 in the pellet fraction was dependent on the presence of liposomes. The detection of sHO-1 in the pellet fraction suggests that HO-1 can interact with lipid membranes even in the absence of the membrane-anchoring region, likely reflecting weak or nonspecific associations of the soluble portion of HO-1 with the lipid bilayer. This observation also highlights a limitation of the present reconstitution model and suggests that further refinement will be required to more precisely distinguish specific membrane anchoring from nonspecific membrane association. Our findings support a model in which membrane structures may contribute to the buffering and processing of excess heme, supporting a spatial model of heme processing. Membrane anchoring of HO-1 therefore appears to be not merely a localization determinant but a critical element in the spatial regulation of intracellular heme homeostasis.

Membrane association is expected to be important for the canonical catalytic activity of HO-1, whereas recent studies suggest that HO-1 can be released from the ER membrane through proteolytic cleavage and subsequently translocate to the nucleus, where it may exert non-canonical functions. − The present study shows that sHO-1 can still associate with membranes even in the absence of the membrane-anchoring region, suggesting that nuclear translocation of HO-1 may require additional transport factors. ,, Clarifying the mechanisms underlying HO-1 release from membranes and its non-canonical nuclear functions will require integrated top-down and bottom-up approaches.

Supplementary Material

ao6c05685_si_001.pdf (686.3KB, pdf)

Acknowledgments

This work was supported in part by MEXT/JSPS KAKENHI (grant numbers JP23K04964 and JP26K08567) and the Takeda Science Foundation to JT. The authors acknowledge Mr. M. Tokunaga and Ms. Y. Sumita for preliminary experiments on hGAPDH and liposome preparation.

Glossary

Abbreviations

CO

carbon monoxide

CPR

cytochrome P450 reductase

CYP

cytochrome P450

ER

endoplasmic reticulum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GST

glutathione S-transferase

HO

heme oxygenase

HRP

horseradish peroxidase

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

SUVs

small unilamellar vesicles

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05685.

  • Figure S1: amino acid and nucleotide sequences of mbHO-1; Figure S2: SDS-PAGE analysis of purified mbHO-1, sHO-1, and hGAPDH; Figure S3:Heme binding curves for the data of fluorescent intensity changes at 565 nm as a function of added hemin concentrations; Figure S4:fluorescence spectra and fluorescence images of sedimented POPC liposomes labeled with BODIPY 493/503 (PDF)

#.

R.M. and Y.K. contributed equally. The study conception and design were carried out by J.T. Data collection and analysis were performed by R.M., Y.K., T.K., and J.T. The first draft of the manuscript was written by J.T., M.S., and H.S. All authors approved the submitted manuscript.

The authors declare no competing financial interest.

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