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Heme Oxygenase 1 (HMOX1) is a cytoprotective enzyme, exhibiting the highest activity in the spleen, catalyzing the heme ring breakdown into products of biological significance- biliverdin, CO, and Fe 2+ . In vascular cells, HMOX1 possesses strong anti-apoptotic, antioxidant, anti-proliferative, anti-inflammatory, and immunomodulatory actions. The majority of these activities are crucial for the prevention of atherogenesis. Single amino acid substitutions in proteins generated by missense non-synonymous single nucleotide polymorphism (nsSNPs) in the protein-encoding regions of genes are potent enough to cause significant medical challenges due to the alteration of protein structure and function. The current study aimed at characterizing and analyzing high-risk nsSNPs associated with the human HMOX1 gene. Preliminary screening of the total available 288 missense SNPs was performed through the lens of deleteriousness and stability prediction tools. Finally, a total of seven nsSNPs (Y58D, A131T, Y134H, F166S, F167S, R183S and M186V) were found to be most deleterious by all tools that are present at highly conserved positions. Molecular dynamics simulations (MDS) analysis explained the mutational effects on the dynamic action of the wild-type and mutant proteins. In a nutshell, R183S (rs749644285) was identified as a highly detrimental mutation that could significantly render the enzymatic activity of HMOX1. The finding of this computational analysis might help subject the experimental confirmatory analysis to characterize the role of nsSNPs in HMOX1. Communicated by Ramaswamy H. Sarma.

Human heme oxygenase-1 (hHO-1) plays a crucial role in human physiology because of its ability to metabolize free heme. The heme degradation products, biliverdin and bilirubin, were shown to have protective antioxidant properties in cells. In the context of cancer, hHO-1 function grants cancer cells defense from standard chemotherapy treatments, leading to the development of azole-based inhibitors that target hHO-1 for potential anticancer therapy. This work reports experimental and theoretical characterization of interactions between three azole-based inhibitors and the active site of hHO-1. It was found that all three compounds have K d values within the μM order. The electronic absorption and resonance Raman (rR) spectra indicated that they bind to the ferric heme and coordinate through a nitrogen atom. rR measurements revealed varying effects of inhibitors on the geometry of heme vinyl groups in the ferric form of hHO-1. Changes in peripheral group orientation are known to affect heme redox potential, and consequently can reflect the inhibitory properties of studied azoles. The subsequent docking studies showed that inhibitors with lower K d values are located close to two vinyl groups, while the compound with higher K d is situated near only one, consistent with the rR studies. Finally, the rR studies of the CO adducts showed that the inhibitors bind to the heme in a reversible manner. Altogether, the combination of ligand binding studies, UV-Vis and rR spectroscopies, as well as computational approach revealed an importance of the steric hindrance imposed by the inhibitor's side chain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

In this work, the first mutual prodrug of 5-fluorouracil and heme oxygenase1 inhibitor (5-FU/HO-1 hybrid) has been designed, synthesised, and evaluated for its in vitro chemical and enzymatic hydrolysis stability. Predicted in silico physicochemical properties of the newly synthesised hybrid ( 3 ) demonstrated a drug-like profile with suitable Absorption, Distribution, Metabolism, and Excretion (ADME) properties and low toxic liabilities. Preliminary cytotoxicity evaluation towards human prostate (DU145) and lung (A549) cancer cell lines demonstrated that 3 exerted a similar effect on cell viability to that produced by the reference drug 5-FU. Among the two tested cancer cell lines, the A549 cells were more susceptible for 3 . Of note, hybrid 3 also had a significantly lower cytotoxic effect on healthy human lung epithelial cells (BEAS-2B) than 5-FU. Altogether our results served as an initial proof-of-concept to develop 5-FU/HO-1 mutual prodrugs as potential novel anticancer agents.

Heme oxygenase-1 (HO-1) has attracted accumulating attention for its antioxidant enzymatic activity. However, the exact regulatory role of its non-enzymatic activity in the cardiovascular system remains unaddressed. Here, we show that HO-1 was accumulated in the nuclei of stress-induced senescent endothelial cells, and conferred protection against endothelial senescence independent of its enzymatic activity. Overexpression of ΔHO-1, a truncated HO-1 without transmembrane segment (TMS), inhibited H 2 O 2 -induced endothelial senescence. Overexpression of ΔHO-1 H25A , the catalytically inactive form of ΔHO-1, also exhibited anti-senescent effect. In addition, infection of recombinant adenovirus encoding ΔHO-1 with three nuclear localization sequences (NLS), alleviated endothelial senescence induced by knockdown of endogenous HO-1 by CRISPR/Cas9. Moreover, repression of HO-1 nuclear translocation by silencing of signal peptide peptidase (SPP), which is responsible for enzymatic cleavage of the TMS of HO-1, exacerbated endothelial senescence. Mechanistically, nuclear HO-1 interacted with NPM1 N-terminal portion, prevented NPM1 translocation from nucleolus to nucleoplasm, thus disrupted NPM1/p53/MDM2 interactions and inhibited p53 activation by NPM1, finally resisted endothelial senescence. This study provides a novel understanding of HO-1 as a promising therapeutic strategy for vascular senescence-related cardiovascular diseases., (© 2021. The Author(s).)

Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin-porphyrin FRET break-apart probe to detect HO-1 activity, Fe-L 1 . We designed Fe-L 1 to "break-apart" upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC-MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe-L 2 , we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

Heme oxygenase (HO)-1, a rate-limiting enzyme in heme catabolism, results in the formation of equivalent amounts of biliverdin (BV), carbon monoxide (CO) and ferrous iron (Fe 2+ ). Previous studies have revealed that HO-1 plays an important role in immune responses. However, the mechanism underlying the immune responses against bacterial infection of teleost HO-1 remains enigmatic. To decipher the mechanisms, we have cloned and characterized the HO-1 gene of grass carp (designated as GcHO-1) in this research. The results showed that the open reading frame (ORF) of GcHO-1 was 819 bp, which encoded a putative protein of 272 amino acids. The deduced amino acid sequence phylogenetically shared the highest identity with other teleosts, and contained two domains of heme-oxygenase and a single-pass transmembrane domain. The mRNA expressions of GcHO-1 in healthy grass carp have widely existed in examined tissues in the following order of spleen > head-kidney > middle head-kidney > intestines > liver > gills > heart > muscle > brain. Besides, the mRNA and protein transcription of GcHO-1 were both significantly up-regulated in the liver and head-kidney tissues after Staphylococcus aureus and Aeromonas hydrophila infection. In addition, overexpression of GcHO-1 in kidney cell line (CIK) cells of grass carp could reduce the expression of inflammatory cytokines (IL-1β, IL-8, TNFα, CCL1 and IL-6). Herein, we demonstrate that GcHO-1 plays an anti-inflammatory role in innate immunity. Our results shed new light on the mechanisms underlying the antibacterial immunity of teleost., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Upregulation of Heme Oxygenase-1 (HO-1) has been widely implicated in cancer growth and chemoresistance. This explains the numerous drug discovery efforts aimed at mitigating its pro-carcinogenic roles till date. In a recent study, two selective azole-based HO-1 inhibitors (Cpd1 and Cpd2) were synthesized, which exhibited differential inhibitory potencies of ~200μm. Interestingly, variations in the affinities of these compounds were determined by their positioning across specific regions of the HO-1 binding domain, pin-pointing a pharmacological interrelationship that remains unresolved. Therefore, in this study, using molecular dynamics simulations and binding free energy calculations, we investigate how dynamical orientations of these compounds influence their binding affinities at the active HO-1 domain. Findings revealed favorable binding for the bromobenzene and imidazole substituents of Cpd1 at the western and eastern regions of the HO-1 active domain. The constituent hydroxyl group was coordinated by residues Asp140 and Arg136 over the simulation period. On the contrary, stable binding of the bromobenzene and imidazole substituents were negated by the optimal orientations of the benzyl substituent, which extended into the northeastern region. These were supported by the displacement of Asp140 and Arg136, crucial for hydrogen bond formation in Cpd1. Also, we observed that Cpd2 exhibited high deviations indicative of an unstable binding relative to Cpd1. This further supports the presumption that Cpd2 was systematically oriented away from the active HO-1 region, a phenomenon that was due to the optimal motions of the benzyl group at the northeastern regions. The highlight of our findings is that the benzyl substituent in Cpd2 elicited negative effects on HO-1, vis a vis, instability, displacement of crucial residues, and low binding energy when compared to Cpd1. Findings pave the way for future drug discovery efforts related to HO-1 inhibition in cancer therapy.

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, 'How are the HO-1 and P450 systems organized in the endoplasmic reticulum?' Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site. Outside their catalytic cores are two regions unique to HO2: a 20-amino acid-long N-terminal extension and a C-terminal domain containing two heme regulatory motifs (HRMs) that bind heme independently of the core. Both HO isoforms contain a C-terminal hydrophobic membrane anchor; however, their sequences diverge. Here, using hydrogen-deuterium exchange MS, size-exclusion chromatography, and sedimentation velocity, we investigated how these divergent regions impact the dynamics and structure of the apo and heme-bound forms of HO1 and HO2. Our results reveal that heme binding to the catalytic cores of HO1 and HO2 causes similar dynamic and structural changes in regions (proximal, distal, and A6 helices) within and linked to the heme pocket. We observed that full-length HO2 is more dynamic than truncated forms lacking the membrane-anchoring region, despite sharing the same steady-state activity and heme-binding properties. In contrast, the membrane anchor of HO1 did not influence its dynamics. Furthermore, although residues within the HRM domain facilitated HO2 dimerization, neither the HRM region nor the N-terminal extension appeared to affect HO2 dynamics. In summary, our results highlight significant dynamic and structural differences between HO2 and HO1 and indicate that their dissimilar C-terminal regions play a major role in controlling the structural dynamics of these two proteins., (© 2019 Kochert et al.)

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl 2 . In the present study, CdCl 2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl 2 -induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl 2 -induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl 2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A 3D quantitative structure-activity relationship (3D-QSAR) model for predicting the activity of heme oxygenase 1 (HO-1) inhibitors was constructed with the aim of providing a useful tool for the identification, design, and optimization of novel HO-1 inhibitors. The model was built using a set of 222 HO-1 inhibitors recovered from the Heme Oxygenase Database (HemeOxDB) and developed with the software Forge. The present model showed high statistical quality, as confirmed by its robust predictive potential and satisfactory descriptive capability. The drawn-up 3D map enables prompt visual comprehension of the electrostatic, hydrophobic, and shaping features underlying the interactions with HO-1 inhibitors. A theoretical approach for the generation of new lead compounds was performed by means of scaffold-hopping analysis. For the first time, a 3D-QSAR model is reported for this target, and was built with a number of chemically diverse HO-1 inhibitors; the model also accounts well for individual ligand affinities. The new model contains all of the inhibitors published to date with high potency toward the selected target and contains a complete pharmacophore description of the binding cavity of HO-1. These features will ensure application in accelerating the identification of new potent and selective HO-1 inhibitors., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alternative splicing is a routine phenomenon which greatly increases the diversity of proteins in eukaryotic cells. In humans, most multi-exonic genes are alternatively spliced and their splice variants confer distinct functions. Heme oxygenase-1 (HO-1, 32 kDa) is an inducible stress responsive protein, which possesses multiple functions in many cellular processes. In the current study, we identified a novel alternative splice isoform of 14 kDa HO-1 generated through exclusion of exon 3, and it is highly expressed in immortalized cells. In contrast to nuclear accumulation of the full-length 32 kDa HO-1, the novel 14 kDa HO-1 isoform is retained in the cytoplasm under ultraviolet (UV) irradiation. Interestingly, the 14 kDa HO-1 is shown to promote cell proliferation and an increase in relative telomere lengths in vivo and in vitro. Thus, we are pioneer to report and confirm the presence of a novel splice form of HO-1 and its distinct role in modulating telomere length and tumor growth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To elucidate the anti-inflammatory mechanism of Curcuma sp., we investigated whether dehydrocurdione, a sesquiterpene contained in Curcuma sp., induces heme oxygenase (HO)-1, an antioxidative enzyme, in RAW 264.7 macrophages. Dehydrocurdione was extracted from the rhizome of Curcuma sp., and its purity was verified by high performance liquid chromatography. Treatment with 10-100 μM dehydrocurdione transiently and concentration-dependently increased HO-1 mRNA and protein levels. Docking simulation suggested the presence of the Michael reaction between dehydrocurdione and Kelch-like ECH-associated protein (Keap)1 keeping nuclear factor-erythroid2-related-factor (Nrf)2, a transcription factor, in the cytoplasm. Nrf2 that was definitely free from Keap1 was detected in the nuclei after dehydrocurdione treatment. Subsequently, the HO-1 E2 enhancer, a target of Nrf2, was activated, resulting in HO-1 expression. Also, an investigation using 6-shogaol and 6-gingerol supported the concept that the α, β-unsaturated carbonyl structure plays an important role in the interaction with Keap1. Dehydrocurdione suppressed lipopolysaccharide-induced NO release, a marker of inflammation. Clarification of the HO-1 synthesis increase mechanism revealed in this study will help contribute to the development of novel phytotherapeutic strategies against inflammation-associated diseases., (Copyright © 2018 John Wiley & Sons, Ltd.)

Carbon monoxide (CO) is attracting increasing attention because of its role as a gasotransmitter with cytoprotective and homeostatic properties. Carbon monoxide releasing molecules (CORMs) are spatially and temporally controlled CO releasers that exhibit superior and more effective pharmaceutical traits than gaseous CO because of their chemistry and structure. Experimental and preclinical research in animal models has shown the therapeutic potential of inhaled CO and CORMs, and the biological effects of CO and CORMs have also been observed in preclinical trials via the genetic modulation of heme oxygenase-1 (HO-1). In this review, we describe the pharmaceutical use of CO and CORMs, methods of detecting CO release, and developments in CORM design and synthesis. Many valuable clinical CORMs formulated using macromolecules and nanomaterials are also described.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a cluster of oxidative stress-inducible genes in cells. Here, we aimed to investigate whether trehalose (Tre) protects primary rat proximal tubular (rPT) cells against cadmium (Cd)-induced oxidative stress via Nrf2 antioxidant pathway. Data showed that Tre treatment inhibited Nrf2 nuclear translocation and restored the decline in Kelch-like ECH-associated protein 1 (Keap1) protein level in Cd-exposed rPT cells. Moreover, Cd-activated Nrf2 target genes, including phase II detoxifying enzymes, that is, NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1, direct antioxidant proteins, that is, glutathione peroxidase, superoxide dismutase, catalase, and glutathione biosynthesis-related proteins, that is, glutamatecysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase, were all downregulated by co-treatment with Tre. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced oxidative stress in rPT cells by inhibiting the Nrf2-Keap1 signaling pathway., (© 2017 Wiley Periodicals, Inc.)

The discovery of biocompatible or bioactive nanoparticles for medicinal applications is an expensive and time-consuming process that may be significantly facilitated by incorporating more rational approaches combining both experimental and computational methods. However, it is currently hindered by two limitations: (1) the lack of high-quality comprehensive data for computational modeling and (2) the lack of an effective modeling method for the complex nanomaterial structures. In this study, we tackled both issues by first synthesizing a large library of nanoparticles and obtained comprehensive data on their characterizations and bioactivities. Meanwhile, we virtually simulated each individual nanoparticle in this library by calculating their nanostructural characteristics and built models that correlate their nanostructure diversity to the corresponding biological activities. The resulting models were then used to predict and design nanoparticles with desired bioactivities. The experimental testing results of the designed nanoparticles were consistent with the model predictions. These findings demonstrate that rational design approaches combining high-quality nanoparticle libraries, big experimental data sets, and intelligent computational models can significantly reduce the efforts and costs of nanomaterial discovery.

It has recently been shown that signal peptide peptidase (SPP) can catalyze the intramembrane cleavage of heme oxygenase-1 (HO-1) that leads to translocation of HO-1 into the cytosol and nucleus. While there is consensus that translocated HO-1 promotes tumor progression and drug resistance, the physiological signals leading to SPP-mediated intramembrane cleavage of HO-1 and the specificity of the process remain unclear. In this study, we used co-immunoprecipitation and confocal laser scanning microscopy to investigate the translocation mechanism of HO-1 and its regulation by SPP. We show that HO-1 and the closely related HO-2 isoenzyme bind to SPP under normoxic conditions. Under hypoxic conditions SPP mediates intramembrane cleavage of HO-1, but not HO-2. In experiments with an inactive HO-1 mutant (H25A) we show that translocation is independent of the catalytic activity of HO-1. Studies with HO-1 / HO-2 chimeras indicate that the membrane anchor, the PEST-domain and the nuclear shuttle sequence of HO-1 are necessary for full cleavage and subsequent translocation under hypoxic conditions. In the presence of co-expressed exogenous SPP, the anchor and the PEST-domain are sufficient for translocation. Taken together, we identified the domains involved in HO-1 translocation and showed that SPP-mediated cleavage is isoform-specific and independent of HO-activity. A closer understanding of the translocation mechanism of HO-1 is of particular importance because nuclear HO-1 seems to lead to tumor progression and drug resistance.

Significance: Several lines of evidence suggest that hematological malignancies exhibit an altered redox balance homeostasis that can lead to the activation of various survival pathways that, in turn, lead to the progression of disease and chemoresistance. Among these pathways, the heme oxygenase-1 (HO-1) pathway is likely to play a major role. HO catalyzes the enzymatic degradation of heme with the simultaneous release of carbon monoxide (CO), ferrous iron (Fe 2+ ), and biliverdin. This review focuses on the role of HO-1 in various hematological malignancies and the possibility of exploiting such targets to improve the outcome of well-established chemotherapeutic regimens. Recent Advances and Critical Issues: Interestingly, the inhibition of the expression of HO-1 (e.g., with siRNA) or HO activity (with competitive inhibitors) contributes to the increased efficacy of chemotherapy and improves the outcome in animal models. Furthermore, some hematological malignancies (e.g., chronic myeloid leukemia and multiple myeloma) have served to explore the non-canonical functions of HO-1, such as the association between nuclear compartmentalization and genetic instability and/or chemoresistance., Future Directions: The HO system may serve as an important tool in the field of hematological malignancies because it can be exploited to counteract chemoresistance and to monitor the outcome of bone marrow transplants and may be an additional target for combined therapies. Antioxid. Redox Signal. 27, 363-377.

Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

An imbalance between oxidative stress and antioxidant activity plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke, a major risk factor of COPD, induces cellular oxidative stress, but levels of antioxidants such as heme oxygenase-1 (HO-1) are reduced in individuals with severe COPD. In this study, we evaluated the molecular mechanism of reduced HO-1 expression in human bronchial epithelial cells. We found that cigarette smoke extract (CSE) increases HO-1 levels via activation of NFE2-related factor 2 (Nrf2). However, pretreating cells with the protease neutrophil elastase (NE) suppressed the CSE-induced expression of HO-1 mRNA and protein. NE also decreased the sirtuin 1 (SIRT1) level, but did not inhibit CSE-induced nuclear translocation and DNA-binding activity of Nrf2. Transfection of cells with a Myc/His-tagged SIRT1 expression vector completely blocked the NE-mediated suppression of HO-1 expression. We further noted that the NE-induced down-regulation of SIRT1 was not due to decreased transcription or proteasomal/lysosomal degradation or loss of solubility. Immunofluorescence staining revealed that NE enters the cell cytoplasm, and we observed that NE directly cleaved SIRT1 in vitro , indicating that SIRT1 levels are decreased via direct degradation by internalized NE. Of note, we observed decreased SIRT1 levels in NE-treated primary human bronchial epithelial cells and in lung homogenates from both smokers and patients with COPD. In conclusion, NE suppresses CSE-induced HO-1 expression by cleaving SIRT1. This finding indicates the importance of cross-talk between oxidative stress and protease responses in the pathogenesis of COPD., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Heme is an efficient source of iron in the diet, and heme preparations are used to prevent and cure iron deficiency anemia in humans and animals. However, the molecular mechanisms responsible for heme absorption remain only partially characterized. Here, we employed young iron-deficient piglets as a convenient animal model to determine the efficacy of oral heme iron supplementation and investigate the pathways of heme iron absorption. The use of bovine hemoglobin as a dietary source of heme iron was found to efficiently counteract the development of iron deficiency anemia in piglets, although it did not fully rebalance their iron status. Our results revealed a concerted increase in the expression of genes responsible for apical and basolateral heme transport in the duodenum of piglets fed a heme-enriched diet. In these animals the catalytic activity of heme oxygenase 1 contributed to the release of elemental iron from the protoporphyrin ring of heme within enterocytes, which may then be transported by the strongly expressed ferroportin across the basolateral membrane to the circulation. We hypothesize that the well-recognized high bioavailability of heme iron may depend on a split pathway mediating the transport of heme-derived elemental iron and intact heme from the interior of duodenal enterocytes to the bloodstream.

Oxidative stress is considered as an important mediator in the progression of metabolic disorders. The objective of this study was to investigate the potential hepatoprotective effects and mechanisms of bovine casein glycomacropeptide hydrolysates (GHP) on hydrogen peroxide (H₂O₂)-induced oxidative damage in HepG2 cells. Results showed that GHP significantly blocked H₂O₂-induced intracellular reactive oxygen species (ROS) generation and cell viability reduction in a dose-dependent manner. Further, GHP concentration-dependently induced heme oxygenase-1 (HO-1) expression and increased nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation. Moreover, pretreatment of GHP increased the activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2), which were shown to contribute to Nrf2-mediated HO-1 expression. Taken together, GHP protected HepG2 cells from oxidative stress by activation of Nrf2 and HO-1 via p38 MAPK and ERK1/2 signaling pathways. Our findings indicate that bovine casein glycomacropeptide hydrolysates might be a potential ingredient in the treatment of oxidative stress-related disorders and further studies are needed to investigate the protective effects in vivo., Competing Interests: The authors declare no conflict of interest.

The basal transcription of heme oxygenase-1 (HO-1) regulation is dependent upon a GT repeat germ line polymorphism (GTn) in the promoter of the HO-1 gene. We determined the prognostic value of HO-1 promoter polymorphism on the natural postoperative course of complete resected oesophageal cancer. Genomic DNA from 297 patients was amplified by polymerase chain reaction and sequenced. The results were correlated with clinicopathological parameters, disseminated tumour cells in bone marrow (DTC) and clinical outcome. Depending on short allele with <25 and long allele with ≥25, GTn repeats three genotypes (SS, SL and LL) were defined. A diverse role of GTn was evident in squamous cell carcinoma (SCC) and adenocarcinoma (AC). In SCC, the SS genotype presented less advanced tumours with lower rate DTC in bone marrow and relapse compared with L-allele carriers. In contrast, AC patients with the SS genotype displayed a complete opposing tumour characteristic. The disease-free (DFS) and overall survival (OS) in SCC patients was markedly reduced in LL genotypes (p < 0.001). In AC contrarily the SS genotype patients displayed the worst DFS and OS (p < 0.001). GTn is a strong prognostic factor with diverse prognostic value for recurrence and survival in AC and SCC., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Heme oxygenase (HO) is a ubiquitous enzyme with key roles in inflammation, cell signaling, heme disposal, and iron acquisition. HO catalyzes the oxidative conversion of heme to biliverdin (BV) using a conserved histidine to coordinate the iron atom of bound heme. This His-heme interaction has been regarded as being essential for enzyme activity, because His-to-Ala mutants fail to convert heme to biliverdin in vitro. We probed a panel of proximal His mutants of cyanobacterial, human, and plant HO enzymes using a live-cell activity assay based on heterologous co-expression in Escherichia coli of each HO mutant and a fluorescent biliverdin biosensor. In contrast to in vitro studies with purified proteins, we observed that multiple HO mutants retained significant activity within the intracellular environment of bacteria. X-ray crystallographic structures of human HO1 H25R with bound heme and additional functional studies suggest that HO mutant activity inside these cells does not involve heme ligation by a proximal amino acid. Our study reveals unexpected plasticity in the active site binding interactions with heme that can support HO activity within cells, suggests important contributions by the surrounding active site environment to HO catalysis, and can guide efforts to understand the evolution and divergence of HO function.

This study investigated whether sulforaphane (SFN), a compound found in cruciferous vegetables, could attenuate the progression of post-myocardial infarction (MI) cardiac remodeling. Male Wistar rats (350 g) were allocated to four groups: SHAM (n=8), SHAM+SFN (n=7), MI (n=8) and MI+SFN (n=5). On the third day after surgery, cardiac function was assessed and SFN treatment (5 mg/kg/day) was started. At the end of 25 days of treatment, cardiac function was assessed and heart was collected to measure collagen content, oxidative stress and protein kinase. MI and MI+SFN groups presented cardiac dysfunction, without signs of congestion. Sulforaphane reduced fibrosis (2.1-fold) in infarcted rats, which was associated with a slight attenuation in the cardiac remodeling process. Both infarcted groups presented increases in the oxidative markers xanthine oxidase and 4-hydroxinonenal, as well as a parallel increase in the antioxidant enzymes glutathione peroxidase and superoxide dismutase. Moreover, sulforaphane stimulated the cytoprotective heme oxygenase-1 (HO-1) (38%). Oxidative markers correlated with ERK 1/2 activation. In the MI+SFN group, up-regulation of ERK 1/2 (34%) and Akt (35%), as well as down-regulation of p38 (52%), was observed. This change in the prosurvival kinase balance in the MI+SFN group was related to a down-regulation of apoptosis pathways (Bax/Bcl-2/caspase-3). Sulforaphane was unable to modulate autophagy. Taken together, sulforaphane increased HO-1, which may generate a redox environment in the cardiac tissue favorable to activation of prosurvival and deactivation of prodeath pathways. In conclusion, this natural compound contributes to attenuation of the fibrotic process, which may contribute to mitigation against the progression of cardiac remodeling postinfarction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Significance: Acute kidney injury (AKI) and chronic kidney disease (CKD) represent a considerable burden in healthcare. The heme oxygenase (HO) system plays an important role in regulating oxidative stress and is protective in a variety of human and animal models of kidney disease. Preclinical studies of the HO system have led to the development of several clinical trials targeting the enzyme or its products., Recent Advances: Connection of HO, ferritin, and other proteins involved in iron regulation has provided important insight into mechanisms of damage in AKI. Also, HO-1 expression is important in the pathogenesis of hypertension, diabetic kidney disease, and progression to end-stage renal disease., Critical Issues: Despite intriguing discoveries, no drugs targeting the HO system have been translated to the clinic. Meanwhile, treatments for AKI and CKD are urgently needed. Many factors have likely contributed to challenges in clinical translation, including variation in animal models, difficulties in obtaining human tissue, and complexity of the disease processes being studied., Future Directions: The HO system represents a promising avenue of investigation that may lead to targeted therapeutics. Tissue-specific gene modulation, widening the scope of animal studies, and continued clinical research will provide valuable insight into the role HO plays in kidney homeostasis and disease. Antioxid. Redox Signal. 25, 165-183.

Sulforaphane, a natural isothiocyanate, demonstrates cardioprotection associated with its capacity to stimulate endogenous antioxidants and to inhibit inflammation. The aim of this study was to investigate whether sulforaphane is capable of attenuating oxidative stress and inflammatory responses through the TLR4/MyD88/NFκB pathway, and thereby could modulate post-ischemic ventricular function in isolated rat hearts submitted to ischemia and reperfusion. Male Wistar rats received sulforaphane (10 mg·kg(-1)·day(-1)) or vehicle i.p. for 3 days. Global ischemia was performed using isolated hearts, 24 h after the last injection, by interruption of the perfusion flow. The protocol included a 20 min pre-ischemic period followed by 20 min of ischemia and a 20 min reperfusion. Although no changes in mechanical function were observed, sulforaphane induced a significant increase in superoxide dismutase and heme oxygenase-1 expression (both 66%) and significantly reduced reactive oxygen species levels (7%). No differences were observed for catalase and glutathione peroxidase expression or their activities, nor for thioredoxin reductase, glutaredoxin reductase and glutathione-S-transferase. No differences were found in lipid peroxidation or TLR4, MyD88, and NF-κB expression. In conclusion, although sulforaphane was able to stimulate endogenous antioxidants modestly, this result did not impact inflammatory signaling or cardiac function of hearts submitted to ischemia and reperfusion.

In our previous study, l-carnitine was shown to have cytoprotective effect against hydrogen peroxide (H2O2)-induced injury in human normal HL7702 hepatocytes. The aim of this study was to investigate whether the protective effect of l-carnitine was associated with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway. Our results showed that pretreatment with l-carnitine augmented Nrf2 nuclear translocation, DNA binding activity and heme oxygenase-1 (HO-1) expression in H2O2-treated HL7702 cells, although l-carnitine treatment alone had no effect on them. Analysis using Nrf2 siRNA demonstrated that Nrf2 activation was involved in l-carnitine-induced HO-1 expression. In addition, l-carnitine-mediated protection against H2O2 toxicity was abrogated by Nrf2 siRNA, indicating the important role of Nrf2 in l-carnitine-induced cytoprotection. Further experiments revealed that l-carnitine pretreatment enhanced the phosphorylation of Akt in H2O2-treated cells. Blocking Akt pathway with inhibitor partly abrogated the protective effect of l-carnitine. Moreover, our finding demonstrated that the induction of Nrf2 translocation and HO-1 expression by l-carnitine directly correlated with the Akt pathway because Akt inhibitor showed inhibitory effects on the Nrf2 translocation and HO-1 expression. Altogether, these results demonstrate that l-carnitine protects HL7702 cells against H2O2-induced cell damage through Akt-mediated activation of Nrf2 signaling pathway.

During a discovery project of potential inhibitors for three proteins, TNF-α, RANKL and HO-1, implicated in the pathogenesis of rheumatoid arthritis, significant amounts of purified proteins were required. The application of statistically designed experiments for screening and optimization of induction conditions allows rapid identification of the important factors and interactions between them. We have previously used response surface methodology (RSM) for the optimization of soluble expression of TNF-α and RANKL. In this work, we initially applied RSM for the optimization of recombinant HO-1 and a 91% increase of protein production was achieved. Subsequently, we slightly modified a published incomplete factorial approach (called IF1) in order to evaluate the effect of three expression variables (bacterial strains, induction temperatures and culture media) on soluble expression levels of the three tested proteins. However, soluble expression yields of TNF-α and RANKL obtained by the IF1 method were significantly lower (<50%) than those obtained by RSM. We further modified the IF1 approach by replacing the culture media with induction times and the resulted method called IF-STT (Incomplete Factorial-Stain/Temperature/Time) was validated using the three proteins. Interestingly, soluble expression levels of the three proteins obtained by IF-STT were only 1.2-fold lower than those obtained by RSM. Although RSM is probably the best approach for optimization of biological processes, the IF-STT is faster, it examines the most important factors (bacterial strain, temperature and time) influencing protein soluble expression in a single experiment, and can be used in any recombinant protein expression project as a starting point., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway defends cells against oxidative stress and regulates the cellular redox balance. Activation of this pathway induces a variety of antioxidant enzymes, resulting in the protection of our bodies against oxidative damage. It has been reported that aged garlic extract (AGE), a garlic preparation that is rich in water-soluble cysteinyl moieties, reduces oxidative stress and helps to ameliorate of cardiovascular, renal and hepatic diseases. We hypothesized that AGE enhances the expression of antioxidant enzymes via the Nrf2-ARE pathway in human umbilical vein endothelial cells in culture. Gene expression of antioxidant enzymes was measured using real-time polymerase chain reaction. Nuclear accumulation of Nrf2 and antioxidant enzymes expression were evaluated using western blotting analyses. We found that AGE promoted the accumulation of Nrf2 into the nucleus in a time- and dose-dependent manner and increased the gene expression and polypeptide level of heme oxygenase-1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM). Moreover, the effect of AGE in elevating the gene expression of HO-1 and GCLM was found to be mediated via Nrf2 activation in human umbilical vein endothelial cells. Taken together, these observations suggest that AGE induces the expression of HO-1 and GCLM, which are antioxidant enzymes, via activation of the Nrf2-ARE signaling pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The two isoforms of human heme oxygenase (HO1 and HO2) catalyze oxidative degradation of heme to biliverdin, Fe, and CO. Unlike HO1, HO2 contains two C-terminal heme regulatory motifs (HRMs) centered at Cys265 and Cys282 that act as redox switches and, in their reduced dithiolate state, bind heme (Fleischhacker et al., Biochemistry , 2015 , 54 , 2693 - 2708 ). Here, we describe cryoreduction/annealing and electron paramagnetic resonance spectroscopic experiments to study the structural features of the oxyheme moiety in HO2 and to elucidate the initial steps in heme degradation. We conclude that the same mechanism of heme hydroxylation to α-meso-hydroxyheme is employed by both isoforms and that the HRMs do not affect the physicochemical properties of the oxy-Fe(II) and HOO-Fe(III) states of HO2. However, the absorption spectrum of oxy-Fe(II)-HO2 is slightly blue-shifted relative to that of HO1. Furthermore, heme hydroxylation proceeds three times more slowly, and the oxy-Fe(II) state is 100-fold less stable in HO2 than in HO1. These distinctions are attributed to slight structural variances in the two proteins, including differences in equilibrium between open versus closed conformations. Kinetic studies revealed that heme oxygenation by HO2 occurs solely at the catalytic core in that a variant of HO2 lacking the C-terminal HRM domain exhibits the same specific activity as one containing both the catalytic core and HRM domain; furthermore, a truncated variant containing only the HRM region binds but cannot oxidize heme. In summary, HO1 and HO2 share similar catalytic mechanisms, and the HRMs do not play a direct role in the HO2 catalytic cycle.

Specific reactive oxygen species (ROS) from different sources, might lead to different and even opposite, cellular effects. We studied the production of specific ROS resulting from the exposure of human umbilical veins endothelial cells (HUVEC) to H2O2 derived from the natural antioxidant epigallocathechin gallate (EGCG) or from the exposure to IL-1β using a fluorogenic probe and flow cytometry, and evaluated by western blot analysis and immunocytochemistry the associated expression of transcription factors sensitive to both inflammatory and oxidative stress, such as NF-κB and Nrf2, and some downstream activated genes such as cyclooxygenase-2 (COX-2) and hemeoxygenase 1 (HO-1). The results obtained showed that exogenously-generated H2O2 induce anti-inflammatory and antioxidant effects in HUVECs counteracting the pro-inflammatory and pro-oxidant effect of IL-1β related to the production of superoxide anions. The underlying mechanisms resulting from the extracellular production of H2O2, include (1) Nrf2 nuclear translocation and the enhanced expression of antioxidant enzymes such as HO-1, and (2) the previously unreported inhibition of NF-κB and COX-2 expression. Overall, these findings provide evidence that the production of specific reactive oxygen species finely tunes endothelial cell function and might be relevant for the reappraisal of the effects of exogenous antioxidants in the context of cardiovascular diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Heme oxygenase-1 (HO-1) inhibition is associated with antitumor activity. Imidazole-based analogues show effective and selective inhibitory potency of HO-1. In this work, five single-crystal structures of four imidazole-based compounds are presented, with an in-depth structural analysis. In order to study the influence of the conformation of the ligands on binding to protein, conformational data from crystallography are compared with quantum mechanics analysis and molecular docking studies. Molecular docking of imidazole-based analogues in the active site of HO-1 is in good agreement with the experimental structures. Inhibitors interact with the heme cofactor and a hydrophobic pocket (Met34, Phe37, Val50, Leu147 and Phe214) in the HO-1 binding site. An alternate binding mode can be hypothesized for some inhibitors in the series.

Naturally occurring phenolic compounds are widely found in plants. Here, the phenolic composition and hepatoprotective effect of the butanolic extract (BE) from Nelumbo nucifera leaves against H2O2-induced hepatic damage in cultured hepatocytes were investigated. BE showed high total phenol and flavonoid contents, and major phenolic compounds are quercetin, catechin, ferulic acid, rutin, and protocatechuic acid by HPLC analysis. BE effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) cation radicals (IC50 values of 5.21 μg mL(-1) for DPPH and 6.22 μg mL(-1) for ABTS(+)) and showed strong reducing power. Pretreatment of BE prior to 650 μM H2O2 exposure markedly increased cell viability and suppressed H2O2-induced intracellular reactive oxygen species generation and AAPH-induced cell membrane lipid peroxidation. In addition, BE up-regulated intracellular glutathione levels under normal and oxidative stress conditions. Notably, the hepatoprotective effect of BE was directly correlated with the increased expression of superoxide dismutase-1 (SOD-1) by 0.62-fold, catalase (CAT) by 0.42-fold, and heme oxygenase-1 (HO-1) by 2.4-fold. Pretreatment of BE also increased the nuclear accumulation of Nrf2 by 8.1-fold indicating that increased SOD-1, CAT, and HO-1 expressions are Nrf2-mediated.

Contact sensitizers induce phenotypic and functional changes in dendritic cells (DC) that enhance their antigen-presenting capacity and, ultimately, modulate the T cell response. To evaluate if there is a similar effect of drugs causing T-cell-mediated cutaneous adverse drug reactions (CADR), we studied the in vitro effect of drugs on THP-1 cells, a cell line widely used to evaluate the early molecular and cellular events triggered by contact sensitizers. The effect of allopurinol, oxypurinol, ampicillin, amoxicillin, carbamazepine and sodium valproate, at EC30 concentrations, was evaluated on p38 MAPK activation, by Western Blot, and on the expression of genes coding for DC maturation markers, pro-inflammatory cytokine/chemokines and hemeoxygenase 1 (HMOX1), by real-time RT-PCR. Results were compared with lipopolysaccharide (LPS), a DC maturation stimulus, and the strong contact sensitizer, 1-fluoro-2,4-dinitrobenzene (DNFB). All drugs studied significantly upregulated HMOX1 gene transcription and all, except the anticonvulsants, also upregulated IL8. Allopurinol and oxypurinol showed the most intense effect, in a magnitude similar to DNFB and superior to betalactams. Transcription of CD40, IL12B and CXCL10 genes by drugs was more irregular. Moreover, like DNFB, all drugs activated p38 MAPK, although significantly only for oxypurinol. Like contact sensitizers, drugs that cause non-immediate CADR activate THP-1 cells in vitro, using different signalling pathways and affecting gene transcription with an intensity that may reflect the frequency and severity of the CADR they cause. Direct activation of antigen-presenting DC by systemic drugs may be an important early step in the pathophysiology of non-immediate CADR., (Copyright © 2014 John Wiley & Sons, Ltd.)

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1-232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes the oxidative degradation of heme. Since free heme is toxic to cells, rapid degradation of heme is important for maintaining cellular health. There have been useful mechanistic studies of the HO reaction based on crystal structures; however, how HO-1 recognizes heme is not completely understood because the crystal structure of heme-free rat HO-1 lacks electron densities for A-helix that ligates heme. In this study, we characterized conformational dynamics of HO-1 using NMR to elucidate the mechanism by which HO-1 recognizes heme. NMR relaxation experiments showed that the heme-binding site in heme-free HO-1 fluctuates in concert with a surface-exposed loop and transiently forms a partially unfolded structure. Because the fluctuating loop is located over 17 Å distal from the heme-binding site and its conformation is nearly identical among different crystal structures including catalytic intermediate states, the function of the loop has been unexamined. In the course of elucidating its function, we found interesting mutations in this loop that altered activity but caused little change to the conformation. The Phe79Ala mutation in the loop changed the conformational dynamics of the heme-binding site. Furthermore, the heme binding kinetics of the mutant was slower than that of the wild type. Hence, we concluded that the distal loop is involved in the regulation of the conformational change for heme binding through the conformational fluctuations. Similar to other enzymes, HO-1 effectively promotes its function using the identified distal sites, which might be potential targets for protein engineering.

Aims. This study was aimed at investigating whether elevation of heme oxygenase-1 (HO-1) expression could lead to restoring intestinal tight junction (TJ) function in a rat liver transplantation model. Methods. Intestinal mucosa injury was induced by orthotopic autologous liver transplantation (OALT) on male Sprague-Dawley rats. Hemin (a potent HO-1 activator) and zinc-protoporphyrin (ZnPP, a HO-1 competitive inhibitor), were separately administered in selected groups before OALT. The serum and intestinal mucosa samples were collected at 8 hours after the operation for analysis. Results. Hemin pretreatment significantly reduced the inflammation and oxidative stress in the mucosal tissue after OALT by elevating HO-1 protein expression, while ZnPP pretreatment aggravated the OALT mucosa injury. Meanwhile, the restriction on the expression of tight junction proteins zonula occludens-1 and occludin was removed after hemin pretreatment. These molecular events led to significant improvement on intestinal barrier function, which was proved to be through increasing nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and reducing nuclear translocation of nuclear factor kappa-B (NF-κB) in intestinal injured mucosa. Summary. Our study demonstrated that elevation of HO-1 expression reduced the OALT-induced intestinal mucosa injury and TJ dysfunction. The HO-1 protective function was likely mediated through its effects of anti-inflammation and antioxidative stress.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting that exploitation of these cellular systems may offer new therapeutic avenues in a variety of diseases. Starting from a CO-releasing compound and a chemical scaffold exhibiting electrophilic characteristics (esters of fumaric acid), we report the synthesis of hybrid molecules that simultaneously activate Nrf2 and liberate CO. These hybrid compounds, which we termed "HYCOs", release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide here the first example of a new class of pharmacologically active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Heme oxygenase1 (HO1) is involved in protecting plants from environmental stimuli. In this study, a sunflower (Helianthus annuus L.) HO1 gene (HaHO1) was cloned and sequenced. It was confirmed that HaHO1 encodes a precursor protein of 32.93 kDa with an N-terminal plastid transit peptide which was validated by subcellular localization. The amino acid sequence of HaHO1 shared high homology with other plant HO1s. The predicted three-dimensional structure showed a high degree of structural conservation as compared to the known HO1 crystal structures. Phylogenetic analysis revealed that HaHO1 clearly grouped with the plant HO1-like sequences. Moreover, the purified recombinant mature HaHO1 expressed in Escherichia coli exhibits HO activity. Thus, it was concluded that HaHO1 encodes a functional HO1 in sunflower. Additionally, HaHO1 gene was ubiquitously expressed in all tested tissues, and induced differentially during different growth stages after germination, and could be differentially induced by several stresses and hemin treatment. For example, a pretreatment with a low concentration of NaCl (25 mM) could lead to the induction of HaHO1 gene expression and thereafter a salinity acclamatory response. Above cytoprotective effect could be impaired by the potent HO1 inhibitor zinc protoporphyrin IX (ZnPPIX), which was further rescued by the addition of 50% carbon monoxide aqueous solution (in particular) or bilirubin, two catalytic by-products of HO1, respectively. Similarly, a HO1 inducer, hemin, could mimic the salinity acclamatory response. Together, these findings strongly suggested that the up-regulation of HaHO1 might be required for the observed salinity acclimation in sunflower plants.

We investigated the protective effects of lucidone, a naturally occurring cyclopentenedione isolated from the fruits of Lindera erythrocarpa Makino, against free-radical and inflammation stimulator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress in human keratinocyte (HaCaT) cells, with the aim of revealing the possible mechanisms underlying the protective efficacy. Lucidone pretreatment (0.5-10 μg/mL) markedly increased HaCaT cell viability and suppressed AAPH-induced reactive oxygen species (ROS) generation, lipid peroxidation, and DNA damage. Notably, the antioxidant potential of lucidone was directly correlated with the increased expression of an antioxidant gene, heme oxygenase 1 (HO-1), which was followed by the augmentation of the nuclear translocation and transcriptional activation of NF-E2-related factor-2 (Nrf2), with or without AAPH. Nrf2 knockdown diminished the protective effects of lucidone. We also observed that lucidone pretreatment inhibited AAPH-induced inflammatory chemokine prostaglandin E₂ (PGE₂) production and the expression of cyclooxygenase-2 (COX-2) in HaCaT cells. Lucidone treatment also significantly inhibited AAPH-induced nuclear factor-κB (NF-κB) activation and suppressing the degradation of inhibitor-κB (I-κB). Furthermore, lucidone significantly diminished AAPH-induced COX-2 expression through the down-regulation of the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Therefore, lucidone may possess antioxidant and anti-inflammatory properties and may be useful for the prevention of free radical-induced skin damage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: There are no published randomized data on secondary prevention in humans about whether aspirin affects nitric oxide (NO) formation. In patients with chronic stable coronary disease, we tested whether aspirin at clinically relevant doses increases NO formation., Materials and Methods: In a randomized, double-blind trial, 37 patients from 2 cardiology office practices were assigned to daily doses of 81, 162.5, 325, 650, or 1300 aspirin for 12 weeks. Primary prespecified outcome measures were changes in heme oxygenase (HO-1), a downstream target of NO formation, and asymmetrical dimethyl arginine (ADMA), a competitive inhibitor of NO synthase., Results: There were no significant differences for HO-1 or ADMA between any of the clinically relevant doses of aspirin tested, so all were combined. For HO-1, there was a significant increase (10.29 ± 2.44, P < .001) from baseline (15.37 ± 1.85) to week 12 (25.66 ± 1.57). The mean ratio (MR) of week 12 to baseline for HO-1 was significantly higher than 1.0 (1.67, confidence interval [CI] from 1.60 to 1.74, P < .001). For ADMA, there was a significant decrease (-0.24 ± 0.11, P < .001) from baseline (0.78 ± 0.08) to week 12 (0.54 ± 0.07). The MR of week 12 to baseline for ADMA was significantly lower than 1.0 (0.69, CI from 0.66 to 0.73, P < .001)., Conclusions: In patients with chronic stable coronary disease, all clinically relevant daily doses of aspirin tested, from 81 to 1300 mg, produce similar and statistically significant increases in HO-1 and decreases in ADMA. These are the first randomized data on secondary prevention patients. These data support the hypothesis that aspirin has additional beneficial effects mediated through NO formation. Further research, including direct randomized comparisons on atherosclerosis using noninvasive techniques as well as on occlusive vascular disease events, is necessary.

This work deals with dioxygen (O2 ) binding sites and pathways through inducible human heme oxygenase (HO-1). The experimentally known distal binding site 1, and sites 2-3 above it, could be reproduced by means of non-deterministic random-acceleration molecular-dynamics (RAMD) simulations. In addition, RAMD revealed the proximal binding site 5, a deeply-seated binding site 4, which lies behind heme, as well as a few gates communicating with the external medium. In getting from site 1 to the main gate, which lies on the protein front opposed to site 4, O2 follows chiefly the shortest direct pathway. Less frequently, O2 visits intermediate sites 2, 4, or 5 along longer pathways. A similarity between HO-1, myoglobin, and cytoglobin in using, for diatomic gas delivery, the direct shortest pathway from the heme center to the surrounding medium, is emphasized. Otherwise, comparing other proteins and diatomic gases, each system reveals its peculiarities as to sites, gates, and pathways. Thus, relating these properties to the physiological functions of the proteins remains in general a challenge for future studies., (Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.)

Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigate the cytoprotective effects and the molecular mechanism of genistein in oxidative stress-induced injury using an endothelial cell line (EA.hy926). An oxidative stress model was established by incubating endothelial cells with H₂O₂. According to the present results, genistein pretreatment protected endothelial cells against H₂O₂-induced decreases in cell viability and increases in apoptosis. Genistein also prevented the inhibition of B-cell lymphoma 2 and the activation of caspase-3 induced by H₂O₂. Genistein increased superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels and attenuated the decrease in these antioxidants during oxidative stress. We also found that genistein induced the promoter activity of both nuclear factor erythroid 2-related factor 2 (Nrf2) and PPARγ. Additionally, genistein induced the nuclear translocation of Nrf2 and PPARγ. While genistein caused the up-regulation of both Nrf2 and PPARγ, it also activated and up-regulated the protein expression and transcription of a downstream protein, haem oxygenase-1 (HO-1). Moreover, the use of Nrf2 small interfering RNA transfection and HO-1- or PPARγ-specific antagonists (Znpp and GW9662, respectively) blocked the protective effects of genistein on endothelial cell viability during oxidative stress. Therefore, we conclude that oxidative stress-induced endothelial cell injury can be attenuated by treatment with genistein, which functions via the regulation of the Nrf2 and PPARγ signalling pathway. Additionally, the endogenous antioxidants SOD, CAT and GSH appear to play a role in the antioxidant activity of genistein. The present findings suggest that the beneficial effects of genistein involving the activation of cytoprotective antioxidant genes may represent a novel strategy in the prevention and treatment of cardiovascular endothelial damage.

The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties and may play a role in several disease states, making it an enticing therapeutic target. Traditionally, the metalloporphyrins have been used as competitive HO inhibitors owing to their structural similarity with the substrate, heme. However, given heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non-selectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated, which provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies.