Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin.

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.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

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.

A Coumarin-Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1.

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-L1. We designed Fe-L1 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-L2, 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.

The immune function of heme oxygenase-1 from grass carp (Ctenopharyngodon idellus) in response to bacterial infection.

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 (Fe2+). 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-1ß, 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.

Novel mutual prodrug of 5-fluorouracil and heme oxygenase-1 inhibitor (5-FU/HO-1 hybrid): design and preliminary in vitro evaluation.

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.

Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.

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.

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer.

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.

Exogenous neutrophil elastase enters bronchial epithelial cells and suppresses cigarette smoke extract-induced heme oxygenase-1 by cleaving sirtuin 1.

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.

A novel heme oxygenase-1 splice variant, 14kDa HO-1, promotes cell proliferation and increases relative telomere length.

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.

Alleviation of cadmium-induced oxidative stress by trehalose via inhibiting the Nrf2-Keap1 signaling pathway in primary rat proximal tubular cells.

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.

Curcuma sp.-derived dehydrocurdione induces heme oxygenase-1 through a Michael reaction between its α, ß-unsaturated carbonyl and Keap1.

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, ß-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.

Comparison of the Mechanisms of Heme Hydroxylation by Heme Oxygenases-1 and -2: Kinetic and Cryoreduction Studies.

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.

In-Cell Enzymology To Probe His-Heme Ligation in Heme Oxygenase Catalysis.

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.

Diverse prognostic value of the GTn promoter polymorphism in squamous cell and adeno carcinoma of the oesophagus.

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.

A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli.

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.

Sulforaphane improves oxidative status without attenuating the inflammatory response or cardiac impairment induced by ischemia-reperfusion in rats.

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.

l-carnitine protects human hepatocytes from oxidative stress-induced toxicity through Akt-mediated activation of Nrf2 signaling pathway.

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.

Distal regulation of heme binding of heme oxygenase-1 mediated by conformational fluctuations.

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.

Antiinflammatory and antioxidant effects of H2O2 generated by natural sources in Il1ß-treated human endothelial cells.

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-1ß 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-1ß 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.

Systemic drugs inducing non-immediate cutaneous adverse reactions and contact sensitizers evoke similar responses in THP-1 cells.

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.