Heme Degradation Pathway Research Articles

Direct Ingestion of Oxidized Red Blood Cells (Efferocytosis) by Hepatocytes.

Both hepatic iron accumulation and hemolysis have been identified as independent prognostic factor in alcohol-related liver disease (ALD); however, the mechanisms still remain poorly understood. We here demonstrate that hepatocytes are able to directly ingest aged and ethanol-primed red blood cells (RBCs), a process termed efferocytosis. Efferocytosis of RBCs was directly studied in vitro and observed by live microscopy for real-time visualization. RBCs pretreated with either CuSO4 or ethanol following co-incubation with Huh7 cells and murine primary hepatocytes. Heme oxygenase-1 (HO-1) and other targets were measured by q-PCR. As shown by live microscopy, oxidized RBCs, but not intact RBCs, are rapidly ingested by both Huh7 cells and murine primary hepatocytes within 10 minutes. In some cases, more than 10 RBCs were seen within hepatocytes, surrounding the nucleus. RBC efferocytosis also rapidly induces HO1, its upstream regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin, indicating efficient heme degradation. Preliminary data further suggest that hepatocyte efferocytosis of oxidized RBCs is, at least in part, mediated by scavenging receptors such as ASGPR1. Of note, pretreatment of RBCs with ethanol but also heme and bilirubin also initiated efferocytosis. In a cohort of heavy human drinkers, a significant correlation of hepatic ASGPR1 with the heme degradation pathway was observed. We here demonstrate that hepatocytes can directly ingest and degrade oxidized RBCs through efferocytosis, a process that can be also triggered by ethanol, heme and bilirubin. Our findings are highly suggestive for a novel mechanism of hepatic iron overload in ALD patients.

Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration.

Chemotherapy, notably docetaxel (Doc), stands as the primary treatment for castration-resistant prostate cancer (CRPC). However, its efficacy is hindered by side effects and chemoresistance. Hypoxia in prostate cancer (PC) correlates with chemoresistance to Doc-induced apoptosis via Heme Oxygenase-1 (HO-1) modulation, a key enzyme in heme metabolism. This study investigated targeting heme degradation pathway via HO-1 inhibition to potentiate the therapeutic efficacy of Doc in PC. Utilizing diverse PC cell lines, we evaluated HO-1 inhibition alone and with Doc on viability, apoptosis, migration, and epithelial- to- mesenchymal transition (EMT) markers and elucidated the underlying mechanisms. HO-1 inhibition significantly reduced PC cell viability under hypoxic and normoxic conditions, enhancing Doc-induced apoptosis through interconnected mechanisms, including elevated reactive oxygen species (ROS) levels, glutathione cycle disruption, and modulation of Signal Transducer and Activator of Transcription 1 (STAT1) pathway. The interplay between STAT1 and HO-1 suggests its reliance on HO-1 activation. Additionally, a decrease in cell migration and downregulation of EMT markers (vimentin and snail) were observed, indicating attenuation of mesenchymal phenotype. In conclusion, the combination of HO-1 inhibition with Doc holds promise for improving therapeutic outcomes and advancing clinical management in PC.

Evaluation of Rapanone and Nectandrin B as novel inhibitors for targeting the metastatic regulator protein BACH1 using breast cancer cell line Mcf-7

Cancer formation is defined as the unrestrained proliferation of cells due to various factors acting as a causing agent. A limited number of over-expressed transcription factors are contributed to the development of numerous types of cancer. The metastatic regulator protein BTB And CNC Homology 1 (BACH1) is Cap ‘N’ Collar (CNC) and it belongs to a basic region leucine zipper (bZIP) family. The presence of the least level concentration of intracellular heme BACH1 forms heterodimers with musculo aponeurotic fibrosarcoma (sMAF) proteins and inhibits or induces the target gene expression. Based on the previous studies, BACH1 plays a critical player in the conditions of senescence and oxidative stress, cycling of cell life, heme degradation pathway and cancer, especially in metastasis. Discovering new anti-cancer drugs (identification of bioactive compounds) stages finally needs to inhibit the target protein. This present study is aimed to screen and identify stability, binding affinity and analysis of pharmacokinetics of selected compounds through structural screening, ADMET, DFT and MESP. From this study, it is revealed that Rapanone and Nectandrin B have the potential to alter the degree of gene expression via binding with the BACH1 allosteric region which will further change the degree of expression of BACH1 downstream target genes involved in the regulation of cancer progression particularly in metastasis. The two plant origin compounds Rapanone and Nectandrin B might be novel candidates for developing anti-cancer drugs. The predicted compounds were further validated through in-vitro experimental approaches. Communicated by Ramaswamy H. Sarma.

Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid

5-ALA-mediated photodynamic therapy (PDT) has been developed around the heme biosynthesis physiological pathway. It is based on the external supplementation of 5 aminolevulinic acid (5-ALA), increasing the activity of the heme pathway and leading to a significant protoporphyrin IX (PpIX) accumulation. Interestingly, this metbolite accumulation is predominant in cancer cells, induced by a highly active metabolism, therefore limiting off-target side effects and increasing therapy specificity. Nevertheless, the intrinsic mechanism responsible of PpIX accumulation on cells following PDT is still unknown, limiting clinical therapy translation. In order to further understand the mechanisms behind 5-ALA-induced PDT, in this study we aimed to evaluate the proteome changes reported on the physiological heme pathway, in response to an external 5-ALA supplementation. We studied two different scenarios following 5-ALA treatment, 5-ALA accumulation (5-ALA metabolization into the heme pathway blocked with inhibitors) and accumulation of PpIX (normal heme pathway with 5-ALA supplementation). Therefore, we were able to characterize enzymatic changes and to describe bottlenecks in the pathway. Following mass spectrometry analysis, we reported significant differences between 5-ALA and PpIX effects on heme biosynthesis and regulation of degradation. 5-ALA accumulation significantly decreased porphobilinogen deaminase (HMBS) expression, while phorphyrins accumulation (PpIX) upregulated heme synthesis, specifically HMBS and uroporphyrinogen decarboxylase (UROD), and enhanced the enzymatic level of the heme degradation pathway, including Heme oxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA). Interestingly, porphyrins induced a significant downregulation effect on oxygen-dependent coproporphyrinogen-III oxidase (CPOX). In conclusion, in this study we demonstrated that porphyrins play the most relevant role in heme biosynthesis modulation, while 5-ALA alone (PDT substrate) is not responsible of the main changes observed in this pathway during PDT treatment. Understanding heme enzyme modulation would help to design a more rational approach for patient treatment in the clinic. AIM: Effect of 5-ALA and porphyrins on the different Heme biosynthesis and degradation enzymes.

Enzymological and structural characterization of Arabidopsis thaliana heme oxygenase-1.

Arabidopsis thaliana heme oxygenase‐1 (AtHO‐1), a metabolic enzyme in the heme degradation pathway, serves as a prototype for study of the bilin‐related functions in plants. Past biological analyses revealed that AtHO‐1 requires ferredoxin‐NADP+ reductase (FNR) and ferredoxin for its enzymatic activity. Here, we characterized the binding and degradation of heme by AtHO‐1, and found that ferredoxin is a dispensable component of the reducing system that provides electrons for heme oxidation. Furthermore, we reported the crystal structure of heme‐bound AtHO‐1, which demonstrates both conserved and previously undescribed features of plant heme oxygenases. Finally, the electron transfer pathway from FNR to AtHO‐1 is suggested based on the known structural information.

HEME Degradation Enzyme Biliverdin IXb Reductase Is Required for Stem Cell Glutamine Metabolism

Bioenergetic requirements of pluripotent stem cells (PSC) vary with lineage fate, and cellular adaptations rely largely on substrate availability and mitochondrial function to balance TCA-derived anabolic and redox-regulated antioxidant functions. Heme (protoporphyrin IX complexed with iron) serves as an indispensable co-factor for all aerobic cells, and its cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles, the penultimate step regulated by two non-redundant biliverdin reductases (IXa, BLVRA and IXb, BLVRB) retaining isomeric specificity and NAD(P)H-dependent redox coupling linked to BR antioxidant function. Thus, heme biosynthesis (initiated by the condensation of glycine and succinyl CoA) can be considered a linear pathway that utilizes cataplerotic TCA-derived carbon (derived from glucose and glutamine) linked to the heme degradation pathway and bilirubin excretion. Given the overlapping glucose/glutamine requirements in both heme synthesis and PSC bioenergetics, we studied consequences of dysregulated heme metabolism by targeted BLVRB disruption in iPSCs derived from human umbilical cord CD34+ hematopoietic cells. Compared to control iPSCs, BLVRB-deficient iPSCs had no reciprocal BLVRA induction, no alterations in heme oxygenase expression, and no differences in isomeric BV accumulation, although phenotypically defective in antioxidant handling with exaggerated apoptotic cell death. To better delineate metabolic pathway perturbations causally implicated in BLVRB- deficient antioxidant function and cell viability, we generated gene/metabolite clusters and an extended network plot formulated on RNASeq data coupled with liquid chromatography/mass spectrometric-based targeted metabolomics profiling of 290 intracellular metabolites. Metabolic enrichment with topological pathway analysis identified a limited number of defective pathways involving purine/pyrimidine nucleotide synthesis, redox homeostasis (pentose phosphate pathway, PPP) and TCA cycle intermediates (a-ketoglutarate, fumarate, and malate). Mitochondrial staining using TMRE as a functional and quantifiable marker of the mitochondrial transmembrane potential (DYm) identified no defect across the genotypes, although BLVRB -deficient iPSCs demonstrated defective basal and maximal oxygen consumption rates (OCR) restricted to glutamine (but not glucose) utilization. Metabolic flux quantification using [U6-13C] glucose revealed high-level and comparable (>97%) fractional labeling of 13C lactate across the genotypes, consistent with an active glycolytic metabolism that was comparable between control and BLVRB-/- iPSCs. In contrast, isotopic tracing using [U-13C5] glutamine demonstrated statistically-diminished glutamate-derived aKG accumulation (along with downstream TCA metabolites fumarate and malate isotopomers), results confirming rewired glutamine TCA entry, and reliance on glucose as a preferred substrate. Since the parallel PPP pathway utilizes glycolysis as the preferred substrate for generating both reducing equivalents (NADPH) and essential nucleotide component ribose-5-phosphate, we characterized three-dimensional embryoid body (EB) formation in the presence of the PPP pathway inhibitor 6-aminonicotinamide (6-AN), establishing statistically-decreased EB size (p<0.001) compared to control iPSCs. These data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism, and also suggest in principle that BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization, a key substrate for cancer and hematopoietic metabolism. DisclosuresNo relevant conflicts of interest to declare.

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

The heme molecule serves as an essential prosthetic group for oxygen transport and storage proteins, as well for cellular metabolic enzyme activities, including those involved in mitochondrial respiration, xenobiotic metabolism, and antioxidant responses. Dysfunction in both heme synthesis and degradation pathways can promote human disease. Heme is a pro-oxidant via iron catalysis that can induce cytotoxicity and injury to the vascular endothelium. Additionally, heme can modulate inflammatory and immune system functions. Thus, the synthesis, utilization and turnover of heme are by necessity tightly regulated. The microsomal heme oxygenase (HO) system degrades heme to carbon monoxide (CO), iron, and biliverdin-IXα, that latter which is converted to bilirubin-IXα by biliverdin reductase. Heme degradation by heme oxygenase-1 (HO-1) is linked to cytoprotection via heme removal, as well as by activity-dependent end-product generation (i.e., bile pigments and CO), and other potential mechanisms. Therapeutic strategies targeting the heme/HO-1 pathway, including therapeutic modulation of heme levels, elevation (or inhibition) of HO-1 protein and activity, and application of CO donor compounds or gas show potential in inflammatory conditions including sepsis and pulmonary diseases.

Chimeric Protein IPath® with Chelating Activity Improves Atlantic Salmon's Immunity against Infectious Diseases.

Infection processes displayed by pathogens require the acquisition of essential inorganic nutrients and trace elements from the host to survive and proliferate. Without a doubt, iron is a crucial trace metal for all living organisms and also a pivotal component in the host–parasite interactions. In particular, the host reduces the iron available to face the infectious disease, increasing iron transport proteins’ expression and activating the heme synthesis and degradation pathways. Moreover, recent findings have suggested that iron metabolism modulation in fish promotes the immune response by reducing cellular iron toxicity. We hypothesized that recombinant proteins related to iron metabolism could modulate the fish’s immune system through iron metabolism and iron-responsive genes. Here a chimeric iron transport protein (IPath®) was bioinformatically designed and then expressed in a recombinant bacterial system. The IPath® protein showed a significant chelating activity under in vitro conditions and biological activity. Taking this evidence, a vaccine candidate based on IPath® was evaluated in Atlantic salmon challenged with three different fish pathogens. Experimental trials were conducted using two fish groups: one immunized with IPath® and another injected with adjutant as the control group. After 400 accumulated thermal units (ATUs), two different infection trials were performed. In the first one, fish were infected with the bacterium Aeromonas salmonicida, and in a second trial, fish were exposed to the ectoparasite Caligus rogercresseyi and subsequently infected with the intracellular bacterium Piscirickettsia salmonis. Fish immunized with IPath® showed a significant delay in the mortality curve in response to A. salmonicida and P. salmonis infections. However, no significant differences between infected and control fish groups were observed at the end of the experiment. Notably, sea lice burden reduction was observed in vaccinated Atlantic salmon. Transcriptional analysis evidenced a high modulation of iron-homeostasis-related genes in fish vaccinated with IPath® compared to the control group during the infection. Moreover, increasing expression of Atlantic salmon IgT was associated with IPath® immunization. This study provides evidence that the IPath® protein could be used as an antigen or booster in commercial fish vaccines, improving the immune response against relevant pathogens for salmon aquaculture.

Xanthine Oxidase Has a Protective Role during Heme Crisis By Binding and Degrading Heme

Xanthine oxidase (XO) is a key enzyme in the purine degradation pathway, catalyzing the catabolism of hypoxanthine to xanthine and xanthine to uric acid. A byproduct of these reactions is the generation of the reactive oxygen species (ROS), hydrogen peroxide and superoxide. XO is produced primarily in the liver; however, following hepatic stressors such as inflammation, hypoxia, or ischemia, XO is released from the liver and enters the circulation. XO can then bind distal endothelium via electrostatic interactions with glycosaminoglycans (GAGs). Current dogma believes that XO plays a harmful role in pathologies due to the increase in ROS production that can alter signaling pathways and damage endothelial cells. XO activity has been shown to be elevated in a number of hemolytic conditions including, sickle cell disease, malaria, and sepsis; however, the involvement of XO in these pathological conditions has not been fully elucidated. These conditions result in increased hemolysis, releasing free heme and hemoglobin into the circulation, inducing an inflammatory response, and damaging endothelial cells. Identifying the involvement of XO during heme crisis could improve our understanding of the pathologies associated with hemolytic conditions and lead to the identification or development of more effective treatment options. We hypothesized that XO has damaging properties under basal conditions; however, the presence of XO is crucial and protective during heme crisis by serving as a secondary mechanism of heme degradation when canonical heme degradation pathways are saturated. In order to explore the role of XO in heme crisis, we developed a novel heme crisis model in which we pre-treated mice with the clinically relevant dose of 10 mg/kg/day febuxostat, an FDA approved XO inhibitor, for five days in drinking water. Following inhibition of XO, the mice were challenged with two identical doses of hemin one hour apart and monitored for 24 hours. We observed a 20-fold increase in XO activity in the mice treated with 50 μmol/kg hemin. This increase was completely inhibited in the mice treated with febuxostat. Surprisingly, the febuxostat treated mice had worsened survival compared to the untreated mice. This suggests a protective role for XO during heme crisis. We hypothesized that XO has a protective role by preventing platelet activation and degrading excess free heme. To investigate this hypothesis, we used flow cytometry to quantify heme-induced platelet activation. Healthy human platelets were isolated and treated with 2.5 μM hemin, 10 mU/mL XO, 200 μM hypoxanthine, and 20 μM febuxostat. We observed 72% activation with heme alone, while incubation with XO and hypoxanthine resulted in almost complete prevention of platelet activation (16%). We were also able to partially restore platelet activation (45%) when febuxostat was added. Based on these results, we hypothesize that XO binds GAGs on the platelet surface and degrades heme in order to protect platelets from heme-induced activation. To assess the ability of XO to degrade heme, we tested whether XO binds heme. We performed computational modeling in which we identified a potential heme binding site in the FAD domain of XO with a kd = 128 nM. We confirmed heme-XO binding by performing a heme binding assay. We incubated heme (25 μM) alone, heme + XO (50 μM), and heme + XO + hypoxanthine (100 μM) for 20 minutes. Heme binding was assessed by dot blotting in nitrocellulose followed by chemiluminescent detection and dot density quantification. We observed a 2-fold increase in dot density when heme and XO were incubated and a 4-fold increase with heme, XO, and hypoxanthine. These results support a potential heme-XO interaction that is amplified when the enzyme is active. Lastly, we measured XO's ability to degrade heme using UV visible spectrophotometry. We incubated hemin, XO, and hypoxanthine together and measured the absorbance over 20 minutes. We observed a decrease in absorbance at 618 nm, indicative of heme degradation. In conclusion, contrary to the current dogma, we have identified a potential protective role for XO during hemolytic crisis. We found that febuxostat treatment worsened survival in heme challenged mice, XO prevented heme-induced platelet activation, identified a potential heme-XO binding site, and observed XO-induced heme degradation. XO may have a protective role in hemolytic conditions by serving as a secondary mechanism of heme degradation. Disclosures No relevant conflicts of interest to declare.

Role of heme oxygenase-1 in human placenta on iron supply to fetus

IntroductionTo date, details on how iron is supplied from the mother to the fetus through the placenta have remained unclear. Recently, increasing evidence has shown that heme oxygenase (HO)-1, which is an inducible isoform of the rate-limiting enzyme in the heme degradation pathway, may be involved in the effective reutilization of iron. In this study, we examined the distribution and gene expression of HO-1 in the villous tissue of human placenta at various periods of pregnancy. MethodsUsing the placenta of 38 samples for which consent was obtained, chronological changes in the localization of HO-1 protein were examined by histological examination. RT-PCR was also performed to examine the expression of HO-1, transferrin receptor-1, and ferroportin 1. Ferric iron in the tissues was analyzed by Prussian blue staining. ResultsImmunohistochemical studies showed that HO-1 protein was exclusively expressed in trophoblastic cells throughout gestation. In the miscarriage placenta in the first trimester, ho-1 mRNA levels were significantly higher than normal. Placenta with fetal death (miscarriage) in the first and second trimester indicate significantly higher ratio of ho-1 gene for iron production to the fpn-1 gene for iron excretion than normal. These suggest that the role of HO-1 with various physiological functions is changing throughout pregnancy. DiscussionThese findings suggest that HO-1 in placenta plays an important role in iron supplying system in the second trimester to support fetal development.

Bilirubin metabolism and its role in atherosclerosis

Hemoglobin is not an guiltless bystander of the pathophysiology in a number of atherosclerotic diseases. Heme, which is released from hemoglobin or other heme proteins, triggers various pathophysiological consequence, including heme stress as well as intracellular stress. Although heme serves key functions and is tightly controlled, high levels of free heme, which may occur in various pathophysiological conditions, are may hazardous via pro-oxidant, pro-inflammatory, and cytotoxic effects. &#x0D; Heme oxygenases are heat shock protein enzymes that use heme as a substrate and function as an essential antioxidant adaptive response by all human cells. A major function of heme oxygenases is clearance of heme that accumulate in tissues due to erythrocyte turnover. The potentially toxic free heme is converted by heme oxygenases into carbon monoxide, iron, and biliverdin, the latter of which is reduced to bilirubin. In literature the heme degradation pathway has been demonstrated to play a protective role against the development of atherosclerosis. Because growing evidence suggests that oxidative stress is involved in atherosclerosis.&#x0D; This review documents the roles of bilurubin in atherosclerosis and focuses on the clinical significance as a potential therapeutic target in atherosclerotic diseases, such as coronary artery disease.

A Role of the Heme Degradation Pathway in Shaping Prostate Inflammatory Responses and Lipid Metabolism

The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1β levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli- or phosphate-buffered saline-treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1β expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1β levels in part via an ACSL1-mediated pathway.

Biliverdin reductase deficiency triggers an endothelial-to-mesenchymal transition in human endothelial cells

Endothelial dysfunction accompanied by the loss of endothelial cell phenotype plays an essential role in cardiovascular diseases. Here, we report that knockdown of biliverdin reductase (BVR), the enzyme of the heme degradation pathway converting biliverdin to bilirubin, shifts endothelial phenotype of the primary human aortic endothelial cells (HAECs) to mesenchymal-like one. It is reflected by the loss of endothelial markers and angiogenic response, with concomitant acquiring of mesenchymal markers, increased migratory capacity and metalloproteinase activity. BVR-deficiency induces the activity of Nrf2 transcription factor and increases heme oxygenase-1 (HO-1) level, which is accompanied by the reduction of cellular heme content, increase in a free iron fraction and oxidative stress. Accordingly, the phenotype of BVR-deficient cells can be mimicked by hemin or iron overload. Depletion of HO-1 in BVR-deficient ECs abrogates the increase in intracellular free iron and oxidative stress, preventing the loss of endothelial markers. Treatment of BVR-deficient cells with bilirubin does not rescue the endothelial phenotype of HAECs. Unlike BLVRA mRNA level, the expression of HMOX1, HMOX1:BLVRA ratio and HO-1 protein level positively correlate with abdominal aortic aneurysm size in clinical samples. Collectively, the non-enzymatic activity of BVR contributes to the maintenance of healthy endothelial phenotype through the prevention of HO-1-dependent iron-overload, oxidative stress and subsequent endothelial-to-mesenchymal transition (EndMT).

Hemozoin produced by mammals confers heme tolerance.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.

BILIVERDIN REDUCTASE B/ FLAVIN REDUCTASE (BLVRB/FLR) EXHIBITS HEME-REGULATED, NADPH-DEPENDENT REDUCTASE ACTIVITY AND CONFERS CYTOPROTECTION IN DEVELOPING ERYTHROID CELLS

Biliverdin reductase B (BLVRB), also known as flavin reductase (FLR), is especially expressed in cells having high levels of heme synthesis (e.g. developing erythroid cells, fetal and adult liver). BLVRB exhibits NADPH-dependent reductase activity toward non-alpha isomers of biliverdin, flavins and redox cycling agents, and also has pharmacological importance in the treatment of methemoglobinemia. Nonetheless, the physiological function of BLVRB is obscure. In this work, the functional role of BLVRB in developing erythroid cells in relation to its heme-associated properties is elucidated. Recombinant mouse BLVRB binds ferric heme with a submicromolar affinity in association with inhibition of the NADPH-dependent reductase activity while displaying heme peroxidase activity. The interaction of BLVRB with heme in developing erythroid cells (MEL) was demonstrated by proteomic analysis of heme peroxidase-associated proteins. Upon induction of erythroid differentiation of MEL cells, the BLVRB protein expression is markedly up-regulated. Knockdown of BLVRB in the induced MEL cells aggravates intracellular oxidation and cytotoxicity mediated by thiol depleting agent and heme synthesis inhibitor, suggesting that BLVRB may maintain the cell viability under oxidative stress or heme deficient conditions. Our study suggests that BLVRB/FLR, which is usually regarded as a biliverdin reducing enzyme in the heme degradation pathway, is an erythroid active, heme-regulated NADPH-dependent reductase. It confers cytoprotection in developing erythroid cells under oxidative stress conditions in connection with cellular heme levels. This work was supported by General Research Fund (17160916) and Health and Medical Research Fund (04151846).

Control of Redox Homeostasis in Tick Blood Feeding

Introduction: Ticks are hematophagous ectoparasites that cause significate economical losses. Acaricide application is the main method to tick control. However, it causes environmental contamination and selects resistant ticks. The immunological control represents a suitable method to replace or complement acaricide application. During its life cycle, female ticks ingest large amounts of blood, which contains toxic components able to damage biomolecules. Understanding which molecular mechanisms and proteins are involved in avoid damages caused by blood intake in ticks and other hematophagous arthropods could help to found potential candidate antigens to compose an anti-tick vaccine. Review: Hemoglobin comprises almost 20% of mammalian blood proteins, its hydrolysis during tick digestion increases total free heme that can potentially generate reactive oxygen species (ROS), which easily oxidize lipids, proteins and DNA, modifying they structure and function. Lipids are more susceptible to high ROS levels. It can lead to membrane instability and cell death. Protein modifications caused by ROS can promote the protein loss of function and cell disturbance, however, it can also work as post-translational modifications, acting in cell signaling processes. DNA presents more efficient protective mechanisms against ROS, but damages can lead to double helix rupture. Oxidative stress is defined as a disturbance in the balance between the production and elimination of ROS, in favor of ROS production, leading to a disruption in redox homeostasis and/or molecular damage. Despite the well-recognized heme oxidative power and its already demonstrated cytotoxicity, ticks are able to feed on blood, controlling the redox homeostasis without causing oxidative stress. This occurs because ticks developed physiological adaptations to transport, store, metabolize and secrete toxic components from the diet. The strategies, such as heme compartmentalization in specialized organelles, and heme and iron carrying proteins are shared with most of other hematophagous. Interesting, heme degradation, a mechanism commonly described in hematophagous arthropods, showed to be absent in ticks. Moreover, there is a lack of key enzymes from the heme degradation pathway in tick genomes. As expected, antioxidant enzymes are often involved in homeostasis redox control. They act in a convenient way, eliminating physiological and non-physiological produced ROS. Despite their essential role in non-hematophagous organisms, antioxidant enzymes were associated with blood ingestion during arthropod hematophagous process, demonstrating their role in avoiding oxidative stress caused by blood intake. Catalase showed to be essential to heme detoxification in Rhipicephalus microplus tick, and diverse antioxidant enzymes are up-regulated after blood intake in a variety of hematophagous parasites. If in one hand ROS is responsible to cell damage and oxidative stress, on the other hand, several works revealed the fundamental role of ROS in cell signaling and function, demonstrating the sophisticated regulation that is necessary to redox homeostasis control.Conclusions: In the course of arthropods evolution, blood-feeding life style represent a special strategy to acquire energy and biosynthetic precursors. Together with this advantage, hematophagous organisms needed to develop molecular mechanisms to control toxic components ingested with blood. In this context, several works reviewed here identified the role of proteins and enzymes in the control of free heme released during hematophagy and in redox homeostasis, in order to avoid oxidative stress caused by blood intake. This knowledge represents an important contribution in the search for new candidate targets to develop efficient tick control methods.

Yeast as a protein source with health beneficial properties in diets for smoltifying Atlantic salmon (Salmo salar L.)

Sea lice are copepodid ectoparasites that produce high economic losses and environmental issues, thus impacting the salmon aquaculture worldwide. Atlantic salmon (Salmo salar) from Northern and Southern Hemispheres are primarily parasitized by Lepeophtheirus salmonis and Caligus rogercresseyi, respectively. To cope L. salmonis infestation, studies suggest that Atlantic salmon can restrict iron availability as a mechanism of nutritional immunity. However, no molecular studies of iron regulation from salmonids infected with C. rogercresseyi have been reported. The aim of this study was to determine if there are differences in the regulation of iron metabolism in Atlantic salmon infested with L. salmonis or C. rogercresseyi. For comparisons, skin and head kidney were profiled using qPCR of 15 genes related to iron regulation in Atlantic salmons infected with each sea louse species in Norway and Chile, respectively. Prior to infestation, no significant differences were observed between fish group. However, genes involved in iron transport and Heme biosynthesis were highly upregulated in Atlantic salmon infested with L. salmonis. Interestingly, hepcidin and Heme oxygenase, a component of the Heme degradation pathway, were upregulated during C. rogercresseyi infestation. Oxidative stress related genes were also evaluated, showing higher transcription activity in the head kidney than in the skin of Atlantic salmon infested with L. salmonis. These comparative results suggest pathogen-specific responses in infected Atlantic salmon, where iron metabolism is primarily regulated during the infestation with L. salmonis than C. rogercresseyi. Feeding behavior, for instance haematophagy, of the infesting sea lice species in relation to iron modulation is discussed.

56 - Excess circulating free heme triggers immense elevation in plasma xanthine oxidase activity

Heme, an iron-containing compound and critical prosthetic group for metalloproteins, is found most commonly as an element of hemoglobin. Excess circulating heme is a key driver of hemolytic diseases including thalassemia, malaria, sickle cell disease, sepsis as well as a side effect of transplantation and cardiac bypass. Severe hemolysis (heme crisis) saturates heme degradation pathways such as hemopexin leading to abundant circulating free heme that can directly generate reactive oxygen species (ROS) and/or stimulate elevation in enzymatic sources of ROS, ultimately leading to overt tissue damage. A key enzymatic source of ROS reported to be upregulated in sickle cell disease is xanthine oxidase (XO). As such, we investigated the relationship between circulating XO activity and heme crisis in mice. Both 24 hour survival and plasma XO activity were assessed in C57BL6/J mice injected (tail-vein) with hemin (0-100 μmol/kg). Hemin dose-dependently reduced survival (100% (25 μmol/kg), 87% (50 μmol/kg), 12% (70 μmol/kg) and 0% (100 μmol/kg)). Hematological analysis revealed an increase in monocyte recruitment and diminished platelet counts with increasing doses of hemin. Doses of 50 and 70 μmol/kg hemin elevated plasma XO activity 20-fold and 39-fold, respectively compared to controls and correlated with substantial loss of hepatic XO. Histological analysis (H&E staining) also indicated that liver tissue from mice injected with 50 μmol/kg hemin demonstrated significant alterations in tissue organization. In conclusion, heme crisis induces extensive hepatocellular release of XO into the circulation where it may actively participate in the pathologic process. To validate this assumption, we have generated a liver-specific XO knockout and are currently investigating mechanisms underpinning heme crisis-associated hepatocellular release of XO as well as the impact of circulating XO on cardiovascular function. These ongoing experiments may serve to identify novel clinical strategies to address heme crisis.

Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity.

Heme catabolism is an important biochemical process that many bacterial pathogens utilize to acquire iron. However, tetrapyrrole catabolites can be reactive and often require further processing for transport out of the cell or conversion to another useful cofactor. In previous work, we presented in vitro evidence of an anaerobic heme degradation pathway in Escherichia coli O157:H7. Consistent with reactions that have been reported for other radical S-adenosyl-l-methionine methyltransferases, ChuW transfers a methyl group to heme by a radical-mediated mechanism and catalyzes the β-scission of the porphyrin macrocycle. This facilitates iron release and the production of a new linear tetrapyrrole termed "anaerobilin". In this work, we describe the structure and function of ChuY, an enzyme expressed downstream from chuW within the same heme utilization operon. ChuY is structurally similar to biliverdin reductase and forms a dimeric complex in solution that reduces anaerobilin to the product we have termed anaerorubin. Steady state analysis of ChuY exhibits kinetic cooperativity that is best explained by a random addition mechanism with a kinetically preferred path for initial reduced nicotinamide adenine dinucleotide phosphate binding.

An Integrated Clinico-transcriptomic Approach Identifies a Central Role of the Heme Degradation Pathway for Septic Complications after Trauma.

The present study was aimed to identify mechanisms linked to complicated courses and adverse events after severe trauma by a systems biology approach. In severe trauma, overwhelming systemic inflammation can result in additional damage and the development of complications, including sepsis. In a prospective, longitudinal single-center study, RNA samples from circulating leukocytes from patients with multiple injury (injury severity score ≥17 points; n = 81) were analyzed for dynamic changes in gene expression over a period of 21 days by whole-genome screening (discovery set; n = 10 patients; 90 samples) and quantitative RT-PCR (validation set; n = 71 patients, 517 samples). Multivariate correlational analysis of transcripts and clinical parameters was used to identify mechanisms related to sepsis. Transcriptome profiling of the discovery set revealed the strongest changes between patients with either systemic inflammation or sepsis in gene expression of the heme degradation pathway. Using quantitative RT-PCR analyses (validation set), the key components haptoglobin (HP), cluster of differentiation (CD) 163, heme oxygenase-1 (HMOX1), and biliverdin reductase A (BLVRA) showed robust changes following trauma. Upregulation of HP was associated with the severity of systemic inflammation and the development of sepsis. Patients who received allogeneic blood transfusions had a higher incidence of nosocomial infections and sepsis, and the amount of blood transfusion as source of free heme correlated with the expression pattern of HP. These findings indicate that the heme degradation pathway is associated with increased susceptibility to septic complications after trauma, which is indicated by HP expression in particular.