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Sickle cell disease (SCD) is a genetic disorder predominantly affecting individuals of African descent, with a significant global health burden. SCD is characterized by intravascular hemolysis, driven by the polymerization of mutated hemoglobin within red blood cells (RBCs), leading to vascular inflammation, organ damage, and heme toxicity. Clinical manifestations include acute pain crises, hemolytic anemia, and multi-organ dysfunction, imposing substantial morbidity and mortality challenges. Current therapeutic strategies mitigate these complications by increasing the concentration of RBCs with normal hemoglobin via transfusion, inducing fetal hemoglobin, restoring nitric oxide signaling, inhibiting platelet-endothelium interaction, and stabilizing hemoglobin in its oxygenated state. While hydroxyurea and gene therapies show promise, each faces distinct challenges. Hydroxyurea's efficacy varies among patients, and gene therapies, though effective, are limited by issues of accessibility and affordability. An emerging frontier in SCD management involves harnessing endogenous clearance mechanisms for hemolysis products. A recent work by Heggland et al. showed that CD-36-like proteins mediate heme absorption in hematophagous ectoparasite, a type of parasite that feeds on the blood of its host. This discovery underscores the need for further investigation into scavenger receptors (e.g., CD36, SR-BI, SR-BII) for their possible role in heme uptake and detoxification in mammalian species. In this review, we discussed current SCD therapeutics and the specific stages of pathophysiology they target. We identified the limitations of existing treatments and explored potential future developments for novel SCD therapies. Novel therapeutic targets, including heme scavenging pathways, hold the potential for improving outcomes and reducing the global burden of SCD. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a genetic disorder predominantly affecting individuals of African descent, with a significant global health burden. SCD is characterized by intravascular hemolysis, driven by the polymerization of mutated hemoglobin within red blood cells (RBCs), leading to vascular inflammation, organ damage, and heme toxicity. Clinical manifestations include acute pain crises, hemolytic anemia, and multi-organ dysfunction, imposing substantial morbidity and mortality challenges. Current therapeutic strategies mitigate these complications by increasing the concentration of RBCs with normal hemoglobin via transfusion, inducing fetal hemoglobin, restoring nitric oxide signaling, inhibiting platelet-endothelium interaction, and stabilizing hemoglobin in its oxygenated state. While hydroxyurea and gene therapies show promise, each faces distinct challenges. Hydroxyurea's efficacy varies among patients, and gene therapies, though effective, are limited by issues of accessibility and affordability. An emerging frontier in SCD management involves harnessing endogenous clearance mechanisms for hemolysis products. A recent work by Heggland et al. showed that CD-36-like proteins mediate heme absorption in hematophagous ectoparasite, a type of parasite that feeds on the blood of its host. This discovery underscores the need for further investigation into scavenger receptors (e.g., CD36, SR-BI, SR-BII) for their possible role in heme uptake and detoxification in mammalian species. In this review, we discussed current SCD therapeutics and the specific stages of pathophysiology they target. We identified the limitations of existing treatments and explored potential future developments for novel SCD therapies. Novel therapeutic targets, including heme scavenging pathways, hold the potential for improving outcomes and reducing the global burden of SCD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xue and Li.)

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.

Hemorrhagic stroke is a major cause of morbidity and mortality worldwide. Secondary mechanisms of brain injury adversely affect functional outcome in patients after intracranial hemorrhage. Potential drivers of intracranial hemorrhage-related secondary brain injury are hemoglobin and its downstream degradation products released from lysed red blood cells, such as free heme. We established a mouse model with stereotactic striatal injection of heme-albumin to gain insights into the toxicity mechanisms of free heme in the brain and assess the therapeutic potential of heme binding and biochemical neutralization by hemopexin. We defined the dose-dependent transcriptional effect of heme or heme-hemopexin exposure 24 h after injection by spatial transcriptome analysis of lesion-centered coronal cryosections. The spatial transcriptome was interpreted in a multimodal approach along with histology, magnetic resonance imaging, and behavioral data and reported in the associated research article “Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain” [1].The spatially resolved transcriptome dataset made available here is intended for continued analysis of free heme toxicity in the brain, which is of potential pathophysiological and therapeutic significance in the context of a wide range of neurovascular and neurodegenerative diseases.

The common bed bug Cimex lectularius (Linnaeus 1758) is an ectoparasite that feeds preferably on human blood, being considered an important public health issue. Blood-feeding is a challenging process for hematophagous organisms, and one of the inherent risks with this kind of diet is the liberation of high doses of free heme after the digestion of hemoglobin. In order to deal with this potent cytotoxic agent, such organisms have acquired different defense mechanisms. Here, we use UV–visible spectrophotometry and infrared spectroscopy to show that C. lectularius crystalizes free heme to form the much less dangerous compound, hemozoin. According to our results, the peak of formation of hemozoin in the intestinal contents occurred 4–5 days after the blood meal, primarily in the posterior midgut. The quantification of the rate of conversion of heme to hemozoin revealed that at the end of digestion all the heme was in the form of hemozoin. Inhibition of the synthesis of hemozoin using the anti-malarial drug quinine led to an increase in both catalase activity in the intestinal epithelium and the mortality of the bed bugs, indicating that the insects were unable to cope with the oxidative stress generated by the overload of free heme. The data presented here show for the first time how C. lectularius deals with free heme, and how the process of formation of hemozoin is essential for the survival of these insects. Since resistance to insecticides is a common feature among field populations of bed bugs, there is an urgent need to develop alternative control methods. Thus, targeting the synthesis of hemozoin emerges as a possible novel strategy to fight bed bugs. [Display omitted] • The bed bug Cimex lectularius synthesizes hemozoin in its intestinal contents. • Hemozoin is found mainly in the posterior midgut. • By the end of the digestive process, all free heme is transformed into hemozoin. • Inhibition of hemozoin synthesis likely leads to insect death due to heme overload. [ABSTRACT FROM AUTHOR]

For pathogenic bacteria, host-derived heme represents an important metabolic cofactor and a source for iron. However, high levels of heme are toxic to bacteria. We have previously shown that excess heme has a growth-inhibitory effect on the Gram-positive foodborne pathogen Listeria monocytogenes, and we have learned that the LhrC1-5 family of small RNAs, together with the two-component system (TCS) LisRK, play a role in the adaptation of L. monocytogenes to heme stress conditions. However, a broader knowledge on how this pathogen responds to heme toxicity is still lacking. Here, we analyzed the global transcriptomic response of L. monocytogenes to heme stress. We found that the response of L. monocytogenes to excess heme is multifaceted, involving various strategies acting to minimize the toxic effects of heme. For example, heme exposure triggers the SOS response that deals with DNA damage. In parallel, L. monocytogenes shuts down the transcription of genes involved in heme/iron uptake and utilization. Furthermore, heme stress resulted in a massive increase in the transcription of a putative heme detoxification system, hrtAB, which is highly conserved in Gram-positive bacteria. As expected, we found that the TCS HssRS is required for heme-mediated induction of hrtAB and that a functional heme efflux system is essential for L. monocytogenes to resist heme toxicity. Curiously, the most highly up-regulated gene upon heme stress was lmo1634, encoding the Listeria adhesion protein, LAP, which acts to promote the translocation of L. monocytogenes across the intestinal barrier. Additionally, LAP is predicted to act as a bifunctional acetaldehyde-CoA/alcohol dehydrogenase. Surprisingly, a mutant lacking lmo1634 grows well under heme stress conditions, showing that LAP is not required for L. monocytogenes to resist heme toxicity. Likewise, a functional ResDE TCS, which contributes to heme-mediated expression of lmo1634, is not required for the adaptation of L. monocytogenes to heme stress conditions. Collectively, this study provides novel insights into the strategies employed by L. monocytogenes to resist heme toxicity. Our findings indicate that L. monocytogenes is using heme as a host-derived signaling molecule to control the expression of its virulence genes, as exemplified by lmo1634.

The LhrC family of small regulatory RNAs (sRNAs) is known to be induced when the foodborne pathogen Listeria monocytogenes is exposed to infection-relevant conditions, such as human blood. Here we demonstrate that excess heme, the core component of hemoglobin in blood, leads to a strong induction of the LhrC family members LhrC1–5. The heme-dependent activation of lhrC1–5 relies on the response regulator LisR, which is known to play a role in virulence and stress tolerance. Importantly, our studies revealed that LhrC1–5 and LisR contribute to the adaptation of L. monocytogenes to excess heme. Regarding the regulatory function of the sRNAs, we demonstrate that LhrC1–5 act to down-regulate the expression of known LhrC target genes under heme-rich conditions: oppA, tcsA, and lapB, encoding surface exposed proteins with virulence functions. These genes were originally identified as targets for LhrC-mediated control under cell envelope stress conditions, suggesting a link between the response to heme toxicity and cell envelope stress in L. monocytogenes. We also investigated the role of LhrC1–5 in controlling the expression of genes involved in heme uptake and utilization: lmo2186 and lmo2185, encoding the heme-binding proteins Hbp1 and Hbp2, respectively, and lmo0484, encoding a heme oxygenase-like protein. Using in vitro binding assays, we demonstrated that the LhrC family member LhrC4 interacts with mRNAs encoded from lmo2186, lmo2185, and lmo0484. For lmo0484, we furthermore show that LhrC4 uses a CU-rich loop for basepairing to the AG-rich Shine–Dalgarno region of the mRNA. The presence of a link between the response to heme toxicity and cell envelope stress was further underlined by the observation that LhrC1–5 down-regulate the expression of lmo0484 in response to the cell wall-acting antibiotic cefuroxime. Collectively, this study suggests a role for the LisR-regulated sRNAs LhrC1–5 in a coordinated response to excess heme and cell envelope stress in L. monocytogenes.

The LhrC family of small regulatory RNAs (sRNAs) is known to be induced when the foodborne pathogen Listeria monocytogenes is exposed to infection-relevant conditions, such as human blood. Here we demonstrate that excess heme, the core component of hemoglobin in blood, leads to a strong induction of the LhrC family members LhrC1-5. The heme-dependent activation of lhrC1-5 relies on the response regulator LisR, which is known to play a role in virulence and stress tolerance. Importantly, our studies revealed that LhrC1-5 and LisR contribute to the adaptation of L. monocytogenes to excess heme. Regarding the regulatory function of the sRNAs, we demonstrate that LhrC1-5 act to down-regulate the expression of known LhrC target genes under heme-rich conditions: oppA, tcsA, and lapB, encoding surface exposed proteins with virulence functions. These genes were originally identified as targets for LhrC-mediated control under cell envelope stress conditions, suggesting a link between the response to heme toxicity and cell envelope stress in L. monocytogenes. We also investigated the role of LhrC1-5 in controlling the expression of genes involved in heme uptake and utilization: lmo2186 and lmo2185, encoding the heme-binding proteins Hbp1 and Hbp2, respectively, and lmo0484, encoding a heme oxygenase-like protein. Using in vitro binding assays, we demonstrated that the LhrC family member LhrC4 interacts with mRNAs encoded from lmo2186, lmo2185, and lmo0484. For lmo0484, we furthermore show that LhrC4 uses a CU-rich loop for basepairing to the AG-rich Shine-Dalgarno region of the mRNA. The presence of a link between the response to heme toxicity and cell envelope stress was further underlined by the observation that LhrC1-5 down-regulate the expression of lmo0484 in response to the cell wallacting antibiotic cefuroxime. Collectively, this study suggests a role for the LisR-regulated sRNAs LhrC1-5 in a coordinated response to excess heme and cell envelope stress in L. monocytogenes. [ABSTRACT FROM AUTHOR]

Heme is a complex of iron and the tetrapyrrole protoporphyrin IX with essential functions in aerobic organisms. Heme is the prosthetic group of hemoproteins such as hemoglobin andmyoglobin, which are crucial for reversible oxygen binding and transport. By contrast, high levels of free heme, which may occur in various pathophysiological conditions, are toxic via pro-oxidant, pro-inflammatory and cytotoxic effects. The toxicity of heme plays a major role for the pathogenesis of prototypical hemolytic disorders including sickle cell disease and malaria. Moreover, there is increasing appreciation that detrimental effects of heme may also be critically involved in diseases, which usually are not associated with hemolysis such as severe sepsis and atherosclerosis. In mammalians homeostasis of heme and its potential toxicity are primarily controlled by two physiological systems. First, the scavenger protein hemopexin (Hx) non-covalently binds extracellular free heme with high affinity and attenuates toxicity of heme in plasma. Second, heme oxygenases (HOs), in particular the inducible HO isozyme, HO-1, can provide antioxidant cytoprotection via enzymatic degradation of intracellular heme. This review summarizes current knowledge on the pathophysiological role of heme for various diseases as demonstrated in experimental animal models and in humans. The functional significance of Hx and HOs for the regulation of heme homeostasis is highlighted. Finally, the therapeutic potential of pharmacological strategies that apply Hx and HO-1 in various clinical settings is discussed. [ABSTRACT FROM AUTHOR]

Heme iron (HI) has been widely used as a food additive and supplement to support iron fortification. However, no sufficient toxicological data to evaluate the safety of HI have been reported. In the current study, we performed a 13-week subchronic toxicity study of HI in male and female Crl:CD(SD) rats. Rats were orally administered HI in the diet at concentrations of 0%, 0.8%, 2%, and 5%. Observations of general condition, body weight (bw) and food consumption, urinalysis, hematology, serum biochemistry, and macroscopic and histopathological examination were performed. The results showed that HI had no adverse effects on any of the examined parameters. Therefore, we concluded that the no-observed-adverse-effect level (NOAEL) for HI was estimated to be 5% for both sexes (2,890 mg/kg bw/day for males and 3,840 mg/kg bw/day for females). Since the iron content of HI used in this study was in a range of 2.0-2.6%, iron content at NOAEL for HI was calculated to be 57.8-75.1 mg/kg bw/day for males and 76.8-99.8 mg/kg bw/day for females., 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 Elsevier Ltd. All rights reserved.)

Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host sources, particularly hemoglobin, and both heme acquisition and synthesis are important for pathogenesis. Paradoxically, excess heme is toxic to bacteria and pathogens must rely on heme detoxification strategies. Heme is a key nutrient in the struggle for survival between host and pathogen, and its study has offered significant insight into the molecular mechanisms of bacterial pathogenesis. [ABSTRACT FROM AUTHOR]

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.

Abstract: Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (β-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria. [Copyright &y& Elsevier]

Hemorrhagic stroke is a major cause of morbidity and mortality worldwide. Secondary mechanisms of brain injury adversely affect functional outcome in patients after intracranial hemorrhage. Potential drivers of intracranial hemorrhage-related secondary brain injury are hemoglobin and its downstream degradation products released from lysed red blood cells, such as free heme. We established a mouse model with stereotactic striatal injection of heme-albumin to gain insights into the toxicity mechanisms of free heme in the brain and assess the therapeutic potential of heme binding and biochemical neutralization by hemopexin. We defined the dose-dependent transcriptional effect of heme or heme-hemopexin exposure 24 h after injection by spatial transcriptome analysis of lesion-centered coronal cryosections. The spatial transcriptome was interpreted in a multimodal approach along with histology, magnetic resonance imaging, and behavioral data and reported in the associated research article "Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain" [1]. The spatially resolved transcriptome dataset made available here is intended for continued analysis of free heme toxicity in the brain, which is of potential pathophysiological and therapeutic significance in the context of a wide range of neurovascular and neurodegenerative diseases., Competing Interests: RMB, KA, MH, and DJS are inventors on a provisional patent application for the use of Hpx in aneurysmal subarachnoid hemorrhage., (© 2022 The Author(s). Published by Elsevier Inc.)

Fetal rat anemia from flumioxazin, an N-phenylimide herbicide, is caused by suppression of heme synthesis resulting from inhibition of protoporphyrinogen oxidase (PPO). A series of studies to investigate the effects of flumioxazin have revealed that developmental toxicity is caused in rats but not in rabbits, and the adverse effects are not likely to occur in humans. In this study, as a final weight-of-evidence approach for assessing the human safety of flumioxazin, we compared the toxic potential of inhibition of heme synthesis leading to anemia between human and rat embryonic erythroid cells, which were degenerated as the target of flumioxazin in the rat developmental toxicity. To obtain embryonic erythroid cells, we established respective differentiation methods for embryonic erythroid cells from both human and rat pluripotent stem cells. Derived human and rat embryonic erythroid cells were treated with flumioxazin or dihydroartemisinin (DHA), an anti-malarial drug that causes reduction of embryonic erythroid cells and leads to anemia without species differences. In the human embryonic erythroid cells, DHA inhibited cell proliferation and heme synthesis, whereas there were no effects on heme content or cell proliferation with flumioxazin. In the rat embryonic erythroid cells, however, a dose-related reduction in heme synthesis occurred with treatment of flumioxazin and of DHA. These results confirmed that flumioxazin has no effect on heme synthesis in human embryonic erythroid cells. The present data were in accordance with the results of previous studies and demonstrated that there are no concerns in humans regarding the developmental toxicity of flumioxazin observed in rats.

The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address this important issue, we analyzed the impact of heme iron (0.25 µmol/g diet) on the intestinal microbiota, gut inflammation and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 µmol/g diet) was used as reference. 16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.

Microorganisms encounter toxicities inside the host. Many pathogens exist as subpopulations to maximize survivability. Subpopulations of Staphylococcus aureus include antibiotic-tolerant small colony variants (SCVs). These mutants often emerge following antibiotic treatment but can be present in infections prior to antibiotic exposure. We hypothesize that haem toxicity in the host selects for respiration-deficient S. aureus SCVs in the absence of antibiotics. We demonstrate that some but not all respiration-deficient SCV phenotypes are more protective than the haem detoxification system against transient haem exposure, indicating that haem toxicity in the host may contribute to the dominance of menaquinone-deficient and haem-deficient SCVs prior to antibiotic treatment.

As an abundant protein in milk and blood serum, bovine serum albumin (BSA) contains various sites to bind a lot of bioactive components, generating BSA-monoligand complex. Demonstration of the interaction between BSA and bioactive components (such as heme, flavonoids) is important to develop effective carrier for the protection of bioactive ligands and to reduce cytotoxicity of heme. Herein, the bindings of BSA to quercetin and/or heme were investigated by multispectroscopic and molecular docking methods. The fluorescence of protein was significantly quenched by both quercetin and heme in a static mode (i.e., generation of BSA-ligand complex). Although quercetin had lower affinity to protein than heme, the interactions of both compounds with protein did locate in site I (i.e., subdomain IIA). BSA-diligand complex was successfully generated after the coaddition of quercetin and heme. The cytotoxicity of free heme to endothelial cells was reduced in the BSA-diligand complex relative to that of heme or BSA-monoligand complex, while the stability of bioactive quercetin was promoted in the complex relative to free flavonoid. The complex provided a better inhibition on the cytotoxicity of heme than BSA-monoligand complex, in which the copresence of quercetin played a vital role.

Intracerebral hemorrhage (ICH) is a serious medical problem, and effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme, which is mainly released from hemoglobin, plays a vital role in neurotoxicity. However, the specific mechanism involved in heme-mediated neurotoxicity has not been well studied. In this study, we investigated the neurotoxicity of heme in neurons. Neurons were treated with heme, and cell death, autophagy, and endoplasmic reticulum (ER) stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also assessed. We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacological inhibitors (3-methyladenine) or RNA interference of essential autophagy genes (BECN1 and ATG5) decreased heme-induced cell death in neurons. Moreover, the ER stress activator thapsigargin increased cell autophagy and the cell death ratio following heme treatment. Autophagy promoted heme-induced cell apoptosis and cell death through the BECN1/ATG5 pathway. Our findings suggest that heme potentiates neuronal autophagy via ER stress, which in turn induces cell death via the BECN1/ATG5 pathway. Targeting ER stress-mediated autophagy might be a promising therapeutic strategy for ICH.

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.

Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α 1 -microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.

Both infectious as non-infectious inflammation can cause placental dysfunction and pregnancy complications. During the first trimester of human gestation, when palatogenesis takes place, intrauterine hematoma and hemorrhage are common phenomena, causing the release of large amounts of heme, a well-known alarmin. We postulated that exposure of pregnant mice to heme during palatogenesis would initiate oxidative and inflammatory stress, leading to pathological pregnancy, increasing the incidence of palatal clefting and abortion. Both heme oxygenase isoforms (HO-1 and HO-2) break down heme, thereby generating anti-oxidative and -inflammatory products. HO may thus counteract these heme-induced injurious stresses. To test this hypothesis, we administered heme to pregnant CD1 outbred mice at Day E12 by intraperitoneal injection in increasing doses: 30, 75 or 150 μmol/kg body weight (30H, 75H or 150H) in the presence or absence of HO-activity inhibitor SnMP from Day E11. Exposure to heme resulted in a dose-dependent increase in abortion. At 75H half of the fetuses where resorbed, while at 150H all fetuses were aborted. HO-activity protected against heme-induced abortion since inhibition of HO-activity aggravated heme-induced detrimental effects. The fetuses surviving heme administration demonstrated normal palatal fusion. Immunostainings at Day E16 demonstrated higher numbers of ICAM-1 positive blood vessels, macrophages and HO-1 positive cells in placenta after administration of 75H or SnMP + 30H. Summarizing, heme acts as an endogenous "alarmin" during pregnancy in a dose-dependent fashion, while HO-activity protects against heme-induced placental vascular inflammation and abortion.

Serum albumin binds avidly to heme to form heme-serum albumin complex and can protect against the potentially toxic effects of heme. Rutin is a glycoside of the bioflavonoid quercetin with various protective effects due to its antioxidant ability. Clarification of the interaction mechanisms between serum albumin and bioactive components (such as heme and flavonoid) is important to develop effective carriers for encapsulation of heme and suppression of its toxicity. In this study, bindings of bovine serum albumin (BSA) to heme and/or rutin were investigated by experimental and molecular docking techniques. The fluorescence of BSA was quenched by both heme and rutin in static mode (i.e. formation of BSA-monoligand complexes), which was confirmed by Stern-Volmer calculations. Although heme showed higher affinity to BSA than rutin, the interactions of both components with BSA did locate within subdomain IIA (site I). BSA-diligand complexes were successfully formed after the simultaneous addition of heme and rutin. Bioactive rutin in the BSA-diligand complex still kept strong free radical scavenging activity compared to free rutin or BSA-monoligand complex. Hydrogen peroxide (H 2 O 2 )-induced heme degradation and free iron release was inhibited upon BSA binding and further decreased in BSA-diligand complexes. Consistently, the cytotoxicity of heme and oxidative stress in endothelial cells was decreased in the BSA-diligand complexes relative to those of heme or BSA-heme complex, where the co-presence of rutin played an important role. These results suggest the possibility and advantage of developing BSA-based carriers for the suppression of heme toxicity in their biomedical applications., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: RP, XY, NR, JZ, LJ, EN, AR, WS, JJ, HS, MA, LG, MH, MR, JP, DB No competing interests declared, IH IH is the President and Founder of Rakta Therapeutics Inc (College Park, MD), which is a company involved in the development of heme transporter-related diagnostics. He declares no other competing financial interests.

Serum albumin (SA) has been shown to act as a heme scavenger in hemolysis and can protect cell against the toxic effect of heme. However, the mechanism of SA in heme detoxification is not well understood. Interestingly, increasing studies indicate that heme/H 2 O 2 -dependent reaction is unlikely to be the principal cause of heme toxicity in excessive intravascular hemolysis conditions. Moreover, high levels of NO 2 - and NO 3 - were also found in patients with severe hemolytic diseases, which seem to involve in heme toxic effect as well. Therefore, we proposed that studying the protection mechanism of SA against the heme/H 2 O 2 /NO 2 - -induced cytotoxicity may be more consistent with free heme-associated disorder pathologies. In this study, we tested the hypotheses that tyrosine residues of bovine serum albumin (BSA) play a prominent role in detoxifying heme in SH-SY5Y cells. Both BSA and tyrosine modified BSA (BSA-T) were used to explore this protective mechanism. Most of cellular injury (oxidative and nitrative damage) induced by heme/H 2 O 2 /NO 2 - were prevented by pretreatment with an equimolar concentration of BSA or BSA-T, and BSA was found more efficient than BSA-T. Meanwhile, BSA or BSA-T binding to heme is not accompanied by a decrease of heme's peroxidase activity. Collectively, these data suggest that the protecting effect of BSA against heme-induced damage in the intravascular hemolysis diseases is not accomplished by preventing the primary reactivity of heme with H 2 O 2 , but by trapping radical through special residues such as tyrosine to render other important protein less damaged.

Hemolytic diseases are associated with elevated levels of circulating free heme that can mediate endothelial dysfunction directly via redox reactions with biomolecules or indirectly by upregulating enzymatic sources of reactive species. A key enzymatic source of these reactive species is the purine catabolizing enzyme, xanthine oxidase (XO) as the oxidation of hypoxanthine to xanthine and subsequent oxidation of xanthine to uric acid generates superoxide (O 2 •- ) and hydrogen peroxide (H 2 O 2 ). While XO has been studied for over 120 years, much remains unknown regarding specific mechanistic roles for this enzyme in pathologic processes. This gap in knowledge stems from several interrelated issues including: 1) lethality of global XO deletion and the absence of tissue-specific XO knockout models have coalesced to relegate proof-of-principle experimentation to pharmacology; 2) XO is mobile and thus when upregulated locally can be secreted into the circulation and impact distal vascular beds by high-affinity association to the glycocalyx on the endothelium; and 3) endothelial-bound XO is significantly resistant (> 50%) to inhibition by allopurinol, the principle compound used for XO inhibition in the clinic as well as the laboratory. While it is known that circulating XO is elevated in hemolytic diseases including sickle cell, malaria and sepsis, little is understood regarding its role in these pathologies. As such, the aim of this review is to define our current understanding regarding the effect of hemolysis (free heme) on circulating XO levels as well as the subsequent impact of XO-derived oxidants in hemolytic disease processes., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Heme is central to functions of many biologically important enzymes (hemoproteins). It is an assembly of four porphyrin rings joined through methylene bridges with a central Fe (II). Heme is present in all cells, and its synthesis and degradation balance its amount in the cell. The deregulations of heme networks and incorporation in hemoproteins lead to pathogenic state. This article addresses the detailed structure, biosynthesis, degradation, and transportation associated afflictions to heme. The article is followed by its roles in various diseased conditions where it is produced mainly as the cause of increased hemolysis. It manifests the symptoms in diseases as it is a pro-oxidant, pro-inflammatory and pro-hemolytic agent. We have also discussed the genetic defects that tampered with the biosynthesis, degradation, and transportation of heme. In addition, a brief about the largest hemoprotein group of enzymes- Cytochrome P450 (CYP450) has been discussed with its roles in drug metabolism., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

For pathogenic bacteria, host-derived heme represents an important metabolic cofactor and a source for iron. However, high levels of heme are toxic to bacteria. We have previously shown that excess heme has a growth-inhibitory effect on the Gram-positive foodborne pathogen Listeria monocytogenes , and we have learned that the LhrC1-5 family of small RNAs, together with the two-component system (TCS) LisRK, play a role in the adaptation of L. monocytogenes to heme stress conditions. However, a broader knowledge on how this pathogen responds to heme toxicity is still lacking. Here, we analyzed the global transcriptomic response of L. monocytogenes to heme stress. We found that the response of L. monocytogenes to excess heme is multifaceted, involving various strategies acting to minimize the toxic effects of heme. For example, heme exposure triggers the SOS response that deals with DNA damage. In parallel, L. monocytogenes shuts down the transcription of genes involved in heme/iron uptake and utilization. Furthermore, heme stress resulted in a massive increase in the transcription of a putative heme detoxification system, hrtAB , which is highly conserved in Gram-positive bacteria. As expected, we found that the TCS HssRS is required for heme-mediated induction of hrtAB and that a functional heme efflux system is essential for L. monocytogenes to resist heme toxicity. Curiously, the most highly up-regulated gene upon heme stress was lmo1634 , encoding the Listeria adhesion protein, LAP, which acts to promote the translocation of L. monocytogenes across the intestinal barrier. Additionally, LAP is predicted to act as a bifunctional acetaldehyde-CoA/alcohol dehydrogenase. Surprisingly, a mutant lacking lmo1634 grows well under heme stress conditions, showing that LAP is not required for L. monocytogenes to resist heme toxicity. Likewise, a functional ResDE TCS, which contributes to heme-mediated expression of lmo1634 , is not required for the adaptation of L. monocytogenes to heme stress conditions. Collectively, this study provides novel insights into the strategies employed by L. monocytogenes to resist heme toxicity. Our findings indicate that L. monocytogenes is using heme as a host-derived signaling molecule to control the expression of its virulence genes, as exemplified by lmo1634 .

Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease., (© 2018 Sweeny et al.)

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

Severe hypertension can lead to malignant hypertension (MH) with renal thrombotic microangiopathy and hemolysis. The role of plasma heme release in this setting is unknown. We aimed at evaluating the effect of a mild plasma heme increase by hemin administration in angiotensin II (AngII)-mediated hypertensive rats. Prevalence of MH and blood pressure values were similar in AngII and AngII + hemin groups. MH rats displayed a decreased renal blood flow (RBF), increased renal vascular resistances (RVR), and increased aorta and interlobar arteries remodeling with a severe renal microcirculation assessed by peritubular capillaries (PTC) rarefaction. Hemin-treated rats with or without AngII displayed also a decreased RBF and increased RVR explained only by PCT rarefaction. In AngII rats, RBF was similar to controls (with increased RVR). PTC density appeared strongly correlated to tubular damage score (rho = -0.65, p < 0.0001) and also renal Heme Oygenase-1 (HO-1) mRNA (rho = -0.67, p < 0.0001). HO-1 was expressed in PTC and renal tubules in MH rats, but only in PTC in other groups. In conclusion, though increased plasma heme does not play a role in triggering or aggravating MH, heme release appears as a relevant toxic mediator leading to renal impairment, primarily through PTC endothelial dysfunction rather than direct tubular toxicity.

Transition metal ions (Zn(II), Cu(II)/(I), Fe(III)/(II), Mn(II)) are essential for life and participate in a wide range of biological functions. Cellular Zn(II) levels must be high enough to ensure that it can perform its essential roles. Yet, since Zn(II) binds to ligands with high avidity, excess Zn(II) can lead to protein mismetallation. The major targets of mismetallation, and the underlying causes of Zn(II) intoxication, are not well understood. Here, we use a forward genetic selection to identify targets of Zn(II) toxicity. In wild-type cells, in which Zn(II) efflux prevents intoxication of the cytoplasm, extracellular Zn(II) inhibits the electron transport chain due to the inactivation of the major aerobic cytochrome oxidase. This toxicity can be ameliorated by depression of an alternate oxidase or by mutations that restrict access of Zn(II) to the cell surface. Conversely, efflux deficient cells are sensitive to low levels of Zn(II) that do not inhibit the respiratory chain. Under these conditions, intracellular Zn(II) accumulates and leads to heme toxicity. Heme accumulation results from dysregulation of the regulon controlled by PerR, a metal-dependent repressor of peroxide stress genes. When metallated with Fe(II) or Mn(II), PerR represses both heme biosynthesis (hemAXCDBL operon) and the abundant heme protein catalase (katA). Metallation of PerR with Zn(II) disrupts this coordination, resulting in depression of heme biosynthesis but continued repression of catalase. Our results support a model in which excess heme partitions to the membrane and undergoes redox cycling catalyzed by reduced menaquinone thereby resulting in oxidative stress., Competing Interests: The authors have declared that no competing interests exist.

Placental malaria (PM) is a complication associated with malaria infection during pregnancy that often leads to abortion, premature delivery, intrauterine growth restriction and low birth weight. Increased levels of circulating free heme, a by-product of Plasmodium-damaged erythrocytes, is a major contributor to inflammation, tissue damage and loss of blood brain barrier integrity associated with fatal experimental cerebral malaria. However, the role of heme in PM remains unknown. Proliferation and apoptosis of trophoblasts and fusion of the mononucleated state to the syncytial state are of major importance to a successful pregnancy. In the present study, we examined the effects of heme on the viability and fusion of a trophoblast-derived cell line (BeWo). Results indicate that heme induces apoptosis in BeWo cells by activation of the STAT3/caspase-3/PARP signaling pathway. In the presence of forskolin, which triggers trophoblast fusion, heme inhibits BeWo cell fusion through activation of STAT3. Understanding the effects of free plasma heme in pregnant women either due to malaria, sickle cell disease or other hemolytic diseases, will enable identification of high-risk women and may lead to discovery of new drug targets against associated adverse pregnancy outcome.

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by "loose" (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity--CO and bilirubin--have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity.

The massive erythrocyte lysis caused by the Group A Streptococcus (GAS) suggests that the β-hemolytic pathogen is likely to encounter free heme during the course of infection. In this study, we investigated GAS mechanisms for heme sensing and tolerance. We compared the minimal inhibitory concentration of heme among several isolates and established that excess heme is bacteriostatic and exposure to sub-lethal concentrations of heme resulted in noticeable damage to membrane lipids and proteins. Pre-exposure of the bacteria to 0.1 μM heme shortened the extended lag period that is otherwise observed when naive cells are inoculated into heme-containing medium, implying that GAS is able to adapt. The global response to heme exposure was determined using microarray analysis revealing a significant transcriptome shift that included 79 up regulated and 84 down regulated genes. Among other changes, the induction of stress-related chaperones and proteases, including groEL/ES (8x), the stress regulators spxA2 (5x) and ctsR (3x), as well as redox active enzymes were prominent. The heme stimulon also encompassed a number of regulatory proteins and two-component systems that are important for virulence. A three-gene cluster that is homologous to the pefRCD system of the Group B Streptococcus was also induced by heme. PefR, a MarR-like regulator, specifically binds heme with stoichiometry of 1:2 and protoporphyrin IX (PPIX) with stoichiometry of 1:1, implicating it is one of the GAS mediators to heme response. In summary, here we provide evidence that heme induces a broad stress response in GAS, and that its success as a pathogen relies on mechanisms for heme sensing, detoxification, and repair.

Large and small aggregates of Aβ peptides, resembling the morphology and dimensions of fibrillar and oligomeric forms of Aβ respectively, relevant to Alzheimer's disease, are stabilized on electrodes using self-assembly. Both of these forms were found to bind redox active Cu and heme, resulting in active sites having distinctive biophysical properties. The reduced metal bound Aβ active sites of both the oligomeric and fibrillar forms of Aβ produce detrimental partially reduced oxygen species (PROS). While the larger aggregates of heme-Aβ produce more PROS in situ, the smaller aggregates of Cu-Aβ produce more PROS. 8-Hydroxy quinoline and methylene blue are inhibitors of Cu and heme bound Aβ respectively, and are shown to efficiently reduce PROS formation in the oligomeric forms. However, these inhibitors are ineffective in reducing the toxicities of the Cu and heme bound Aβ peptides in the fibrils, making them significantly more lethal than the smaller Aβ aggregates.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

The reactive nature of heme enables its use as an enzymatic cofactor while rendering excess heme toxic. The importance of heme detoxification machinery is highlighted by the presence of various types of these homeostatic systems in Gram-positive and Gram-negative microorganisms. A number of pathogens possess orthologs of the HssRS/HrtAB heme detoxification system, underscoring a potential role this system plays in the survival of bacteria in heme-rich environments such as the vertebrate host. In this work, we sought to determine the role of this system in protection against metalloporphyrin heme analogues identified by previous studies as antimicrobial agents. Our findings demonstrate that only toxic metalloporphyrins maximally activate expression of the Staphylococcus aureus heme detoxification system, suggesting that the sensing mechanism of HssRS might require a component of the associated toxicity rather than or in addition to the metalloporphyrin itself. We further establish that only a subset of toxic metalloporphyrins elicit the oxidative damage previously shown to be a significant component of heme toxicity whereas all toxic noniron metalloporphyrins inhibit bacterial respiration. Finally, we demonstrate that, despite the fact that toxic metalloporphyrin treatment induces expression of S. aureus heme detoxification machinery, the HrtAB heme export pump is unable to detoxify most of these molecules. The ineffectiveness of HrtAB against toxic heme analogues provides an explanation for their increased antimicrobial activity relative to heme. Additionally, these studies define the specificity of HssRS/HrtAB, which may provide future insight into the biochemical mechanisms of these systems.

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20(th) gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

HO1 (haem oxygenase 1) and Fpn (ferroportin) are key proteins for iron recycling from senescent red blood cells and therefore play a major role in controlling the bioavailability of iron for erythropoiesis. Although important aspects of iron metabolism in HO1-deficient (Hmox1-/-) mice have already been revealed, little is known about the regulation of Fpn expression and its role in HO1 deficiency. In the present study, we characterize the cellular and systemic factors influencing Fpn expression in Hmox1-/- bone marrow-derived macrophages and in the liver and kidney of Hmox1-/- mice. In Hmox1-/- macrophages, Fpn protein was relatively highly expressed under high levels of hepcidin in culture medium. Similarly, despite high hepatic hepcidin expression, Fpn is still detected in Kupffer cells and is also markedly enhanced at the basolateral membrane of the renal tubules of Hmox1-/- mice. Through the activity of highly expressed Fpn, epithelial cells of the renal tubules probably take over the function of impaired system of tissue macrophages in recycling iron accumulated in the kidney. Moreover, although we have found increased expression of FLVCR (feline leukaemia virus subgroup C receptor), a haem exporter, in the kidneys of Hmox1-/- mice, haem level was increased in these organs. Furthermore, we show that iron/haem-mediated toxicity are responsible for renal injury documented in the kidneys of Hmox1-/- mice.

Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating oxidative and inflammatory injury. Protective mechanisms against free heme are therefore pivotal for cellular survival. We postulated that overexpression of Heme Oxygenase-1 (HO-1) and Breast Cancer Resistance Protein (BCRP) would protect against heme-induced cytotoxicity. HO-1 is a heme-degrading enzyme generating carbon monoxide, iron, and biliverdin/bilirubin, while BCRP is a heme efflux transporter. Human embryonic kidney cells were transduced using a baculovirus system as a novel strategy to efficiently overexpress HO-1 and BCRP. Exposing cells to heme resulted in a dose-dependent increase in reactive oxygen species formation, DNA damage and cell death. Heme-induced cell death was significantly attenuated when cells overexpressed HO-1, BCRP, or both. The protective effects of HO-1 overexpression were most pronounced, while co-treatment with the HO-activity inhibitor tin mesoporphyrin reversed these protective effects. Also cells treated with the anti-oxidants N-acetylcysteine or HO-effector molecule bilirubin showed protection against heme insults, which may explain the increased protection by HO-1 compared to BCRP. In conclusion, both HO-1 and BCRP protect against heme-induced toxicity and may thus form novel therapeutic targets for heme-mediated pathologies.

We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of haem homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study, ChrSA was shown to be crucial for haem resistance of C. glutamicum by activation of the putative haem-detoxifying ABC-transporter HrtBA in the presence of haem. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards haem. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA revealed that phosphorylated ChrA acts as an activator of hrtBA in the presence of haem. The haem oxygenase gene, hmuO, showed a decreased mRNA level in a chrSA deletion mutant but no significant binding of ChrA to the hmuO promoter was observed in vitro. In contrast, activation from P(hmuO) fused to eyfp was almost abolished in an hrrSA mutant, indicating that HrrSA is the dominant system for haem-dependent activation of hmuO in C. glutamicum. Remarkably, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralogous response regulator, whereas chrSA itself seemed to be repressed by HrrA. These data suggest a close interplay of HrrSA and ChrSA at the level of transcription and emphasize ChrSA as a second TCS involved in haem-dependent gene regulation in C. glutamicum, besides HrrSA.