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.

Specialized cells, known as red blood cells, are responsible for transporting oxygen to various organs in the body. Each red blood cell contains over a billion molecules of heme which make up the iron containing portion of the hemoglobin protein that binds and transports oxygen. When red blood cells reach the end of their life, they are degraded, and the heme and iron inside them is recycled to produce new red blood cells. Heme, however, is highly toxic to cells, and can cause severe tissue damage if not properly removed. Scavenger cells called macrophages perform this recycling role in the spleen, liver and bone marrow. Collectively, macrophages can process around five million red blood cells every second or about 100 trillion heme molecules. But, it is unclear how they are able to handle such enormous volumes. Macrophages isolated from human and mice have been shown to transport heme from damaged red blood cells using a protein called HRG1. To investigate the role HRG1 plays in heme-iron recycling, Pek et al. used a gene editing tool known an CRISPR/Cas9 to remove the gene for HRG1 from the macrophages of mice. If HRG1 is a major part of this process, removing the gene should result in a build-up of toxic heme and eventual death of the mouse. But, rather than dying of heme-iron overload as expected, these mutant mice managed to survive. Pek et al. found that despite being unable to recycle heme, these mice were still able to make new red blood cells as long as they had a diet that was rich in iron. However, the darkening color of the spleen, bone marrow, and liver in these HRG1 deficient mice indicated that these mice were still accumulating high levels of heme. Further experiments revealed that these mice protected themselves from toxicity by converting the excess heme into crystals called hemozoin. This method of detoxification is commonly seen in blood-feeding parasites, and this is the first time it has been observed in a mammal. These crystals invite new questions about how mammals recycle heme and what happens when this process goes wrong. The next step is to ask whether humans also start to make hemozoin if the gene for HRG1 is faulty. If so, this could open a new avenue of exploration into treatments for red blood cell diseases like anemia and iron overload.

Coordination between NADPH oxidase and vascular peroxidase 1 promotes dysfunctions of endothelial progenitor cells in hypoxia-induced pulmonary hypertensive rats.

Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.

Streptococcal heme binding protein (Shp) promotes virulence and contributes to the pathogenesis of group A Streptococcus infection.

Streptococcal heme binding protein (Shp) is involved in the process of heme acquisition in group A Streptococcus (GAS). However, no research thus far has examined the contribution of Shp to the virulence of GAS. To this end, we generated an isogenic strain lacking the shp gene (Δshp) and its complemented strain (Δshp-c) using the parent strain MGAS5005 (WT). Deletion of shp increased survival rates and neutrophil recruitment and reduced skin lesion sizes and GAS loads in the blood and the liver, lung, kidney and spleen in subcutaneous infections of mice. These results indicate that Shp significantly contributes to the skin and systemic invasion of GAS. The growth of the Δshp mutant was significantly slower than MGAS5005 and Δshp-c than in non-immune human blood and in incubation with isolated rat neutrophils. Microarray transcriptional analyses found no alteration in expression of virulence genes, indicating that the phenotype of the Δshp mutant was directly linked to the lack of Shp. The findings indicate that Shp significantly contributes to GAS skin invasion, systemic infection and virulence and that these contributions of Shp are mediated by the effects of Shp on systemic GAS growth and neutrophil responses.

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Nitrosative stress causes amino acid auxotrophy in hmp mutant Salmonella Typhimurium.

Cytotoxic nitic oxide (NO) damages various bacterial macromolecules, resulting in abnormal metabolism by mechanisms largely unknown. We show that NO can cause amino acid auxotrophy in Salmonella Typhimurium lacking major NO-metabolizing enzyme, flavohemoglobin Hmp. In NO-producing cultures, supplementation with amino acid pool restores growth of Hmp-deficient Salmonella to normal growth phases, whereas excluding Cys or BCAA Leu, Ile, or Val from amino acid pool reduces growth recovery. Data suggest that, without detoxification, NO might inactivate key enzymes in the biosynthesis pathway of amino acids essential for Salmonella replication in amino acid-limiting host environments.

Hemin-binding protein 35 (HBP35) plays an important role in bacteria-mammalian cells interactions in Porphyromonas gingivalis.

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.

Facilitation of mitochondrial outer and inner membrane permeabilization and cell death in oxidative stress by a novel Bcl-2 homology 3 domain protein.

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

A genetic analysis of nitrosative stress.

Nitrosative stress is induced by pathophysiological levels of nitric oxide (NO) and S-nitrosothiols (e.g., S-nitrosoglutathione, GSNO) and arises, at least in significant part, from the nitrosylation of critical protein Cys thiols (S-nitrosylation) and metallocofactors. However, the mechanisms by which NO and GSNO mediate nitrosative stress are not well understood. Using yeast Saccharomyces cerevisiae strains lacking NO- and/or GSNO-consuming enzymes (flavohemoglobin and GSNO reductase, respectively), we measured the individual and combined effects of NO and GSNO on both cell growth and the formation of protein-bound NO species. Our results suggest an intracellular equilibrium between NO and GSNO, dependent in part on cell-catalyzed release of NO from GSNO (i.e., "SNO-lyase" activity). However, whereas NO induces multiple types of protein-based modifications, levels of which correlate with inhibition of cell growth, GSNO mainly affects protein S-nitrosylation, and the relationship between S-nitrosylation and nitrosative stress is more complex. These data support the idea of multiple classes of protein-SNO, likely reflected in divergent routes of synthesis and degradation. Indeed, a significant fraction of protein S-nitrosylation by NO occurs in the absence of O(2), which is commonly assumed to drive this reaction but instead is apparently dependent in substantial part upon protein-bound transition metals. Additionally, our findings suggest that nitrosative stress is mediated principally via the S-nitrosylation of a subset of protein targets, which include protein SNOs that are stable to cellular glutathione (and thus are not metabolized by GSNO reductase). Collectively, these results provide new evidence for the mechanisms through which NO and GSNO mediate nitrosative stress as well as the cellular pathways of protein S-nitrosylation and denitrosylation involving metalloproteins, SNO lyase(s) and GSNO reductase.

Drug metabolizing enzyme system and heme pathway in hepatocarcinogenesis.

Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre-initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2-allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis.

Modulation of the impaired drug metabolism in sarcoma-180-bearing mice by echitamine chloride.

Echitamine chloride (EC), an indole alkaloid, extracted from the bark of Alstonia scholaris has got highly promising anticancer effect. The effect of this drug on the microsomal drug detoxifying system was studied in sarcoma-180 induced mice. When given sub-cutaneously at a dosage of 5 mg/kg body weight, it was able to alter the impaired drug detoxifying system which was observed in the Sarcoma-180 bearing mice. The levels of microsomal protein, Cyt-P450, Cyt-b5, NADH-Cyt-C-reductase, NADPH-Cyt-C-reductase, and glu-6 phosphatase were determined. The levels of these drug metabolizing enzymes were decreased in S-180 bearing mice. EC treatment corrected to near normal levels of these enzymes and microsomal hemeproteins. In order to understand the mechanism responsible for the decreased protein level and its normalization after treatment with EC, 3H-Phenylalanine incorporation study was carried out. From the results, it is observed that the synthesis of apoproteins is also altered in tumor-bearing animals. All these changes which were observed in tumor-bearing animals were corrected to near normal levels after treatment with EC.