Extracellular Heme Uptake and the Challenge of Bacterial Cell Membranes.

Iron is essential for the survival of most bacteria but presents a significant challenge given its limited bioavailability. Furthermore, the toxicity of iron combined with the need to maintain physiological iron levels within a narrow concentration range requires sophisticated systems to sense, regulate, and transport iron. Most bacteria have evolved mechanisms to chelate and transport ferric iron (Fe3+) via siderophore receptor systems, and pathogenic bacteria have further lowered this barrier by employing mechanisms to utilize the host's hemoproteins. Once internalized, heme is cleaved by both oxidative and nonoxidative mechanisms to release iron. Heme, itself a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such, pathogenic bacteria have evolved sophisticated cell surface signaling and transport systems to obtain heme from the host. In this review, we summarize the structure and function of the heme-sensing and transport systems of pathogenic bacteria and the potential of these systems as antimicrobial targets.

Iron in immune cell function and host defense.

The control over iron availability is crucial under homeostatic conditions and even more in the case of an infection. This results from diverse properties of iron: first, iron is an important trace element for the host as well as for the pathogen for various cellular and metabolic processes, second, free iron catalyzes Fenton reaction and is therefore producing reactive oxygen species as a part of the host defense machinery, third, iron exhibits important effects on immune cell function and differentiation and fourth almost every immune activation in turn impacts on iron metabolism and spatio-temporal iron distribution. The central importance of iron in the host and microbe interplay and thus for the course of infections led to diverse strategies to restrict iron for invading pathogens. In this review, we focus on how iron restriction to the pathogen is a powerful innate immune defense mechanism of the host called "nutritional immunity". Important proteins in the iron-host-pathogen interplay will be discussed as well as the influence of iron on the efficacy of innate and adaptive immunity. Recently described processes like ferritinophagy and ferroptosis are further covered in respect to their impact on inflammation and infection control and how they impact on our understanding of the interaction of host and pathogen.

USP14 regulates heme metabolism and ovarian cancer invasion through BACH1 deubiquitination and stabilization.

The deubiquitinating enzyme USP14 has been established as a crucial regulator in various diseases, including tumors, neurodegenerative diseases, and metabolic diseases, through its ability to stabilize its substrate proteins. Our group has utilized proteomic techniques to identify new potential substrate proteins for USP14, however, the underlying signaling pathways regulated by USP14 remain largely unknown. Here, we demonstrate the key role of USP14 in both heme metabolism and tumor invasion by stabilizing the protein BACH1. The cellular oxidative stress response factor NRF2 regulates antioxidant protein expression through binding to the antioxidant response element (ARE). BACH1 can compete with NRF2 for ARE binding, leading to the inhibition of the expression of antioxidant genes, including HMOX-1. Activated NRF2 also inhibits the degradation of BACH1, promoting cancer cell invasion and metastasis. Our findings showed a positive correlation between USP14 expression and NRF2 expression in various cancer tissues from the TCGA database and normal tissues from the GTEx database. Furthermore, activated NRF2 was found to increase USP14 expression in ovarian cancer (OV) cells. The overexpression of USP14 was observed to inhibit HMOX1 expression, while USP14 knockdown had the opposite effect, suggesting a role for USP14 in regulating heme metabolism. The depletion of BACH1 or inhibition of heme oxygenase 1 (coded by HMOX-1) was also found to significantly impair USP14-dependent OV cell invasion. In conclusion, our results highlight the importance of the NRF2-USP14-BACH1 axis in regulating OV cell invasion and heme metabolism, providing evidence for its potential as a therapeutic target in related diseases.

Heme metabolism mediates the effects of smoking on gut microbiome.

INTRODUCTION: The number of smokers worldwide increased greatly during the past decades and reached 1.14 billion in 2019, becoming a leading risk factor for human health. Tobacco smoking has wide effects on human genetics, epigenetics, transcriptome, and gut microbiome. Although many studies have revealed effects of smoking on host transcriptome, research on the relationship among smoking, host gene expression, and the gut microbiome is limited. METHODS: We first explored transcriptome and metagenome profile differences between smokers and non-smokers. To evaluate the relationship between host gene expression and gut microbiome, we then applied bi-directional mediation analysis to infer causal relationships between smoking, gene expression, and gut microbes. RESULTS: Metagenome and transcriptome analyses revealed 71 differential species and 324 differential expressed genes between smokers and non-smokers. With smoking as an exposure variable, we identified 272 significant causal relationships between gene expression and gut microbes, among which there were 247 genes that mediate the effect of smoking on gut microbes. Pathway-based enrichment analysis showed that these genes were significantly enriched in heme metabolic pathway, which mainly mediated the changes of Bacteroides finegoldii and Lachnospiraceae bacterium 9_1_43BFAA. Additionally, by performing metabolome data analysis in the Integrated Human Microbiome project (iHMP) database, we verified the correlation between the intermediate products of the heme metabolism pathway (porphobilinogen, bilirubin, and biliverdin) and gut microbiome. CONCLUSIONS: By investigating the bi-directional interaction between smoking-related host gene expression and gut microbes, this study provided evidence for the mediation of smoking on gut microbes through co-involvement or interaction of heme metabolism. IMPLICATIONS: By comparing the metagenome and transcriptome sequencing profiles between 34 smokers and 33 age- and gender-matched non-smokers, we are the first to reveal causal relationships among tobacco smoking, host gene expression and gut microbes. These findings offer insight into how smoking affects gut microbes through host gene expression and metabolism, which highlights the importance of heme metabolism in modulating the effects of smoking on gut microbiome.

Mechanisms of Resistance to Photodynamic Therapy (PDT) in Vulvar Cancer.

Photodynamic therapy (PDT) is a valuable treatment method for vulvar intraepithelial neoplasia (VIN). It allows for the treatment of a multifocal disease with minimal tissue destruction. 5-Aminolevulinic acid (5-ALA) is the most commonly used prodrug, which is converted in the heme pathway to protoporphyrin IX (PpIX), an actual photosensitizer (PS). Unfortunately, not all patients treated with PDT undergo complete remission. The main cause of their failure is resistance to anticancer therapy. In many cancers, resistance to various anticancer treatments is correlated with increased activity of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1). Enhanced activity of drug pumps may also affect the effectiveness of therapy. To investigate whether multidrug resistance mechanisms underlie PDT resistance in VIN, porphyrins were isolated from sensitive and resistant vulvar cancer cells and their culture media. APE1 activity was measured, and survival assay after PDT combined with APE1 inhibitor was performed. Our results revealed that resistant cells accumulated and effluxed less porphyrins than sensitive cells, and in response to PDT, resistant cells increased APE1 activity. Moreover, PDT combined with inhibition of APE1 significantly decreased the survival of PDT-resistant cells. This means that resistance to PDT in vulvar cancer may be the result of alterations in the heme synthesis pathway. Moreover, increased APE1 activity may be essential for the repair of PDT-mediated DNA damage, and inhibition of APE1 activity may increase the efficacy of PDT.

People with blood disorders can be more vulnerable during COVID-19 pandemic: A hypothesis paper.

The world has been encountered with COVID-19 pandemic since at the beginning of 2020 and the number of infected people by COVID-19 is increasing every day. Despite various studies conducted by researchers and doctors, no treatment has been developed until now, therefore self-protection and isolation are strongly recommended to stop the spread of the virus. The elderly population and people with chronic diseases such as hypertension, cardiovascular diseases, diabetes, and cancer are categorized as risk groups, however, we suggest that people with hemoglobinopathies or porphyria can be described as risk groups as well. Current in silico studies have revealed that the COVID-19 virus can attack heme and hemoglobin metabolisms which are responsible for the oxygen transport to the tissues, iron metabolism, elevated levels of oxidative stress, and tissue damage. Data of the in silico study have been supported with the biochemistry and hemogram results of the COVID-19 patients, for instance hemoglobin levels decreased and serum ferritin and C-reactive protein levels increased. Indicated biochemistry biomarkers are tightly associated with inflammation, iron overload, and oxidative stress. In conclusion, since people with hemoglobinopathies or porphyria have already impaired heme and hemoglobin metabolism, COVID-19 infection can enhance the adverse effects of impaired hemoglobin metabolism and accelerate the progression of severe symptoms in patients with hemoglobinopathies or porphyria compared to the normal individuals. Thus those people can be considered as a risk group and extra precautions should be applied for them to protect them.

Heme uptake and utilization by hypervirulent Acinetobacter baumannii LAC-4 is dependent on a canonical heme oxygenase (abHemO).

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in critically ill and immune compromised patients. The ability to acquire iron from the hosts iron and heme containing proteins is critical to their survival and virulence. The majority of A. baumannii hypervirulent strains encode a heme uptake system that includes a putative heme oxygenase (hemO). Despite reports indicating A. baumannii can grow on heme direct evidence of extracellular heme uptake and metabolism has not been shown. Through isotopic labeling (13C-heme) we show the hypervirulent A. baumannii LAC-4 metabolizes heme to biliverdin IXα (BVIXα), whereas ATC 17978 that lacks the hemO gene cluster cannot efficiently utilize heme. Expression and purification of the protein encoded by the A. baumannii LAC-4 hemO gene confirmed catalytic conversion of heme to BVIX. We further show inhibition of abHemO with previously characterized P. aeruginosa HemO inhibitors in a fluorescence based assay that couples HemO catalytic activity to the BVIXα binding phytochrome IFP1.4. Furthermore, the hemO gene cluster encodes genes with homology to heme-dependent extra cytoplasmic function (ECF) σ factor systems. The hemophore-dependent ECF system in Pseudomonas aeruginosa has been shown to play a critical role in heme sensing and virulence within the host. The prevalence of a hemO gene cluster in A. baumannii LAC4 and other hypervirulent strains suggests it is required within the host to adapt and utilize heme and is a major contributor to virulence.

Effects of volatile anaesthetics on heme metabolism in a murine genetic model of Acute Intermittent Porphyria. A comparative study with other porphyrinogenic drugs.

BACKGROUND: Acute Intermittent Porphyria (AIP) is an inherited disease produced by a deficiency of Porphobilinogen deaminase (PBG-D). The aim of this work was to evaluate the effects of Isoflurane and Sevoflurane on heme metabolism in a mouse genetic model of AIP to further support our previous proposal for avoiding their use in porphyric patients. A comparative study was performed administering the porphyrinogenic drugs allylisopropylacetamide (AIA), barbital and ethanol, and also between sex and mutation using AIP (PBG-D activity 70% reduced) and T1 (PBG-D activity 50% diminished) mice. METHODS: The activities of 5-Aminolevulinic synthetase (ALA-S), PBG-D, Heme oxygenase (HO) and CYP2E1; the expression of ALA-S and the levels of 5-aminolevulinic acid (ALA) were measured in different tissues of mice treated with the drugs mentioned. RESULTS: Isoflurane increased liver, kidney and brain ALA-S activity of AIP females but only affected kidney AIP males. Sevoflurane induced ALA-S activity in kidney and brain of female AIP group. PBG-D activity was further reduced by Isoflurane in liver male T1; in AIP male mice activity remained in its low basal levels. Ethanol and barbital also caused biochemical alterations. Only AIA triggered neurological signs similar to those observed during human acute attacks in male AIP being the symptoms less pronounced in females although ALA-S induction was greater. Heme degradation was affected. DISCUSSION: Biochemical alterations caused by the porphyrinogenic drugs assayed were different in male and female mice and also between T1 and AIP being more affected the females of AIP group. GENERAL SIGNIFICANCE: This is the first study using volatile anaesthetics in an AIP genetic model confirming Isoflurane and Sevoflurane porphyrinogenicity.

Potential role of heme metabolism in the inducible expression of heme oxygenase-1.

BACKGROUND: The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. METHODS: We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. RESULTS: The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. CONCLUSIONS: We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. GENERAL SIGNIFICANCE: The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.

Pleiotropic effects of 5-aminolevulinic acid in mouse brain.

5-Aminolevulinic acid (ALA) seems to be responsible for the neuropsychiatric manifestations of acute intermittent porphyria (AIP). Our aim was to study the effect of ALA on the different metabolic pathways in the mouse brain to enhance our knowledge about the action of this heme precursor on the central nervous system. Heme metabolism, the cholinergic system, the defense enzyme system, and nitric oxide metabolism were evaluated in the encephalon of CF-1 mice receiving a single (40 mg/kg body mass) or multiple doses of ALA (40 mg/kg, every 48 h for 14 days). We subsequently found ALA accumulation in the encephalon of the mice. ALA also altered the brain cholinergic system. After one dose of ALA, a decrease in superoxide dismutase activity and a reduction in glutathione levels were detected, whereas malondialdehyde levels and catalase activity were increased. Heme oxygenase was also increased as an antioxidant response to protect the encephalon against injury. All nitric oxide synthase isoforms were induced by ALA, these changes were more significant for the inducible isoform in glial cells. In conclusion, ALA affected several metabolic pathways in mouse encephalon. Data indicate that a rapid response to oxidative stress was developed; however, with long-term intoxication, the redox balance was probably restored, thereby minimizing oxidative damage.

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus.

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split ß-barrel conformation in each monomer comprising six ß strands and three α helices. This structure resembles the split ß-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split ß-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.

Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor.

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo. IMPORTANCE: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

Heme metabolism and HO-1 in the pathogenesis and potential intervention of endometriosis.

Endometriosis (EM) is one of the diseases related to retrograded menstruation and hemoglobin. Heme, released from hemoglobin, is degraded by heme oxygenase-1 (HO-1). In EM lesions, heme metabolites regulate processes such as inflammation, redox balance, autophagy, dysmenorrhea, malignancy, and invasion, where macrophages (Mø) play a fundamental role in their interactions. Regulation occurs at molecular, cellular, and pathological levels. Numerous studies suggest that heme is an indispensable component in EM and may contribute to its pathogenesis. The regulatory role of heme in EM encompasses cytokines, signaling pathways, and kinases that mediate cellular responses to external stimuli. HO-1, a catalytic enzyme in the catabolic phase of heme, mitigates heme's cytotoxicity in EM due to its antioxidant, anti-inflammatory, and anti-proliferative properties. Certain compounds may intervene in EM by targeting heme metabolism, guiding the development of appropriate treatments for all stages of endometriosis.

Clinical and molecular analysis of a novel variant in heme oxygenase-1 deficiency: Unraveling its role in inflammation, heme metabolism, and pulmonary phenotype.

Heme oxygenase 1 (HO-1) is the pivotal catalyst for the primary and rate-determining step in heme catabolism, playing a crucial role in mitigating heme-induced oxidative damage. Pathogenic variants in the HMOX1 gene which encodes HO-1, are responsible for a severe, multisystem disease characterized by recurrent inflammatory episodes, organ failure, and an ultimately fatal course. Chronic hemolysis and abnormally low bilirubin levels are cardinal laboratory features of this disorder. In this study, we describe a patient with severe interstitial lung disease, frequent episodes of hyperinflammation non-responsive to immunosuppression, and fatal pulmonary hemorrhage. Employing exome sequencing, we identified two protein truncating variants in HMOX1, c.262_268delinsCC (p.Ala88Profs*51) and a previously unreported variant, c.55dupG (p.Glu19Glyfs*14). Functional analysis in patient-derived lymphoblastoid cells unveiled the complete absence of HO-1 protein expression and a marked reduction in cell viability upon exposure to hemin. These findings confirm the pathogenicity of the identified HMOX1 variants, further underscoring their association with severe pulmonary manifestations . This study describes the profound clinical consequences stemming from disruptions in redox metabolism.

Prognostic value and potential function of a novel heme-related LncRNAs signature in gastric cancer.

Heme is a coordination complex formed by the binding of iron ions and porphyrin rings. Its metabolic processes are associated with various cancers, including gastric cancer (GC). In recent years, long non-coding RNAs (LncRNAs) have been identified as key regulatory factors in GC. However, the role of LncRNAs associated with heme metabolism in GC and their relationship with prognosis have not been reported. In this study, we constructed a novel LncRNAs signature related to heme metabolism (HMlncSig) and validated its prognostic value for predicting the survival of GC patients through training, test, and entire cohorts. Kaplan-Meier analysis demonstrated that patients in the high-risk group had shorter survival times. Univariate and multivariate Cox regression analysis showed that HMlncSig was an independent prognostic indicator for GC patients, regardless of other clinical pathological features. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis and gene set variation analysis pathways showed that the activation of these markers may be involved in tumor progression, influencing the survival of GC patients. The nomogram, based on HMlncSig score and clinical features, demonstrated the strong predictive ability of this signature. Additionally, significant differences were observed between the high-risk and low-risk groups in terms of immune cell subtypes, expression of immune checkpoint genes, and response to chemotherapy and immunotherapy. Through clinical validation, we found that the risk score and heme levels of GC patients were both significantly elevated and correlated with the degree of malignancy. Furthermore, we found that AP000692.1, a key gene in this signature, promoted the proliferation, migration, and invasion of GC cells. In conclusion, our HMlncSig model has significant predictive value for the prognosis of GC patients and can provide clinical guidance for personalized immunotherapy.

Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.

The role of nonesterified fatty acids in cancer biology: Focus on tryptophan and related metabolism.

Plasma nonesterified fatty acids (NEFA) are elevated in cancer, because of decreased albumin levels and of fatty acid oxidation, and increased fatty acid synthesis and lipolysis. Albumin depletion and NEFA elevation maximally release albumin-bound tryptophan (Trp) and increase its flux down the kynurenine pathway, leading to increased production of proinflammatory kynurenine metabolites, which tumors use to undermine T-cell function and achieve immune escape. Activation of the aryl hydrocarbon receptor by kynurenic acid promotes extrahepatic Trp degradation by indoleamine 2,3-dioxygenase and leads to upregulation of poly (ADP-ribose) polymerase, activation of which and also of SIRT1 (silent mating type information regulation 2 homolog 1) could lead to depletion of NAD+ and ATP, resulting in cell death. NEFA also modulate heme synthesis and degradation, changes in which impact homocysteine metabolism and production of reduced glutathione and hydrogen sulphide. The significance of the interactions between heme and homocysteine metabolism in cancer biology has received little attention. Targeting Trp disposition in cancer to prevent the NEFA effects is suggested.

Biogenesis of cytochromes c and c 1 in the electron transport chain of malaria parasites.

Plasmodium malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and a key antimalarial drug target. ETC function requires cytochromes c and c 1 that are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate biogenesis of the mature cytochrome c or c 1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, Plasmodium parasites are like yeast and encode two separate HCCS homologs thought to be specific for heme attachment to cyt c (HCCS) or cyt c 1 (HCC1S). To test the function and specificity of P. falciparum HCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c 1 biogenesis and caused lethal ETC dysfunction that was not reversed by over-expression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in E. coli of cyt c hemylation by P. falciparum HCCS and HCC1S strongly suggest that both homologs are essential for mitochondrial ETC function and have distinct specificities for biogenesis of cyt c and c 1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

Absolute quantification of BACH1 and BACH2 transcription factors in B and plasma cells reveals their dynamic changes and unique roles.

Changes in the absolute protein amounts of transcription factors are important for regulating gene expression during cell differentiation and in responses to changes in the cellular and extracellular environment. However, few studies have focused on the absolute quantification of mammalian transcription factors. In this study, we established an absolute quantification method for the transcription factors BACH1 and BACH2, which are expressed in B cells and regulated by direct heme binding. The method used purified recombinant proteins as controls in Western blotting and was applied to mouse naïve B cells in the spleen, as well as activated B cells and plasma cells. BACH1 was present in naïve B cells at approximately half the levels of BACH2. In activated B cells, BACH1 decreased compared to naïve B cells, while BACH2 increased. In plasma cells, BACH1 increased back to the same extent as in naïve B cells, while BACH2 was not detected. Their target genes Prdm1 and Hmox1 were highly induced in plasma cells. BACH1 was found to undergo degradation with lower concentrations of heme than BACH2. Therefore, BACH1 and BACH2 are similarly abundant in B cells but differ in heme sensitivity, potentially regulating gene expression differently depending on their heme responsiveness.