The clinical relevance of heme detoxification by the macrophage heme oxygenase system.

Heme degradation by the heme oxygenase (HMOX) family of enzymes is critical for maintaining homeostasis and limiting heme-induced tissue damage. Macrophages express HMOX1 and 2 and are critical sites of heme degradation in healthy and diseased states. Here we review the functions of the macrophage heme oxygenase system and its clinical relevance in discrete groups of pathologies where heme has been demonstrated to play a driving role. HMOX1 function in macrophages is essential for limiting oxidative tissue damage in both acute and chronic hemolytic disorders. By degrading pro-inflammatory heme and releasing anti-inflammatory molecules such as carbon monoxide, HMOX1 fine-tunes the acute inflammatory response with consequences for disorders of hyperinflammation such as sepsis. We then discuss divergent beneficial and pathological roles for HMOX1 in disorders such as atherosclerosis and metabolic syndrome, where activation of the HMOX system sits at the crossroads of chronic low-grade inflammation and oxidative stress. Finally, we highlight the emerging role for HMOX1 in regulating macrophage cell death via the iron- and oxidation-dependent form of cell death, ferroptosis. In summary, the importance of heme clearance by macrophages is an active area of investigation with relevance for therapeutic intervention in a diverse array of human diseases.

Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism.

In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

Haem iron versus ferrous iron salts to treat iron deficiency anaemia in Gambian children: protocol for randomised controlled trial {1}.

BACKGROUND: The World Health Organization recommends universal iron supplementation for children aged 6-23 months in countries where anaemia is seen in over 40% of the population. Conventional ferrous salts have low efficacy due to low oral absorption in children with inflammation. Haem iron is more bioavailable, and its absorption may not be decreased by inflammation. This study aims to compare daily supplementation with haem iron versus ferrous sulphate on haemoglobin concentration and serum ferritin concentration after 12 weeks of supplementation. METHODS: This will be a two-arm, randomised controlled trial. Gambian children aged 6-12 months with anaemia will be recruited within a predefined geographical area and recruited by trained field workers. Eligible participants will be individually randomised using a 1:1 ratio within permuted blocks to daily supplementation for 12 weeks with either 10.0 mg of elemental iron as haem or ferrous sulphate. Safety outcomes such as diarrhoea and infection-related adverse events will be assessed daily by the clinical team (see Bah et al. Additional file 4_Adverse event eCRF). Linear regression will be used to analyse continuous outcomes, with log transformation to normalise residuals as needed. Binary outcomes will be analysed by binomial regression or logistic regression, Primary analysis will be by modified intention-to-treat (i.e., those randomised and who ingested at least one supplement dose of iron), with multiple imputations to replace missing data. Effect estimates will be adjusted for baseline covariates (C-reactive protein, alpha-1-acid glycoprotein, haemoglobin, ferritin, soluble transferrin receptor). DISCUSSION: This study will determine if therapeutic supplementation with haem iron is more efficacious than with conventional ferrous sulphate in enhancing haemoglobin and ferritin concentrations in anaemic children aged 6-12 months. TRIAL REGISTRATION: Pan African Clinical Trial Registry PACTR202210523178727.

Tyrosine nitration of glucagon impairs its function: Extending the role of heme in T2D pathogenesis.

New studies raise the possibility that the higher glucagon (GCG) level present in type 2 diabetes (T2D) is a compensatory mechanism to enhance ß-cell function, rather than induce dysregulated glucose homeostasis, due to an important role for GCG that acts directly within the pancreas on insulin secretion by intra-islet GCG signaling. However, in states of poorly controlled T2D, pancreatic α cell mass increases (overproduced GCG) in response to insufficient insulin secretion, indicating decreased local GCG activity. The reason for this decrease is not clear. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. Considering that protein tyrosine nitration in diabetic islets increases and glucose-stimulated insulin secretion (GSIS) decreases, we speculated that heme modulates GSIS by transient interaction with GCG and catalyzing its tyrosine nitration, and the tyrosine nitration may impair GCG activity, leading to loss of intra-islet GCG signaling and markedly impaired insulin secretion. Data presented here elucidate a novel role for heme in disrupting local GCG signaling in diabetes. Heme bound to GCG and induced GCG tyrosine nitration. Two tyrosine residues in GCG were both sensitive to the nitrating species. Further, GCG was also demonstrated to be a preferred target peptide for tyrosine nitration by co-incubation with BSA. Tyrosine nitration impaired GCG stimulated cAMP-dependent signaling in islet ß cells and decreased insulin release. Our results provided a new role of heme for impaired GSIS in the pathological process of diabetes.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration.

Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH-) and hemin (liganded with Cl-)-are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe3. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca2+]i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca2+]i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Comparing the oxidative functions of neutrophil myeloperoxidase and cytochrome P450 enzymes in drug metabolism.

Drug metabolism is an essential process that chemically alters xenobiotic substrates to activate or terminate drug activity. Myeloperoxidase (MPO) is a neutrophil-derived haem-containing enzyme that is involved in killing invading pathogens, although consequentially, this same oxidative activity can produce metabolites that damage host tissue and play a role in various human pathologies. Cytochrome P450s (CYPs) are a superfamily of haem-containing enzymes that are significantly involved in the metabolism of drugs by functioning as monooxygenases and can be induced or inhibited, resulting in significant drug-drug interactions that lead to unanticipated adverse drug reactions. In this review, the functions of drug metabolism of MPO and CYPs are explored, along with their involvement and association for common enzymatic pathways by certain xenobiotics. MPO and CYPs metabolize numerous xenobiotics, although few reported studies have made a direct comparison between both enzymes. Additionally, we employed molecular docking to compare the active site and haem prosthetic group of MPO and CYPs, supporting their similar catalytic activities. Furthermore, we performed LCMS analysis and observed a shared hydroxylated mefenamic acid metabolite produced in both enzymatic systems. A proper understanding of the enzymology and mechanisms of action of MPO and CYPs is of significant importance when enhancing the beneficial functions of drugs in health and diminishing their damaging effects on diseases. Therefore, awareness of drugs and xenobiotic substrates involved in MPO and CYPs metabolism pathways will add to the knowledge base to foresee and prevent potential drug interactions and adverse events.

Development strategy of non-GMO organism for increased hemoproteins in Corynebacterium glutamicum: a growth-acceleration-targeted evolution.

Heme, found in hemoproteins, is a valuable source of iron, an essential mineral. The need for an alternative hemoprotein source has emerged due to the inherent risks of large-scale livestock farming and animal proteins. Corynebacterium glutamicum, regarded for Qualified Presumption of Safety or Generally Recognized as Safe, can biosynthesize hemoproteins. C. glutamicum single-cell protein (SCP) can be a valuable alternative hemoprotein for supplying heme iron without adversely affecting blood fat levels. We constructed the chemostat culture system to increase hemoprotein content in C. glutamicum SCP. Through adaptive evolution, hemoprotein levels could be naturally increased to address oxidative stress resulting from enhanced growth rate. In addition, we used several specific plasmids containing growth-accelerating genes and the hemA promoter to expedite the evolutionary process. Following chemostat culture for 15 days, the plasmid in selected descendants was cured. The evolved strains showed improved specific growth rates from 0.59 h-1 to 0.62 h-1, 20% enhanced resistance to oxidative stress, and increased heme concentration from 12.95 µg/g-DCW to 14.22-15.24 µg/g-DCW. Notably, the putative peptidyl-tRNA hydrolase-based evolved strain manifested the most significant increase (30%) of hemoproteins. This is the first report presenting the potential of a growth-acceleration-targeted evolution (GATE) strategy for developing non-GMO industrial strains with increased bio-product productivity.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Unconventional role of Rab4 in the secretory pathway in Leishmania.

Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4:WT, GFP-LdRab4:Q67L, and GFP-LdRab4:S22N. Our results have shown that overexpression of GFP-LdRab4:Q67L or GFP-LdRab4:S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4:S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4:S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4:S22N is severely compromised in macrophages in comparison to GFP-LdRab4:WT and GFP-LdRab4:Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.

Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

Pseudomonas aeruginosa heme metabolites biliverdin IXß and IXδ are integral to lifestyle adaptations associated with chronic infection.

Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source and away from siderophore uptake provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IXß (BVIXß) and BVIXδ positively regulate the heme-dependent cell surface signaling cascade. We further investigated the role of BVIXß and BVIXδ in cell signaling utilizing allelic strains lacking a functional heme oxygenase (hemOin) or one reengineered to produce BVIXα (hemOα). Compared to PAO1, both strains show a heme-dependent growth defect, decreased swarming and twitching, and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIXß and BVIXδ. Utilizing liquid chromatography-tandem mass spectrometry, we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing, phenazine production, and chemotaxis. Metabolite profiling further revealed increased levels of Pseudomonas quinolone signal and phenazine metabolites. In static conditions, we observed a significant repression of chemosensory pathways and type IV pili biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle. IMPORTANCE: The opportunistic pathogen Pseudomonas aeruginosa causes long-term chronic infection in the airways of cystic fibrosis patients. The ability to scavenge iron and to establish chronic infection within this environment coincides with a switch to utilize heme as the primary iron source. Herein, we show the heme metabolites biliverdin beta and delta are themselves important signaling molecules integrating the switch in iron acquisition systems with cooperative behaviors such as motility and biofilm formation that are essential for long-term chronic infection. These significant findings will enhance the development of viable multi-targeted therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.

Hemophore-like proteins of the HmuY family in the oral and gut microbiome: unraveling the mystery of their evolution.

SUMMARY: Heme (iron protoporphyrin IX, FePPIX) is the main source of iron and PPIX for host-associated pathogenic bacteria, including members of the Bacteroidota (formerly Bacteroidetes) phylum. Porphyromonas gingivalis, a keystone oral pathogen, uses a unique heme uptake (Hmu) system, comprising a hemophore-like protein, designated as the first member of the novel HmuY family. Compared to classical, secreted hemophores utilized by Gram-negative bacteria or near-iron transporter domain-based hemophores utilized by Gram-positive bacteria, the HmuY family comprises structurally similar proteins that have undergone diversification during evolution. The best characterized are P. gingivalis HmuY and its homologs from Tannerella forsythia (Tfo), Prevotella intermedia (PinO and PinA), Bacteroides vulgatus (Bvu), and Bacteroides fragilis (BfrA, BfrB, and BfrC). In contrast to the two histidine residues coordinating heme iron in P. gingivalis HmuY, Tfo, PinO, PinA, Bvu, and BfrA preferentially use two methionine residues. Interestingly, BfrB, despite conserved methionine residue, binds the PPIX ring without iron coordination. BfrC binds neither heme nor PPIX in keeping with the lack of conserved histidine or methionine residues used by other members of the HmuY family. HmuY competes for heme binding and heme sequestration from host hemoproteins with other members of the HmuY family to increase P. gingivalis competitiveness. The participation of HmuY in the host immune response confirms its relevance in relation to the survival of P. gingivalis and its ability to induce dysbiosis not only in the oral microbiome but also in the gut microbiome or other host niches, leading to local injuries and involvement in comorbidities.

Carbon monoxide is involved in melatonin-enhanced drought resistance in tomato seedlings by enhancing chlorophyll synthesis pathway.

BACKGROUND: Drought is thought to be a major abiotic stress that dramatically limits tomato growth and production. As signal molecule, melatonin (MT) and carbon monoxide (CO) can enhance plant stress resistance. However, the effect and underlying mechanism of CO involving MT-mediated drought resistance in seedling growth remains unknown. In this study, tomato (Solanum lycopersicum L. 'Micro-Tom') seedlings were used to investigate the interaction and mechanism of MT and CO in response to drought stress. RESULTS: The growth of tomato seedlings was inhibited significantly under drought stress. Exogenous MT or CO mitigated the drought-induced impairment in a dose-dependent manner, with the greatest efficiency provided by 100 and 500 µM, respectively. But application of hemoglobin (Hb, a CO scavenger) restrained the positive effects of MT on the growth of tomato seedlings under drought stress. MT and CO treatment promoted chlorophyll a (Chl a) and chlorophyll a (Chl b) accumulations. Under drought stress, the intermediate products of chlorophyll biosynthesis such as protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX), potochlorophyllide (Pchlide) and heme were increased by MT or CO, but uroporphyrinogen III (Uro III) content decreased in MT-treated or CO-treated tomato seedlings. Meanwhile, MT or CO up-regulated the expression of chlorophyll and heme synthetic-related genes SlUROD, SlPPOX, SlMGMT, SlFECH, SlPOR, SlChlS, and SlCAO. However, the effects of MT on chlorophyll biosynthesis were almost reversed by Hb. CONCLUSION: The results suggested that MT and CO can alleviate drought stress and facilitate the synthesis of Chl and heme in tomato seedlings. CO played an essential role in MT-enhanced drought resistance via facilitating chlorophyll biosynthesis pathway.

FLVCR1a Controls Cellular Cholesterol Levels through the Regulation of Heme Biosynthesis and Tricarboxylic Acid Cycle Flux in Endothelial Cells.

Feline leukemia virus C receptor 1a (FLVCR1a), initially identified as a retroviral receptor and localized on the plasma membrane, has emerged as a crucial regulator of heme homeostasis. Functioning as a positive regulator of δ-aminolevulinic acid synthase 1 (ALAS1), the rate-limiting enzyme in the heme biosynthetic pathway, FLVCR1a influences TCA cycle cataplerosis, thus impacting TCA flux and interconnected metabolic pathways. This study reveals an unexplored link between FLVCR1a, heme synthesis, and cholesterol production in endothelial cells. Using cellular models with manipulated FLVCR1a expression and inducible endothelial-specific Flvcr1a-null mice, we demonstrate that FLVCR1a-mediated control of heme synthesis regulates citrate availability for cholesterol synthesis, thereby influencing cellular cholesterol levels. Moreover, alterations in FLVCR1a expression affect membrane cholesterol content and fluidity, supporting a role for FLVCR1a in the intricate regulation of processes crucial for vascular development and endothelial function. Our results underscore FLVCR1a as a positive regulator of heme synthesis, emphasizing its integration with metabolic pathways involved in cellular energy metabolism. Furthermore, this study suggests that the dysregulation of heme metabolism may have implications for modulating lipid metabolism. We discuss these findings in the context of FLVCR1a's potential heme-independent function as a choline importer, introducing additional complexity to the interplay between heme and lipid metabolism.

Analyzing the interaction of Helicobacter pylori GAPDH with host molecules and hemin: Inhibition of hemin binding.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme. Apart from its primary role in the glycolytic pathway, in many bacterial species it is found in the extracellular milieu and also on the bacterial surface. Positioning on the bacterial surface allows the GAPDH molecule to interact with many host molecules such as plasminogen, fibrinogen, fibronectin, laminin and mucin etc. This facilitates the bacterial colonization of the host. Helicobacter pylori is a major human pathogen that causes a number of gastrointestinal infections and is the main cause of gastric cancer. The binding analysis of H. pylori GAPDH (HpGAPDH) with host molecules has not been carried out. Hence, we studied the interaction of HpGAPDH with holo-transferrin, lactoferrin, haemoglobin, fibrinogen, fibronectin, catalase, plasminogen and mucin using biolayer interferometry. Highest and lowest binding affinity was observed with lactoferrin (4.83 ± 0.70 × 10-9 M) and holo-transferrin (4.27 ± 2.39 × 10-5 M). Previous studies established GAPDH as a heme chaperone involved in intracellular heme trafficking and delivery to downstream target proteins. Therefore, to get insights into heme binding, the interaction between HpGAPDH and hemin was analyzed. Hemin binds to HpGAPDH with an affinity of 2.10 µM while the hemin bound HpGAPDH does not exhibit activity. This suggests that hemin most likely binds at the active site of HpGAPDH, prohibiting substrate binding. Blind docking of hemin with HpGAPDH also supports positioning of hemin at the active site. Metal ions were found to inhibit the activity of HpGAPDH, suggesting that it also possibly occupies the substrate binding site. Furthermore, with metal-bound HpGAPDH, hemin binding was not observed, suggesting metal ions act as an inhibitor of hemin binding. Since GAPDH has been identified as a heme chaperone, it will be interesting to analyse the biological consequences of inhibition of heme binding to GAPDH by metal ions.

Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.