Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate.

A central problem in biology is to identify gene function. One approach is to infer function in large supergenomic networks of interactions and ancestral relationships among genes; however, their analysis can be computationally prohibitive. We show here that these biological networks are compressible. They can be shrunk dramatically by eliminating redundant evolutionary relationships, and this process is efficient because in these networks the number of compressible elements rises linearly rather than exponentially as in other complex networks. Compression enables global network analysis to computationally harness hundreds of interconnected genomes and to produce functional predictions. As a demonstration, we show that the essential, but functionally uncharacterized Plasmodium falciparum antigen EXP1 is a membrane glutathione S-transferase. EXP1 efficiently degrades cytotoxic hematin, is potently inhibited by artesunate, and is associated with artesunate metabolism and susceptibility in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action of a frontline antimalarial drug.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease.

ABSTRACT: Disordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2-containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Regulation of heme utilization and homeostasis in Candida albicans.

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets.

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis. In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent. In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application.

RNA G4, as an integral branch of G4 structure, possesses distinct interactions with ligands compared to the common DNA G4, thus the investigation of RNA G4/ligand interactions might be considered as a fresh breakthrough to improve the biosensing performance of G4/ligand system. In this study, we comparatively explored the structural and functional mechanisms of RNA G4 and DNA G4 in the interaction with ligands, hemin and thioflavin T (ThT), utilizing the classical PS2.M sequence as a model. We found that although the catalytic performance of RNA G4/hemin system was lower than DNA G4/hemin, RNA G4/ThT fluorescence system exhibited a significant improvement (2∼3-fold) compared to DNA G4/ThT, and adenine modification could further enhance the signaling. Further, by exploring the interaction between RNA G4 and ThT, we deemed that RNA G4 and ThT were stacked in a bimolecular mode compared to single-molecule binding of DNA G4/ThT, thus more strongly limiting the structural spin in ThT excited state. Further, RNA G4/ThT displayed higher environmental tolerance and lower ion dependence than DNA G4/ThT. Finally, we employed RNA G4/ThT as a highly sensitive label-free fluorescent signal output system for in situ imaging of isoforms BCR-ABL e13a2 and e14a2. Overall, this study successfully screened a high-performance RNA G4 biosensing system through systematic RNA G4/ligands interaction studies, which was expected to provide a promising reference for subsequent G4/ligand research.

Cysteine in the R3 Tau Peptide Modulates Hemin Binding and Reactivity.

Tau is a neuronal protein involved in axonal stabilization; however under pathological conditions, it triggers the deposition of insoluble neurofibrillary tangles, which are one of the biomarkers for Alzheimer's disease. The factors that might influence the fibrillation process are i) two cysteine residues in two pseudorepetitive regions, called R2 and R3, which can modulate protein-protein interaction via disulfide cross-linking; ii) an increase of reactive oxygen species affecting the post-translational modification of tau; and iii) cytotoxic levels of metals, especially ferric-heme (hemin), in hemolytic processes. Herein, we investigated how the cysteine-containing R3 peptide (R3C) and its Cys→Ala mutant (R3A) interact with hemin and how their binding affects the oxidative damage of the protein. The calculated binding constants are remarkably higher for the hemin-R3C complex (LogK1 = 5.90; LogK2 = 5.80) with respect to R3A (LogK1 = 4.44; LogK2 < 2), although NMR and CD investigations excluded the direct binding of cysteine as an iron axial ligand. Both peptides increase the peroxidase-like activity of hemin toward catecholamines and phenols, with a double catalytic efficiency detected for hemin-R3C systems. Moreover, the presence of cysteine significantly alters the susceptibility of R3 toward oxidative modifications, easily resulting in peptide dopamination and formation of cross-linked S-S derivatives.

Integrative analysis of transcriptomic and metabolomic profiles reveals abnormal phosphatidylinositol metabolism in follicles from endometriosis-associated infertility patients.

Endometriosis is a common gynecological disorder that causes female infertility. Our recent research found that excessive oxidative stress in ovaries of endometriosis patients induced senescence of cumulus granulosa cells. Here, we analyzed the transcriptomic and metabolomics profiles of follicles in a mouse model of endometriosis and in patients with endometriosis and investigated the potential function of changed metabolites in granulosa cells. RNA-sequencing indicated that both endometriosis lesions and oxidative stress in mice induced abnormalities of reactive oxidative stress, steroid hormone biosynthesis, and lipid metabolism. The mouse model and women with endometriosis showed altered lipid metabolism. Nontargeted metabolite profiling of follicular fluid from endometriosis and male-factor infertility patients by liquid chromatography mass spectrometry identified 55 upregulated and 67 downregulated metabolites. These differential metabolites were mainly involved in steroid hormone biosynthesis and glycerophospholipid metabolism. Phosphatidylinositol (PI 16:0/18:2) was significantly elevated in follicular fluid from endometriosis patients compared with controls (p < 0.05), while lysophosphatidylinositol (LPI 18:2, 20:2, 18:1, 20:3 and 18:3) was reduced (p < 0.05). Upregulated PI and downregulated LPI correlated with oocyte retrieval number and mature oocyte number. LPI inhibited cellular reactive oxidative stress induced by hemin in granulosa cells. Cell proliferation inhibition, senescence, and apoptosis induced by hemin were partially reversed by LPI. Moreover, LPI administration rescued hemin blocking of cumulus-oocyte complex expansion and stimulated expression of ovulation-related genes. Transcriptomic Switching mechanism at 5' end of the RNA transcript sequencing and western blot revealed that LPI effects on granulosa cells were associated with its regulation of MAPK-ERK1/2 signaling, which was suppressed in the presence of hemin. In conclusion, our results revealed the dysregulation of lipid metabolism in endometriotic follicles. LPI may represent a novel agent for in vitro follicular culture that reverses the excessive oxidative stress from endometriotic lesions. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Hemin regulates platelet clearance in hemolytic disease by binding to GPIbα.

Hemolysis is associated with thrombosis and vascular dysfunction, which are the pathological components of many diseases. Hemolytic products, including hemoglobin and hemin, activate platelets (PLT). Despite its activation, the effect of hemolysis on platelet clearance remains unclear, It is critical to maintain a normal platelet count and ensure that circulating platelets are functionally viable. In this study, we used hemin, a degradation product of hemoglobin, as a potent agonist to treat platelets and simulate changes in vivo in mice. Hemin treatment induced activation and morphological changes in platelets, including an increase in intracellular Ca2+ levels, phosphatidylserine (PS) exposure, and cytoskeletal rearrangement. Fewer hemin-treated platelets were cleared by macrophages in the liver after transfusion than untreated platelets. Hemin bound to glycoprotein Ibα (GPIbα), the surface receptor in hemin-induced platelet activation and aggregation. Furthermore, hemin decreased GPIbα desialylation, as evidenced by reduced Ricinus communis agglutinin I (RCA- I) binding, which likely extended the lifetime of such platelets in vivo. These data provided new insight into the mechanisms of GPIbα-mediated platelet activation and clearance in hemolytic disease.

What is the context? Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of several diseases.Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more potent agonist than hemoglobin for directly activating platelets.Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet hemostasis.Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological platelet clearance through liver macrophages and hepatocytes.What is new? In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the influence of hemin at 0-50 µM on platelets in vitro before exploring the mechanism underlying hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.Our analyses suggest that: Hemin bound to GPIbα on the platelet surface with high affinity.Platelet clearance occurred slowly in the liver and spleen after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.What is the impact? This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated platelets activation and clearance in diseases associated with hemolysis.

Breaking Iron Homeostasis: Iron Capturing Nanocomposites for Combating Bacterial Biofilm.

Given the scarcity of novel antibiotics, the eradication of bacterial biofilm infections poses formidable challenges. Upon bacterial infection, the host restricts Fe ions, which are crucial for bacterial growth and maintenance. Having coevolved with the host, bacteria developed adaptive pathways like the hemin-uptake system to avoid iron deficiency. Inspired by this, we propose a novel strategy, termed iron nutritional immunity therapy (INIT), utilizing Ga-CT@P nanocomposites constructed with gallium, copper-doped tetrakis (4-carboxyphenyl) porphyrin (TCPP) metal-organic framework, and polyamine-amine polymer dots, to target bacterial iron intakes and starve them. Owing to the similarity between iron/hemin and gallium/TCPP, gallium-incorporated porphyrin potentially deceives bacteria into uptaking gallium ions and concurrently extracts iron ions from the surrounding bacteria milieu through the porphyrin ring. This strategy orchestrates a "give and take" approach for Ga3+/Fe3+ exchange. Simultaneously, polymer dots can impede bacterial iron metabolism and serve as real-time fluorescent iron-sensing probes to continuously monitor dynamic iron restriction status. INIT based on Ga-CT@P nanocomposites induced long-term iron starvation, which affected iron-sulfur cluster biogenesis and carbohydrate metabolism, ultimately facilitating biofilm eradication and tissue regeneration. Therefore, this study presents an innovative antibacterial strategy from a nutritional perspective that sheds light on refractory bacterial infection treatment and its future clinical application.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

Mechanism of SOX10 in ferroptosis of hippocampal neurons after intracerebral hemorrhage via the miR-29a-3p/ACSL4 axis.

Intracerebral hemorrhage (ICH) is classified as a lethal neurological injury associated with cerebrovascular disorders. Ferroptosis is a unique form of cell death and participates in ICH pathogenesis. Herein, the role of SRY-box transcription factor 10 (SOX10) in ferroptosis of hippocampal neurons after ICH was investigated. The in vitro ICH models were established by treating immortalized mouse hippocampal cell line HT-22 with Hemin. Quantitative real-time polymerase chain reaction and Western blotting revealed that the transcription factor SOX10 and microRNA (miR)-29a-3p were decreased whereas acyl-CoA synthetase long-chain family member 4 (ACSL4) was increased in the ICH cell models. Subsequently, the assays of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the commercial kits, and fluorescent labeling revealed that SOX10 overexpression improved cell viability, decreased the amount of reactive oxygen species (ROS) and Fe2+, and increased the amount of glutathione (GSH) and glutathione peroxidase 4 (GPX4) in ICH models. Thereafter, chromatin immunoprecipitation and dual-luciferase assays showed that SOX10 binding to the miR-29a-3p promoter region increased miR-29a-3p expression, and miR-29a-3p targeted and limited ACSL4 transcription. Rescue experiments showed that miR-29a-3p downregulation or ACSL4 overexpression expedited ferroptosis of Hemin-treated HT-22 cells. Taken together, SOX10 contributed to ferroptosis of hippocampal neurons after ICH via increasing miR-29a-3p to limit ACSL4 transcription.NEW & NOTEWORTHY SOX10 promotes the expression of Mir-29a-3p by binding to the promoter region of Mir-29a-3p, thereby targeting the expression of ACSL4 and inhibiting the iron death of hippocampal neuronal cells in mice with ICH.

Colorimetric Detection of Met Dimerization on Live Cells via Smartphone for High-Sensitivity Sensing of the HGF/Met Signaling Pathway.

Membrane protein dimerization regulates numerous cellular biological processes; therefore, highly sensitive and facile detection of membrane protein dimerization are very crucial for clinical diagnosis and biomedical research. Herein, a colorimetric detection of Met dimerization on live cells via smartphone for high-sensitivity sensing of the HGF/Met signaling pathway was developed for the first time. The Met monomers on live cells were recognized by specific ligands (aptamers) first, and the Met dimerizations triggered the proximity-ligation-assisted catalytic hairpin assembly (CHA) reaction to generate large amounts of G-quadruplex (G4) fragments which can further combine hemin to form G4/hemin DNAzymes possessing the horseradish-peroxidase-like catalytic activity for catalyzing the oxidation of ABTS by H2O2 and producing the colorimetric signal (i.e., color change). The colorimetric detection of Met on live cells was then achieved by image acquisition and processing via a smartphone. As a proof-of-principle, the HGF/Met signaling pathway based on Met-Met dimerization was facile monitored, and the human gastric cancer cells MKN-45 with natural Met-Met dimers were sensitively tested and a wide linear working range from 2 to 1000 cells with a low detection limit of 1 cell was obtained. The colorimetric assay possesses a good specificity and high recovery rate of MKN-45 cells spiked in peripheral blood, which indicates that the proposed colorimetric detection of Met dimerization can be used for convenient observation of the HGF/Met signaling pathway and has extensive application prospects in point-of-care testing (POCT) of Met-dimerization-related tumor cells.

Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

A Yersinia T6SS Effector YezP Engages the Hemin Uptake Receptor HmuR and ZnuABC for Zn2+ Acquisition.

Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn2+ acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn2+ into the periplasm by the YezP-Zn2+ complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn2+ from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. IMPORTANCE Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn2+ acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn2+ into the cytoplasm via the YezP-Zn2+ complex, and elucidation of the complete Zn2+ acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.

Zippered G-quadruplex/hemin DNAzyme: exceptional catalyst for universal bioanalytical applications.

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.

Type-I Hemins and Free Porphyrins from a Western Australian Sponge Isabela sp.

Two novel free porphyrins, isabellins A and B, as well as the known compounds corallistin D and deuteroporphyrin IX were isolated from a marine sponge Isabela sp. LC-MS analysis of the crude extract revealed that the natural products were present both as free porphyrins and iron(III) coordinated hemins, designated isabellihemin A, isabellihemin B, corallistihemin D and deuterohemin IX, respectively. Structures were determined via high-resolution mass spectrometry, UV-Vis spectroscopy and extensive NOESY NMR spectroscopic experiments. The type-I alkyl substitution pattern of isabellin A and isabellihemin A was assigned unambiguously by single crystal X-ray diffraction. Biological evaluation of the metabolites revealed potent cytotoxicity for isabellin A against the NS-1 murine myeloma cell line.

In vitro phenotypic evidence for the utilization of iron from different sources and siderophores production in the fish pathogen Tenacibaculum dicentrarchi.

Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 µM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 µM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.

Exogenous Hemin alleviates cadmium stress in maize by enhancing sucrose and nitrogen metabolism and regulating endogenous hormones.

Cadmium (Cd) stress restricts maize growth and productivity severely. We aimed to investigate the effects of Hemin on the metabolism of sucrose and nitrogen and endogenous hormones in maize under cadmium stress. Maize varieties 'Tiannong 9' (cadmium tolerant) and 'Fenghe 6' (cadmium sensitive) were grown in nutrient solutions to study the effects of Hemin on maize physiological and ecological mechanisms under cadmium stress. The results showed that Hemin mediated the increase of sucrose content and the activities of key enzymes sucrose phosphate synthase (SPS) and sucrose synthase (SS) in maize leaves under cadmium stress. Soluble acid invertase (SAInv) and basic/neutral invertase (A/N-Inv) enzyme activities in leaves were decreased significantly, and sucrose accumulation in leaves was increased. Hemin also mediated the increase of NO3- content in leaves, the decrease of NH4+ content and the increase of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase activity (GOGAT) and glutamate dehydrogenase (GDH) enzyme activities under cadmium stress. The contents of IAA, ZR, and GA in leaves and roots increased, ABA, MeJA, and SA decreased, and IAA/ABA, ZR/ABA, and GA/ABA increased under cadmium stress. Our study showed Hemin can alleviate cadmium stress in maize by enhancing sucrose and nitrogen metabolism and regulating endogenous hormones.

This work further investigates the effects of Hemin on the metabolism of sucrose and nitrogen and endogenous hormones in maize under cadmium stress, which, hopefully, is to guide Hemin application to maize field resilience production. It also explains that Hemin is beneficial for dry matter accumulation and transport, alleviated ammonia toxicity and nitrogen metabolism disorder, and induced the changes of endogenous hormone content and the adaptive hormone ratio balance under cadmium stress.