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.

Depletion assisted hemin affinity (DAsHA) proteomics reveals an expanded landscape of heme-binding proteins in the human proteome.

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

Acidity-Biodegradable Iridium-Coordinated Nanosheets for Amplified Ferroptotic Cell Death Through Multiple Regulatory Pathways.

Ferroptosis-based treatment strategies display the potential to suppress some malignant tumors with intrinsic apoptosis resistance. However, current related cancer treatments are still hampered by insufficient intracellular reactive oxygen species (ROS) levels and Fe2+ contents, posing considerable challenges for their clinical translation. Herein, an intracellular acid-biodegradable iridium-coordinated nanosheets (Ir-Hemin) with sonodynamic therapy (SDT) properties to effectively induce ferroptosis in tumor cells through multiple regulatory pathways are proposed. Under ultrasound (US) irradiation, Ir-Hemin nanosheets act as nanosonosensitizers to effectively generate ROS, subsequently causing the accumulation of lipid peroxides (LPO) and inducing ferroptotic cell death. Furthermore, these Ir-Hemin nanosheets decompose quickly to release hemin and Ir(IV), which deplete intracellular glutathione (GSH) to deactivate the enzyme glutathione peroxidase 4 (GPX4) and initiate the ferroptosis pathway. Specifically, the released hemin enables heme oxygenase 1 (HO-1) upregulation for endogenous ferrous ion supplementation, which compensates for the toxicity concerns brought about by the large uptake of exogenous iron. Surprisingly, Ir-Hemin nanosheets exhibit high tumor accumulation and trigger effective ferroptosis for tumor therapy. These Ir-Hemin nanosheets display pronounced synergistic anticancer efficacy under US stimulation both in vitro and in vivo, providing a strong rationale for the application of ferroptosis in cancer treatment.

Protective Effect of Electroacupuncture on the Barrier Function of Intestinal Injury in Endotoxemia through HO-1/PINK1 Pathway-Mediated Mitochondrial Dynamics Regulation.

Background and Aims: Endotoxemia (ET) is a common critical illness in patients receiving intensive care and is associated with high mortality and prolonged hospital stay. The intestinal epithelial cell dysfunction is regarded as the "engine" of deteriorated ET. Although electroacupuncture (EA) can mitigate endotoxin-induced intestinal epithelial cell dysfunction in ET, the mechanism through which EA improves endotoxin-induced intestinal injury for preventing ET deterioration needs further investigation. Methods: An in vivo ET model was developed by injecting lipopolysaccharide (LPS) in wild-type and PINK1-knockout mice. An in vitro model was also established by incubating epithelial cells in the serum samples obtained from both groups of mice. Hemin and zinc protoporphyrin IX (ZnPP) were applied to activate or inhibit heme oxygenase 1 (HO-1) production. EA treatment was performed for 30 min consecutively for 5 days before LPS injection, and on the day of the experiment, EA was performed throughout the process. Samples were harvested at 6 h after LPS induction for analyzing tissue injury, oxidative stress, ATP production, activity of diamine oxidase (DAO), and changes in the levels of HO-1, PTEN-induced putative kinase 1 (PINK1), mitochondrial fusion and fission marker gene, caspase-1, and interleukin 1 beta (IL-1ß). Results: In the wild-type models (both in vivo and vitro), EA alleviated LPS-induced intestinal injury and mitochondrial dysfunction, as indicated by decreased reactive oxygen species (ROS) production and oxygen consumption rate (OCR) and reduced levels of mitochondrial fission proteins. EA treatment also boosted histopathological morphology, ATP levels, DAO activity, and levels of mitochondrial fusion proteins in vivo and vitro. The effect of EA was enhanced by hemin but suppressed by Znpp. However, EA + AP, Znpp, or hemin had no effects on the LPS-induced, PINK1-knocked out mouse models. Conclusion: EA may improve the HO-1/PINK1 pathway-mediated mitochondrial dynamic balance to protect the intestinal barrier in patients with ET.

Amphiphilic Dendrimer Doping Enhanced pH-Sensitivity of Liposomal Vesicle for Effective Co-delivery toward Synergistic Ferroptosis-Apoptosis Therapy of Hepatocellular Carcinoma.

Ferroptosis, characterized by the accumulation of reactive oxygen species and lipid peroxides, has emerged as an attractive strategy to reverse drug resistance. Of particular interest is the ferroptosis-apoptosis combination therapy for cancer treatment. Herein, a nanoplatform is reported for effective co-delivery of the anticancer drug sorafenib (S) and the ferroptosis inducer hemin (H), toward synergistic ferroptosis-apoptosis therapy of advanced hepatocellular carcinoma (HCC) as a proof-of-concept study. Liposome is an excellent delivery system; however, it is not sufficiently responsive to the acidic tumor microenvironment (TME) for tumor-targeted drug delivery. The pH-sensitive vesicles are therefore developed (SH-AD-L) by incorporating amphiphilic dendrimers (AD) into liposomes for controlled and pH-stimulated release of sorafenib and hemin in the acidic TME, thanks to the protonation of numerous amine functionalities in AD. Importantly, SH-AD-L not only blocked glutathione synthesis to disrupt the antioxidant system, but also increased intracellular Fe2+ and ·OH concentrations to amplify oxidative stress, both of which contribute to enhanced ferroptosis. Remarkably, high levels of ·OH also augmented sorafenib-mediated apoptosis in tumor cells. This study demonstrates the efficacy of ferroptosis-apoptosis combination therapy, as well as the promise of the AD-doped TME-responsive vesicles for drug delivery in combination therapy to treat advanced HCC.

Preclinical pharmacokinetics, biodistribution, excretion, and plasma protein binding study of 58Fe-labeled hemin by ICP-MS.

BACKGROUND: Hemin, a stable form of heme iron, is a potential iron supplement for the treatment of iron deficiency. To date, the pharmacokinetics and in vivo ADME properties of hemin are to be elucidated. METHODS: In this study, a rapid, sensitive, and validated inductively coupled plasma mass spectrometry (ICP-MS) method was used in combination with 58Fe stable isotope labeling to systemically investigate the plasma pharmacokinetics, biodistribution, excretion, and plasma binding profiles of hemin in animals. RESULTS: Results showed that the ICP-MS method is accurate and sensitive enough to quantitatively determine the in vivo disposition process of 58Fe derived from 58Fe-labeled hemin. Following intra-gastric administration, 58Fe was rapidly absorbed in gastrointestinal tract, with Cmax of 41.1 ± 23.1 ng/mL, Tmax of 1.38 ± 0.48 h, and bioavailability of 1.12 ± 0.45 % in beagle dogs. Moreover, 58Fe was distributed to various organs including stomach, small intestine, spleen, and liver, within a few hours after intra-gastric administration in rats. Excretion of 58Fe in rats was predominantly via feces (76.3 ± 15.1 % of dosage), whereas minimally via urine (0.14 ± 0.08 % of dosage). Protein binding study revealed majority of 58Fe in plasma was bound to proteins, with average binding rates of 81.0 % and 92.7 % in human and rat plasma, respectively. CONCLUSION: In conclusion, the present study validated the work-flow of preclinical pharmacokinetic studies of iron-containing drug candidates with using ICP-MS and stable (trace) isotope labeling strategy. It also provided useful information to support the further development of hemin as a drug/nutrition supplement candidate.

Protective effect of heme chloride on hypoxia-induced tissue injury in mice. / æ°¯åŒ–è¡€çº¢ç´ å¯¹ç¼ºæ°§å¯¼è‡´å°é¼ ç»„ç»‡æŸä¼¤çš„ä¿æŠ¤ä½œç”¨.

OBJECTIVES: Heme chloride (Hemin) is an in vitro purified form of natural heme and an important raw material for anti-anemia and antitumor drugs. This study aims to analyze the protective effect of Hemin on tissue damage in low-pressure oxygen chamber simulated plateau hypoxic mice, and explore its role in anti-plateau hypoxia. METHODS: Thirty male BALB/c mice were randomly divided into a blank group, a positive drug group (acetazolomide, 200 mg/kg), a Hemin low-dose group (15 mg/kg), a Hemin medium-dose group (30 mg/kg), and a Hemin high-dose group (60 mg/kg) with intraperitoneal injection. The anti-hypoxic activity of Hemin was explored by atmospheric closed hypoxia experiment and the optimal dose was screened. Thirty-six male BALB/c mice were randomly divided into a blank group, a hypoxia group, a positive drug group, and a Hemin high-dose group. The plasma inflammatory factor levels and oxidative stress indicators malondialdehyde (MDA), glutataione (GSH), and superoxide dismutase (SOD) levels of myocardium, brain, lung, and liver tissues were measured in different groups with hypoxia for 24 h. The degree of histopathological damage of mice was observed with HE staining. The degree of protection of Hemin against tissue hypoxia injury was detected with the hypoxia probe piperidazole. RESULTS: Compared with the blank group, the survival time of mice in the positive drug group, the Hemin medium-dose group, and high-dose group was significantly extended (all P<0.05), with the highest prolongation rate in the Hemin high-dose group. Compared with the hypoxia group, mice in the Hemin high-dose group showed a significant increase in SOD level and GSH content of brain tissue, and a significant decrease in MDA content of lung tissue (all P<0.05). The results of HE staining and hypoxia probe showed that Hemin had a significant protective effect on the damage of liver, heart, brain and lung tissues of mice with hypoxia, and the most obvious effect on that of the brain tissue. CONCLUSIONS: Hemin has an effect of improvement on oxidative stress and inflammatory response caused by hypoxia, and has obvious protective effect on tissue damage caused by hypoxia.

Oxygen-boosted biomimetic nanoplatform for synergetic phototherapy/ferroptosis activation and reversal of immune-suppressed tumor microenvironment.

Photodynamic therapy (PDT) induces apoptosis of cancer cells by generating cytotoxic reactive oxygen species, the therapeutic effect of which, however, is impeded by intrinsic/inducible apoptosis-resistant mechanisms in cancer cells and hypoxia of tumor microenvironment (TME); also, PDT-induced anti-tumor immunity activation is insufficient. To deal with these obstacles, a novel biomimetic nanoplatform is fabricated for the precise delivery of photosensitizer chlorin e6 (Ce6), hemin and PEP20 (CD47 inhibitory peptide), integrating oxygen-boosted PDT, ferroptosis activation and CD47-SIRPα blockade. Hemin's catalase-mimetic activity alleviates TME hypoxia and enhances PDT. The nanoplatform activates ferroptosis via both classical (down-regulating glutathione peroxidase 4 pathway) and non-classical (inducing Fe2+ overload) modes. Besides the role of hemin in consuming glutathione and up-regulating heme oxygenase-1 expression, interestingly, we observe that Ce6 enhance ferroptosis activation via both classical and non-classical modes. The anti-cancer immunity is reinforced by combining PEP20-mediated CD47-SIRPα blockade and PDT-mediated T cell activation, efficiently suppressing primary tumor growth and metastasis. PEP20 has been revealed for the first time to sensitize ferroptosis by down-regulating system Xc-. This work sheds new light on the mechanisms of PDT-ferroptosis activation interplay and bridges immunotherapy and ferroptosis activation, laying the theoretical foundation for novel combinational modes of cancer treatment.

Epigallocatechin-3-gallate suppresses hemin-aggravated colon carcinogenesis through Nrf2-inhibited mitochondrial reactive oxygen species accumulation.

BACKGROUND: Previous studies have presented evidence to support the significant association between red meat intake and colon cancer, suggesting that heme iron plays a key role in colon carcinogenesis. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, exhibits anti-oxidative and anti-cancer effects. However, the effect of EGCG on red meat-associated colon carcinogenesis is not well understood. OBJECTIVES: We aimed to investigate the regulatory effects of hemin and EGCG on colon carcinogenesis and the underlying mechanism of action. METHODS: Hemin and EGCG were treated in Caco2 cells to perform the water-soluble tetrazolium salt-1 assay, lactate dehydrogenase release assay, reactive oxygen species (ROS) detection assay, real-time quantitative polymerase chain reaction and western blot. We investigated the regulatory effects of hemin and EGCG on an azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colon carcinogenesis mouse model. RESULTS: In Caco2 cells, hemin increased cell proliferation and the expression of cell cycle regulatory proteins, and ROS levels. EGCG suppressed hemin-induced cell proliferation and cell cycle regulatory protein expression as well as mitochondrial ROS accumulation. Hemin increased nuclear factor erythroid-2-related factor 2 (Nrf2) expression, but decreased Keap1 expression. EGCG enhanced hemin-induced Nrf2 and antioxidant gene expression. Nrf2 inhibitor reversed EGCG reduced cell proliferation and cell cycle regulatory protein expression. In AOM/DSS mice, hemin treatment induced hyperplastic changes in colon tissues, inhibited by EGCG supplementation. EGCG reduced the hemin-induced numbers of total aberrant crypts and malondialdehyde concentration in the AOM/DSS model. CONCLUSIONS: We demonstrated that EGCG reduced hemin-induced proliferation and colon carcinogenesis through Nrf2-inhibited mitochondrial ROS accumulation.

Lead exposure represses mitochondrial metabolism by activation of heme-binding protein BACH1 in differentiated SH-SY5Y cell.

Exposure to lead (Pb), a known toxin causing developmental neurotoxicity, can impair neurogenesis and oxidative phosphorylation (OXPHOS), but the mechanism is not clarified. In the current study, we aim to explore the effects of Pb on the differentiation of SH-SY5Y cells and investigate the role of heme and heme-binding protein BACH1 during differentiation. We found that Pb exposure caused a shift from OXPHOS to glycolysis, resulting in neurogenesis impairment by decreasing neurite growth and downregulation of PSD95 and Synapsin-1 in differentiated SH-SY5Y cells. Heme reduction mediated this mitochondria metabolism repression caused by Pb depending on BACH1 activation. Hemin supplement alleviated Pb-induced OXPHOS damage and adenosine triphosphate (ATP) reduction in differentiated SH-SY5Y cells, and further protected for Pb-induced damage of synapse. Heme binding factor BACH1 was negatively regulated by heme content and BACH1 knockout rescued the Pb-induced transcription and expression decline of genes related to OXPHOS and abrogated Pb-induced growth inhibition of axon promotion and synapse formation. Collectively, the present study demonstrates that heme deficiency mediates OXPHOS damage caused by Pb through BACH1 activation, resulting in neurogenesis impairment.

Establishment of an Improved ELONA Method for Detecting Fumonisin B1 Based on Aptamers and Hemin-CDs Conjugates.

Fumonisin B1 (FB1) is a strong mycotoxin that is ubiquitous in agricultural products. The establishment of rapid detection methods is an important means to prevent and control FB1 contamination. In this study, an improved enzyme-linked oligonucleotide assay (ELONA) method was designed and tested to detect the contents of FB1 in maize (corn) samples. F10 modified with biotin was bound to an enzyme label plate that was coated with streptavidin (SA) in advance, and carbon dots (CDs) were used to catalyze the color of tetramethylbenzidine (TMB). The complementary chain of F10 was modified with an amino group and coupled with CDs to obtain conjugates. The sample and conjugates were then added to the enzyme plate coated with F10 (an FB1 aptamer). Upon completion of the color reaction, the absorbance was measured at 450 nm. The LOD of this method was 4.30 ng/mL and the LOQ was 13.03 ng/mL. We observed a linear relationship in the FB1 concentration range of 0-100 ng/mL. The standard curve was y = -0.001482 × x + 0.3463, R2 = 0.9918, and the experimental results could be directly measured visually. The recovery of the maize sample was 97.5-99.23% and 94.54-99.25%, and the total detection time was 1 h.

Hemin-incorporating DNA nanozyme enabling catalytic oxygenation and GSH depletion for enhanced photodynamic therapy and synergistic tumor ferroptosis.

Photodynamic therapy (PDT) has emerged as a promising tumor treatment method via light-triggered generation of reactive oxygen species (ROS) to kill tumor cells. However, the efficacy of PDT is usually restricted by several biological limitations, including hypoxia, excess glutathione (GSH) neutralization, as well as tumor resistance. To tackle these issues, herein we developed a new kind of DNA nanozyme to realize enhanced PDT and synergistic tumor ferroptosis. The DNA nanozyme was constructed via rolling circle amplification, which contained repeat AS1411 G quadruplex (G4) units to form multiple G4/hemin DNAzymes with catalase-mimic activity. Both hemin, an iron-containing porphyrin cofactor, and chlorine e6 (Ce6), a photosensitizer, were facilely inserted into G4 structure with high efficiency, achieving in-situ catalytic oxygenation and photodynamic ROS production. Compared to other self-oxygen-supplying tools, such DNA nanozyme is advantageous for high biological stability and compatibility. Moreover, the nanostructure could achieve tumor cells targeting internalization and intranuclear transport of Ce6 by virtue of specific nucleolin binding of AS1411. The nanozyme could catalyze the decomposition of intracellular H2O2 into oxygen for hypoxia relief as evidenced by the suppression of hypoxia-inducible factor-1α (HIF-1α), and moreover, GSH depletion and cell ferroptosis were also achieved for synergistic tumor therapy. Upon intravenous injection, the nanostructure could effectively accumulate into tumor, and impose multi-modal tumor therapy with excellent biocompatibility. Therefore, by integrating the capabilities of O2 generation and GSH depletion, such DNA nanozyme is a promising nanoplatform for tumor PDT/ferroptosis combination therapy.

A G-quadruplex/hemin structure-undamaged method to inhibit peroxidase-mimic DNAzyme activity for biosensing development.

Damaging the structure of the G-quadruplex (G4) to prevent the formation of the G4/hemin complex is presently the only available method to inhibit the activity of the peroxidase-mimic DNAzyme. In this study, a unique intramolecular inhibitory effect of the adjacent base-pair (InE(N:N)), by installing a rationally adjacent base-pair of the G4 core sequence, is proposed for the inhibition of the DNAzyme activity, which eliminates the need to damage the entire G4 structure. Various base pairs show different abilities to inhibit DNAzyme activity. The adjacent adenine: thymine pair possesses the best inhibitory efficiency (17 times). Through detailed investigations of the InE(N:N), it was revealed that the adjacent adenine: thymine pair downregulated the formation of compound I in the catalytic process, thus inhibiting the G4 DNAzyme activity. The mechanism of inhibition indicated that the carbonyl group on the hexatomic ring of the complementary base played an important role. To further reflect the advantages of the proposed strategy, two InE(N:N)-based biosensors were developed for DNA analysis and Uracil-DNA glycosylase (UDG) detection. Compared with existing DNAzyme-based methods, the application of InE(N: N) facilitates the real-time assay and simplifies the design difficulty. Therefore, InE(N:N) provides new insights into the regulation of the DNAzyme activity and offers an efficient approach for the future application of DNAzyme.

Hemin-loaded black phosphorus-based nanosystem for enhanced photodynamic therapy and a synergistic photothermally/photodynamically activated inflammatory immune response.

The biocompatible nanosystem integrating hemin into black phosphorus nanosheets was ingeniously constructed through the easy modified strategy. Taking advantage of the enhanced permeability and retention (EPR) effect, the designed nanosystem could accumulate into the tumor location, leading to attractive cytotoxicity through the enhanced photodynamic therapy (PDT) ascribing to the catalytic oxygen supply and GSH depletion of hemin. Simultaneously, combining PDT and photothermal therapy (PTT) showed an apparent promotion in anti-tumor effect. Moreover, inflammatory response and immune activation amplified anti-tumor effect, which could compensate limitations of exogenous therapy (i.e., limited tissue depth and intensity-dependent curation effect) and potentiate the efficiency of the endogenous immune-activating behavior. Especially, the designed nanosystem degraded followed by being metabolized in the blood circulation. By and large, this constructed nanosystem provides the new insight into designing biocompatible nanomaterials and paves the ideal way for anti-tumor therapy. STATEMENT OF SIGNIFICANCE: Biocompatible nanomaterials-based synergistic tumor therapy offers the potential application prospect. Taking advantage of degradable black phosphorus, the nanosystem integrating hemin into black phosphorus for the enhanced photodynamic therapy and synergistic photothermal-photodynamic activating inflammation-immune response was developed and the results demonstrate that tumor growth was inhibited followed by activating inflammatory factors and leading to satisfactory immune response.

Protein nitration induced by Hemin/NO: A complementary mechanism through the catalytic functions of hemin and NO-scavenging.

Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (●NO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the ●NO-binding affinity of hemin has been studied along with the influence of ●NO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and ●NO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its ●NO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of ●NO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and ●NO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.

Insertion of Hemin into Metal-Organic Frameworks: Mimicking Natural Peroxidase Microenvironment for the Rapid Ultrasensitive Detection of Uranium.

Constructing enzyme-like active sites in mimic enzyme systems is critical for achieving catalytic performances comparable to natural enzymes and can shed light on the natural development of enzymes. In this study, we described a specific hemin-based mimetic enzyme, which was facilely synthesized by the assembly of zeolitic imidazolate framework-l (ZIF-l) and hemin. The obtained hemin-based mimetic enzyme (denoted as ZIF-l-hemin) displayed enhanced peroxidase activity compared to free hemin in solution. Such excellent activity originated from the ZIF-l framework mimicking the active site cavity microenvironment of horseradish peroxidase in terms of axially coordinated histidine and distal histidine. Additionally, the constructed peroxidase mimetic was extremely resistant to a variety of severe circumstances that would normally denature natural enzymes. These characteristics made ZIF-l-hemin a potential platform for the colorimetric sensor of uranium (UO22+) with wide linear ranges (0.25-40 µM) and low limits of detection (0.079 µM). Moreover, the detection mechanism demonstrated that the coordination of uranyl ion with imidazole of ZIF-l-hemin reduced the catalytic efficiency of ZIF-l-hemin. The current work not only proposed a novel approach for fabricating artificial peroxidase but also offered facile colorimetric methods for selective radionuclide detection.

Identification and Tetramer Structure of Hemin-Binding Protein SPD_0310 Linked to Iron Homeostasis and Virulence of Streptococcus pneumoniae.

Iron and iron-containing compounds are essential for bacterial virulence and host infection. Hemin is an important supplement compound for bacterial survival in an iron-deficient environment. Despite strong interest in hemin metabolism, the detailed mechanism of hemin transportation in Gram-positive bacteria is yet to be reported. The results of our study revealed that the homologous proteins of SPD_0310 were significantly conservative in Gram-positive bacteria (P < 0.001), and these proteins were identified as belonging to an uncharacterized protein family (UPF0371). The results of thermodynamic and kinetic studies have shown that SPD_0310 has a high hemin-binding affinity. Interestingly, we found that the crystal structure of SPD_0310 presented a homotetramer conformation, which is required for hemin binding. SPD_0310 can interact with many hemin-binding proteins (SPD_0090, SPD_1609, and GAPDH) located on the cell surface, which contributes to hemin transfer to the cytoplasm. It also has a high affinity with other iron transporters in the cytoplasm (SPD_0226 and SPD_0227), which facilitates iron redistribution in cells. More importantly, the knockout of the spd_0310 gene (Δspd_0310) resulted in a decrease in the iron content and protein expression levels of many bacterial adhesion factors. Moreover, the animal model showed that the Δspd_0310 strain has a lower virulence than the wild type. Based on the crystallographic and biochemical studies, we inferred that SPD_0310 is a hemin intermediate transporter which contributes to iron homeostasis and further affects the virulence of Streptococcus pneumoniae in the host. Our study provides not only an important theoretical basis for the in-depth elucidation of the hemin transport mechanism in bacteria but also an important candidate target for the development of novel antimicrobial agents based on metal transport systems. IMPORTANCE Iron is an essential element for bacterial virulence and infection of the host. The detailed hemin metabolism in Gram-positive bacteria has rarely been studied. SPD_0310 belongs to the UPF0371 family of proteins, and results of homology analysis and evolutionary tree analysis suggested that it was widely distributed and highly conserved in Gram-positive bacteria. However, the function of the UPF0371 family remains unknown. We successfully determined the crystal structure of apo-SPD_0310, which is a homotetramer. We found that cytoplasmic protein SPD_0310 with a special tetramer structure has a strong hemin-binding ability and interacts with many iron transporters, which facilitates hemin transfer from the extracellular space to the cytoplasm. The results of detailed functional analyses indicated that SPD_0310 may function as a hemin transporter similar to hemoglobin in animals and contributes to bacterial iron homeostasis and virulence. This study provides a novel target for the development of antimicrobial drugs against pathogenic Gram-positive bacteria.

Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron.

BACKGROUND: In transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. RESULTS: LIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DISCUSSION: The difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

Heme oxygenase-1/carbon monoxide signaling participates in the accumulation of triterpenoids of Ganoderma lucidum.

Ganoderic triterpenoids (GTs) are the primary bioactive constituents of the Basidiomycotina fungus, Ganoderma lucidum. These compounds exhibit antitumor, anti-hyperlipidemic, and immune-modulatory pharmacological activities. This study focused on GT accumulation in mycelia of G. lucidum mediated by the heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling. Compared with the control, hemin (10 µmol/L) induced an increase of 60.1% in GT content and 57.1% in HO-1 activity. Moreover, carbon monoxide-releasing molecule-2 (CORM-2), CO donor, increased GT content by 56.0% and HO-1 activity by 18.1%. Zn protoporphyrin IX (ZnPPIX), a specific HO-1 inhibitor, significantly reduced GT content by 26.0% and HO-1 activity by 15.8%, while hemin supplementation reversed these effects. Transcriptome sequencing showed that HO-1/CO could function directly as a regulator involved in promoting GT accumulation by regulating gene expression in the mevalonate pathway, and modulating the reactive oxygen species (ROS) and Ca2+ pathways. The results of this study may help enhance large-scale GT production and support further exploration of GT metabolic networks and relevant signaling cross-talk.