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Journal of Inorganic Biochemistry Sep 2024Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental...
Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O binding modulates activity of diguanylate cyclase-containing GCS proteins.
Topics: Phosphorus-Oxygen Lyases; Heme; Bacterial Proteins; Pectobacterium carotovorum; Protein Conformation; Oxygen; Cyclic GMP; Escherichia coli Proteins
PubMed: 38878680
DOI: 10.1016/j.jinorgbio.2024.112638 -
Redox Biology Aug 2024Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are at the genesis of placental disorders observed in preeclampsia, intrauterine growth restriction, and...
Manganese porphyrin-based treatment improves fetal-placental development and protects against oxidative damage and NLRP3 inflammasome activation in a rat maternal hypothyroidism model.
Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are at the genesis of placental disorders observed in preeclampsia, intrauterine growth restriction, and maternal hypothyroidism. In this regard, cationic manganese porphyrins (MnPs) comprise potent redox-active therapeutics of high antioxidant and anti-inflammatory potential, which have not been evaluated in metabolic gestational diseases yet. This study evaluated the therapeutic potential of two MnPs, [MnTE-2-PyP] (MnP I) and [MnT(5-Br-3-E-Py)P]5+ (MnP II), in the fetal-placental dysfunction of hypothyroid rats. Hypothyroidism was induced by administration of 6-Propyl-2-thiouracil (PTU) and treatment with MnPs I and II 0.1 mg/kg/day started on the 8th day of gestation (DG). The fetal and placental development, and protein and/or mRNA expression of antioxidant mediators (SOD1, CAT, GPx1), hypoxia (HIF1α), oxidative damage (8-OHdG, MDA), ERS (GRP78 and CHOP), immunological (TNFα, IL-6, IL-10, IL-1β, IL-18, NLRP3, Caspase1, Gasdermin D) and angiogenic (VEGF) were evaluated in the placenta and decidua on the 18th DG using immunohistochemistry and qPCR. ROS and peroxynitrite (PRX) were quantified by fluorometric assay, while enzyme activities of SOD, GST, and catalase were evaluated by colorimetric assay. MnPs I and II increased fetal body mass in hypothyroid rats, and MnP I increased fetal organ mass. MnPs restored the junctional zone morphology in hypothyroid rats and increased placental vascularization. MnPs blocked the increase of OS and ERS mediators caused by hypothyroidism, showing similar levels of expression of HIFα, 8-OHdG, MDA, Gpx1, GRP78, and Chop to the control. Moreover, MnPs I and/or II increased the protein expression of SOD1, Cat, and GPx1 and restored the expression of IL10, Nlrp3, and Caspase1 in the decidua and/or placenta. However, MnPs did not restore the low placental enzyme activity of SOD, CAT, and GST caused by hypothyroidism, while increased the decidual and placental protein expression of TNFα. The results show that treatment with MnPs improves the fetal-placental development and the placental inflammatory state of hypothyroid rats and protects against oxidative stress and reticular stress caused by hypothyroidism at the maternal-fetal interface.
Topics: Animals; Pregnancy; Female; Rats; Hypothyroidism; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Inflammasomes; Disease Models, Animal; Placenta; Placentation; Antioxidants; Endoplasmic Reticulum Stress; Fetal Development; Manganese; Metalloporphyrins; Endoplasmic Reticulum Chaperone BiP
PubMed: 38870780
DOI: 10.1016/j.redox.2024.103238 -
Analytical Chemistry Jun 2024Biomimetic cytochrome P450 for chemical activation of environmental carcinogens is an efficient model for evaluating their mutagenicity and ultimately acquiring the...
Biomimetic cytochrome P450 for chemical activation of environmental carcinogens is an efficient model for evaluating their mutagenicity and ultimately acquiring the metabolites that cannot be easily accessed by conventional routes of organic synthesis. Different kinds of mutagen derived from polyaromatic hydrocarbons (PAHs) by metalloporphyrin/oxidant model systems have been reported, but the underlying molecular mechanisms are poorly understood. Herein, we have for the first time demonstrated an effective surface-enhanced Raman scattering (SERS) protocol to study the dynamics and biomimetic metabolic behaviors of pyrene (Pyr) in the presence of various oxygen donors. Quantitative information on the relative concentration of Pyr and its metabolites in the biomimetic system can be extracted from the SERS spectra. On the basis of our results, we conclude that the oxidative metabolism of Pyr is highly influenced by the types and concentrations of oxygen donors, leading to the formation of 1-hydroxypyrene and dioxygenated products. Besides, the addition of an appropriate amount of an organic solvent can promote the formation of secondary oxidation products. These results offer valuable insights into the dynamics of PAHs metabolism and the regulation of their metabolic pathways in biomimetic activation. In comparison to traditional liquid chromatography-mass spectrometry, the present SERS approach is more suitable for high-throughput evaluation of the metabolic process and kinetics of PAHs. We anticipate that this approach will enable a more general and comprehensive tracking of metabolic dynamics and molecular mechanisms involved in the biomimetic activation of other xenobiotics, such as procarcinogens, promutagens, and drugs.
Topics: Spectrum Analysis, Raman; Kinetics; Pyrenes; Biomimetics; Polycyclic Aromatic Hydrocarbons; Biomimetic Materials; Surface Properties; Activation, Metabolic; Cytochrome P-450 Enzyme System; Oxidation-Reduction
PubMed: 38869249
DOI: 10.1021/acs.analchem.4c01437 -
The Journal of Physical Chemistry. B Jun 2024Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated -type ferric heme were...
Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated -type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast -1 cytochrome to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.
Topics: Molecular Dynamics Simulation; Lysine; Cytochromes c; Heme; Density Functional Theory; Saccharomyces cerevisiae; Ligands; Saccharomyces cerevisiae Proteins
PubMed: 38864552
DOI: 10.1021/acs.jpcb.4c00625 -
ACS Applied Materials & Interfaces Jun 2024Photothermal therapy (PTT) is a promising clinical antitumor strategy. However, local hyperthermia inevitably induces heat damage to adjacent normal tissues, while...
Photothermal therapy (PTT) is a promising clinical antitumor strategy. However, local hyperthermia inevitably induces heat damage to adjacent normal tissues, while alternative mild-temperature therapy (MPTT, < 45 °C) is also inefficient due to the overexpressed hyperthermia-induced heat shock proteins (HSPs) by cancer cells. Therefore, developing PTT strategies with minimizing damage to healthy tissues with improved cellular temperature sensitivity is extremely valuable for clinical application. Herein, we proposed the strategy of disrupting the intracellular redox environment via destroying the ROS-defending systems to promote MPTT. The gold(III) porphyrin-Fe-tannic acid nanocomplexes (AuTPP@TA-Fe NPs) were achieved via interfacial cohesion and supramolecular assembly of bioadhesive species, which could trigger the Fenton reaction to produce ·OH radicals and downregulation of reductive TrxR enzyme and mitochondrial chaperone protein Hsp60. The aggravation of oxides and the inactivation of Hsp60 provide favorable pathways for impeding the heat shock-induced self-repair mechanism of cancer cells, which strengthens AuTPP@TA-Fe NPs mediated MPTT.
Topics: Humans; Gold; Oxidation-Reduction; Photothermal Therapy; Porphyrins; Animals; Chaperonin 60; Mice; Cell Line, Tumor; Metalloporphyrins; Neoplasms; Metal Nanoparticles
PubMed: 38850233
DOI: 10.1021/acsami.4c04196 -
Structure (London, England : 1993) Jun 2024In a recent issue of Nature, Barends et al. studied the photodissociation of carboxymyoglobin with ultrafast laser pump-probe serial femtosecond crystallography...
In a recent issue of Nature, Barends et al. studied the photodissociation of carboxymyoglobin with ultrafast laser pump-probe serial femtosecond crystallography experiments. They observed significant differences in heme protein structural dynamics for biologically relevant 1-photon excitation relative to high excitation leading to the absorption of several photons per heme.
Topics: Myoglobin; Protein Conformation; Heme; Crystallography, X-Ray; Light; Photons; Models, Molecular
PubMed: 38848681
DOI: 10.1016/j.str.2024.05.008 -
Nature Biotechnology Jun 2024Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining...
Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L and 21.3 g L, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.
PubMed: 38839873
DOI: 10.1038/s41587-024-02267-3 -
Analytica Chimica Acta Jul 2024Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory...
BACKGROUND
Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin.
RESULTS
Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 μM) and hemoglobin (∼0.002-17 μM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme.
SIGNIFICANCE
Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.
Topics: Hemolysis; Heme; Hemoglobins; Humans; Animals; Swine; Chromatography, High Pressure Liquid
PubMed: 38834280
DOI: 10.1016/j.aca.2024.342766 -
Physical Chemistry Chemical Physics :... Jun 2024The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several...
The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several biochemical processes. Human cystathionine β-synthase is a heme-containing, pyridoxal 5'-phosphate (PLP)-dependent enzyme found in this pathway. The heme group does not participate directly in catalysis, but has a regulatory function, whereby CO or NO binding inhibits the PLP-dependent reactions. In this study, we explore the detailed structural changes responsible for inhibition using quantum chemical calculations to validate the experimentally observed bonding patterns associated with heme CO and NO binding and molecular dynamics simulations to explore the medium-range structural changes triggered by gas binding and propagating to the PLP active site, which is more than 20 Å distant from the heme group. Our results support a previously proposed mechanical signaling model, whereby the cysteine decoordination associated with gas ligand binding leads to breaking of a hydrogen bond with an arginine residue on a neighbouring helix. In turn, this leads to a shift in position of the helix, and hence also of the PLP cofactor, ultimately disrupting a key hydrogen bond that stabilizes the PLP in its catalytically active form.
Topics: Cystathionine beta-Synthase; Molecular Dynamics Simulation; Humans; Pyridoxal Phosphate; Gases; Nitric Oxide; Hydrogen Bonding; Carbon Monoxide; Heme; Catalytic Domain; Quantum Theory; Cysteine
PubMed: 38832404
DOI: 10.1039/d4cp01321b -
Mikrochimica Acta Jun 2024CdInS and zinc tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) were synthesized by hydrothermal method, and an organic dye-sensitized inorganic semiconductor ZnTCPP/CdInS...
CdInS and zinc tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) were synthesized by hydrothermal method, and an organic dye-sensitized inorganic semiconductor ZnTCPP/CdInS type II heterojunction was constructed on a fluorine-doped tin oxide (FTO) substrate electrode. A sandwich immunostructure for signal-attenuation photoelectrochemical (PEC) detection of cardiac troponin I (cTnI) was constructed using the ZnTCPP/CdInS/FTO photoanode and a horseradish peroxidase (HRP)-ZnFeO-Ab-bovine serum albumin (BSA) immunolabeling complex. The bioenzyme HRP and the HRP-like nanozyme ZnFeO can co-catalyze the oxidation of 4-chloro-1-naphthol (4-CN) by HO to produce an insoluble precipitate on the photoanode, thus notably reducing the anodic photocurrent for quantitative determination of cTnI. Under the optimal conditions, the photocurrent at 0 V vs. SCE in 0.1 M phosphate buffer solution (pH 7.40) containing 0.1 M ascorbic acid was linear with the logarithm of cTnI concentration from 500 fg mL to 50.0 ng mL, and the limit of detection (LOD, S/N = 3) is 0.15 pg mL. Spiked recoveries were 95.1% ~ 104% for assay of cTnI in human serum samples.
Topics: Troponin I; Humans; Electrochemical Techniques; Immunoassay; Limit of Detection; Tin Compounds; Catalysis; Horseradish Peroxidase; Naphthols; Metalloporphyrins; Electrodes; Hydrogen Peroxide; Serum Albumin, Bovine; Photochemical Processes; Animals; Biosensing Techniques; Semiconductors; Cattle; Sulfides; Porphyrins
PubMed: 38831034
DOI: 10.1007/s00604-024-06436-8