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Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs.
Holt ME, Salay LE, O'Brien E, Barton JK, Chazin WJ
(2018) PLoS One 13: e0209345
MeSH Terms: Binding Sites, Circular Dichroism, Crystallography, X-Ray, DNA, DNA Primase, Molecular Docking Simulation, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Protein Binding, Protein Domains, Protein Structure, Secondary, RNA
Show Abstract · Added March 26, 2019
The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C272-464 construct used for that study and that of a N-terminally shifted p58C266-456 construct and consequently, an assumption that p58C272-464 has abnormal physical and functional properties. To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.
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12 MeSH Terms
Yeast require redox switching in DNA primase.
O'Brien E, Salay LE, Epum EA, Friedman KL, Chazin WJ, Barton JK
(2018) Proc Natl Acad Sci U S A 115: 13186-13191
MeSH Terms: Crystallography, X-Ray, DNA Primase, Electron Transport, Iron-Sulfur Proteins, Models, Molecular, Mutation, Oxidation-Reduction, Protein Conformation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 26, 2019
Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S] species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.
Copyright © 2018 the Author(s). Published by PNAS.
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Substrate Binding Regulates Redox Signaling in Human DNA Primase.
O'Brien E, Holt ME, Salay LE, Chazin WJ, Barton JK
(2018) J Am Chem Soc 140: 17153-17162
MeSH Terms: DNA, DNA Primase, Electrochemical Techniques, Humans, Iron-Sulfur Proteins, Nucleotides, Oxidation-Reduction, Protein Binding, Protein Domains, Transcription Elongation, Genetic, Transcription Initiation, Genetic
Show Abstract · Added March 26, 2019
Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.
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11 MeSH Terms
The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS.
Maklashina E, Rajagukguk S, Iverson TM, Cecchini G
(2018) J Biol Chem 293: 7754-7765
MeSH Terms: Bacterial Proteins, Catalysis, Crystallography, X-Ray, Electron Transport Complex II, Escherichia coli, Flavoproteins, Humans, Models, Molecular, Oxidation-Reduction, Protein Conformation, Protein Subunits, Reactive Oxygen Species
Show Abstract · Added April 1, 2019
Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.
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A Metabolic Basis for Endothelial-to-Mesenchymal Transition.
Xiong J, Kawagishi H, Yan Y, Liu J, Wells QS, Edmunds LR, Fergusson MM, Yu ZX, Rovira II, Brittain EL, Wolfgang MJ, Jurczak MJ, Fessel JP, Finkel T
(2018) Mol Cell 69: 689-698.e7
MeSH Terms: 3-Hydroxyacyl CoA Dehydrogenases, Acetyl Coenzyme A, Acetyl-CoA C-Acyltransferase, Animals, Carbon-Carbon Double Bond Isomerases, Carnitine O-Palmitoyltransferase, Cells, Cultured, Endothelium, Vascular, Enoyl-CoA Hydratase, Epithelial-Mesenchymal Transition, Fatty Acids, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Oxidation-Reduction, Racemases and Epimerases, Signal Transduction, Transforming Growth Factor beta
Show Abstract · Added March 14, 2018
Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2 mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.
Copyright © 2018 Elsevier Inc. All rights reserved.
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20 MeSH Terms
Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.
Albertolle ME, Phan TTN, Pozzi A, Guengerich FP
(2018) Mol Cell Proteomics 17: 889-900
MeSH Terms: Animals, Biocatalysis, Cysteine, Cytochrome P-450 Enzyme System, Humans, Hydrogen Peroxide, Kidney, Mice, Transgenic, Microsomes, Liver, Oxidation-Reduction, Pharmaceutical Preparations, Recombinant Proteins, Staining and Labeling, Sulfenic Acids, Sulfhydryl Compounds
Show Abstract · Added March 14, 2018
The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. (2017) 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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15 MeSH Terms
Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.
Kakimoto K, Murayama N, Takenaka S, Nagayoshi H, Lim YR, Kim V, Kim D, Yamazaki H, Komori M, Guengerich FP, Shimada T
(2019) Xenobiotica 49: 131-142
MeSH Terms: Chromatography, Liquid, Cytochrome P-450 CYP2A6, Cytochrome P-450 Enzyme System, Flavanones, Flavones, Humans, Kinetics, Mass Spectrometry, Molecular Docking Simulation, Oxidation-Reduction
Show Abstract · Added March 14, 2018
1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min and 1.3 min, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.
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10 MeSH Terms
Nicotine Adenine Dinucleotides: The Redox Currency of the Cell.
Fessel JP, Oldham WM
(2018) Antioxid Redox Signal 28: 165-166
MeSH Terms: Humans, Lactic Acid, NAD, Oxidation-Reduction, Pyruvic Acid
Show Abstract · Added March 14, 2018
There has been tremendous and rapidly growing interest in understanding intermediary metabolism as a key aspect of both normal cellular function and as a participant in the molecular pathogenesis of many different complex diseases. This area of research naturally intersects at virtually every level with the substantial and expanding body of knowledge regarding mechanisms of cellular redox balance. In this Forum, the contributing authors address specifically the union of intermediary metabolism and redox biology through detailed consideration of the biochemistry and biology of nicotine adenine dinucleotides, the cell's "redox currency." From technical considerations of how to measure nicotine adenine dinucleotides all the way to detailed treatments of their potential roles in specific disease states, this Forum provides a thorough introduction to a topic that is positioned to be at the heart of the next wave of research in metabolism and redox biology. Antioxid. Redox Signal. 28, 165-166.
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5 MeSH Terms
Limiting Injury During Saphenous Vein Graft Preparation For Coronary Arterial Bypass Prevents Metabolic Decompensation.
Cheung-Flynn J, Song J, Voskresensky I, Wise ES, Liu Y, Xiong Y, Eagle SS, Brophy CM, Flynn CR
(2017) Sci Rep 7: 14179
MeSH Terms: Animals, Coronary Artery Bypass, Energy Metabolism, Homeostasis, Humans, Hydrolysis, Metabolomics, Oxidation-Reduction, Oxidative Stress, Phospholipids, Pressure, Saphenous Vein, Swine, Vascular Grafting
Show Abstract · Added May 14, 2018
Standard harvest and preparation of human saphenous vein (HSV) for autologous coronary and peripheral arterial bypass procedures is associated with injury and increased oxidative stress that negatively affect graft performance. In this study we investigated the global metabolomic profiles of HSV before (unprepared; UP) and after standard vein graft preparation (AP). AP-HSV showed impaired vasomotor function that was associated with increased oxidative stress, phospholipid hydrolysis and energy depletion that are characteristic of mechanical and chemical injury. A porcine model (PSV) was utilized to validate these metabolomic changes in HSV and to determine the efficacy of an improved preparation technique (OP) using pressure-regulated distension, a non-toxic vein marker, and graft storage in buffered PlasmaLyte solution in limiting metabolic decompensation due to graft preparation. Deficits in vasomotor function and metabolic signature observed in AP-PSV could be largely mitigated with the OP procedure. These findings suggest that simple strategies aimed at reducing injury during graft harvest and preparation represents a straightforward and viable strategy to preserve conduit function and possibly improve graft patency.
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14 MeSH Terms
Selenoproteins in Tumorigenesis and Cancer Progression.
Short SP, Williams CS
(2017) Adv Cancer Res 136: 49-83
MeSH Terms: Animals, Antioxidants, Carcinogenesis, Disease Progression, Humans, Neoplasms, Oxidation-Reduction, Oxidative Stress, Selenium, Selenoproteins
Show Abstract · Added April 15, 2019
Selenium is a micronutrient essential to human health and has long been associated with cancer prevention. Functionally, these effects are thought to be mediated by a class of selenium-containing proteins known as selenoproteins. Indeed, many selenoproteins have antioxidant activity which can attenuate cancer development by minimizing oxidative insult and resultant DNA damage. However, oxidative stress is increasingly being recognized for its "double-edged sword" effect in tumorigenesis, whereby it can mediate both negative and positive effects on tumor growth depending on the cellular context. In addition to their roles in redox homeostasis, recent work has also implicated selenoproteins in key oncogenic and tumor-suppressive pathways. Together, these data suggest that the overall contribution of selenoproteins to tumorigenesis is complicated and may be affected by a variety of factors. In this review, we discuss what is currently known about selenoproteins in tumorigenesis with a focus on their contextual roles in cancer development, growth, and progression.
© 2017 Elsevier Inc. All rights reserved.
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