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Glutamine-451 Confers Sensitivity to Oxidative Inhibition and Heme-Thiolate Sulfenylation of Cytochrome P450 4B1.
Albertolle ME, Song HD, Wilkey CJ, Segrest JP, Guengerich FP
(2019) Chem Res Toxicol 32: 484-492
MeSH Terms: Animals, Aryl Hydrocarbon Hydroxylases, Glutamine, Heme, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Oxidation-Reduction, Rabbits, Sulfenic Acids, Sulfhydryl Compounds
Show Abstract · Added March 26, 2019
Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with HO and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.
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10 MeSH Terms
Heme sensing and detoxification by HatRT contributes to pathogenesis during Clostridium difficile infection.
Knippel RJ, Zackular JP, Moore JL, Celis AI, Weiss A, Washington MK, DuBois JL, Caprioli RM, Skaar EP
(2018) PLoS Pathog 14: e1007486
MeSH Terms: Animals, Bacterial Proteins, Clostridium Infections, Clostridium difficile, Genes, Bacterial, Heme, Male, Mice, Mice, Inbred C57BL, Operon, Virulence
Show Abstract · Added April 7, 2019
Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.
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Human mAbs to Staphylococcus aureus IsdA Provide Protection Through Both Heme-Blocking and Fc-Mediated Mechanisms.
Bennett MR, Bombardi RG, Kose N, Parrish EH, Nagel MB, Petit RA, Read TD, Schey KL, Thomsen IP, Skaar EP, Crowe JE
(2019) J Infect Dis 219: 1264-1273
MeSH Terms: Animals, Antibodies, Monoclonal, Antigens, Bacterial, Bacterial Proteins, Disease Models, Animal, Female, Hemeproteins, Humans, Hydrogen Deuterium Exchange-Mass Spectrometry, Mice, Mice, Inbred BALB C, Receptors, Cell Surface, Staphylococcal Infections, Staphylococcus aureus
Show Abstract · Added March 31, 2019
The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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14 MeSH Terms
HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis.
Choby JE, Grunenwald CM, Celis AI, Gerdes SY, DuBois JL, Skaar EP
(2018) mBio 9:
MeSH Terms: Aldehyde Oxidoreductases, Bacterial Proteins, Gene Deletion, Gene Expression, Gene Expression Regulation, Bacterial, Heme, Methyltransferases, Staphylococcus aureus
Show Abstract · Added March 15, 2018
is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy in heme biosynthesis. The first committed enzyme in the heme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by which responds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation of leads to increased heme synthesis. Excess heme synthesis in a Δ mutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates that regulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX. is a leading cause of skin and soft tissue infections, endocarditis, bacteremia, and osteomyelitis, making it a critical health care concern. Development of new antimicrobials against requires knowledge of the physiology that supports this organism's pathogenesis. One component of staphylococcal physiology that contributes to growth and virulence is heme. Heme is a widely utilized cofactor that enables diverse chemical reactions across many enzyme families. relies on many critical heme-dependent proteins and is sensitive to excess heme toxicity, suggesting must maintain proper intracellular heme homeostasis. Because provides heme for heme-dependent enzymes via synthesis from common precursors, we hypothesized that regulation of heme synthesis is one mechanism to maintain heme homeostasis. In this study, we identify that posttranscriptionally regulates heme synthesis by restraining abundance of the first heme biosynthetic enzyme, GtrR, via heme and the broadly conserved membrane protein HemX.
Copyright © 2018 Choby et al.
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8 MeSH Terms
Fur regulation of Staphylococcus aureus heme oxygenases is required for heme homeostasis.
Lojek LJ, Farrand AJ, Weiss A, Skaar EP
(2018) Int J Med Microbiol 308: 582-589
MeSH Terms: Aerobiosis, Bacterial Proteins, Gene Expression Regulation, Bacterial, Heme, Heme Oxygenase (Decyclizing), Homeostasis, Iron, Mixed Function Oxygenases, Oxygenases, Repressor Proteins, Staphylococcus aureus
Show Abstract · Added March 15, 2018
Heme is a cofactor that is essential for cellular respiration and for the function of many enzymes. If heme levels become too low within the cell, S. aureus switches from producing energy via respiration to producing energy by fermentation. S. aureus encodes two heme oxygenases, IsdI and IsdG, which cleave the porphyrin heme ring releasing iron for use as a nutrient source. Both isdI and isdG are only expressed under low iron conditions and are regulated by the canonical Ferric Uptake Regulator (Fur). Here we demonstrate that unregulated expression of isdI and isdG within S. aureus leads to reduced growth under low iron conditions. Additionally, the constitutive expression of these enzymes leads to decreased heme abundance in S. aureus, an increase in the fermentation product lactate, and increased resistance to gentamicin. This work demonstrates that S. aureus has developed tuning mechanisms, such as Fur regulation, to ensure that the cell has sufficient quantities of heme for efficient ATP production through aerobic respiration.
Copyright © 2018 Elsevier GmbH. All rights reserved.
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11 MeSH Terms
Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.
Albertolle ME, Kim D, Nagy LD, Yun CH, Pozzi A, Savas Ü, Johnson EF, Guengerich FP
(2017) J Biol Chem 292: 11230-11242
MeSH Terms: Animals, Catalysis, Cytochrome P-450 CYP4A, Dithiothreitol, Heme, Humans, Hydrogen Peroxide, Hydroxyeicosatetraenoic Acids, Kidney, Liver, Mice, Mice, Transgenic, Oxidation-Reduction, Rats
Show Abstract · Added March 14, 2018
Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents ( dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added HO attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an HO concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 e in kidneys and livers derived from transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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14 MeSH Terms
Structural analyses of sterol 14α-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis.
Hargrove TY, Friggeri L, Wawrzak Z, Qi A, Hoekstra WJ, Schotzinger RJ, York JD, Guengerich FP, Lepesheva GI
(2017) J Biol Chem 292: 6728-6743
MeSH Terms: Animals, Antifungal Agents, Azoles, Candida albicans, Crystallization, Fungal Proteins, Heme, Humans, Kinetics, Ligands, Microbial Sensitivity Tests, Protein Binding, Protein Conformation, Protons, Rats, Sterol 14-Demethylase, Sterols
Show Abstract · Added March 14, 2018
With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: ()-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against and , pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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17 MeSH Terms
The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni.
Johnson JG, Gaddy JA, DiRita VJ
(2016) mBio 7:
MeSH Terms: Animals, Bacterial Proteins, Campylobacter jejuni, Catalase, Chickens, Gastrointestinal Tract, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Heme, Humans, Hydrogen Peroxide, Iron, Mutation
Show Abstract · Added April 26, 2017
Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the heuR mutant being particularly affected during competition with wild-type C. jejuni Transcriptomic analysis identified the chu genes-and those encoding other iron uptake systems-as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni IMPORTANCE: Annually, Campylobacter jejuni causes millions of gastrointestinal infections in the United States, due primarily to its ability to reside within the gastrointestinal tracts of poultry, where it can be released during processing and contaminate meat. In the developing world, humans are often infected by consuming contaminated water or by direct contact with livestock. Following consumption of contaminated food or water, humans develop disease that is characterized by mild to severe diarrhea. There is a need to understand both colonization of chickens, to make food safer, and colonization of humans, to better understand disease. Here we demonstrate that to efficiently colonize a host, C. jejuni requires iron from heme, which is regulated by the protein HeuR. Understanding how HeuR functions, we can develop ways to inhibit its function and reduce iron acquisition during colonization, potentially reducing C. jejuni in the avian host, which would make food safer, or limiting human colonization.
Copyright © 2016 Johnson et al.
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13 MeSH Terms
Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.
Surdel MC, Dutter BF, Sulikowski GA, Skaar EP
(2016) ACS Infect Dis 2: 572-8
MeSH Terms: Bacterial Proteins, Biological Transport, Gene Expression Regulation, Bacterial, Heme, Humans, Nitric Oxide, Nitric Oxide Synthase, Staphylococcal Infections, Staphylococcus aureus
Show Abstract · Added April 8, 2017
Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.
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9 MeSH Terms
Transition Metals and Virulence in Bacteria.
Palmer LD, Skaar EP
(2016) Annu Rev Genet 50: 67-91
MeSH Terms: Animals, Bacteria, Bacterial Infections, Deficiency Diseases, Diet, Heme, Host-Pathogen Interactions, Humans, Iron, Iron Overload, Metals, Siderophores
Show Abstract · Added April 8, 2017
Transition metals are required trace elements for all forms of life. Due to their unique inorganic and redox properties, transition metals serve as cofactors for enzymes and other proteins. In bacterial pathogenesis, the vertebrate host represents a rich source of nutrient metals, and bacteria have evolved diverse metal acquisition strategies. Host metal homeostasis changes dramatically in response to bacterial infections, including production of metal sequestering proteins and the bombardment of bacteria with toxic levels of metals. In response, bacteria have evolved systems to subvert metal sequestration and toxicity. The coevolution of hosts and their bacterial pathogens in the battle for metals has uncovered emerging paradigms in social microbiology, rapid evolution, host specificity, and metal homeostasis across domains. This review focuses on recent advances and open questions in our understanding of the complex role of transition metals at the host-pathogen interface.
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12 MeSH Terms