The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Oxidative stress is a contributing factor in a number of chronic diseases, including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation that occurs during periods of oxidative stress results in the formation of lipid electrophiles, which can modify a multitude of proteins in the cell. 4-Hydroxy-2-nonenal (HNE) is one of the most well-studied lipid electrophiles and has previously been shown to arrest cells at the G1/S transition. Recently, proteomic data have shown that HNE is capable of covalently modifying CDK2, the kinase responsible for the G1/S transition. Here, we identify the sites adducted by HNE using recombinant CDK2 and show that HNE treatment suppresses the kinase activity of the enzyme. We further identify sites of adduction in HNE-treated intact human colorectal carcinoma cells (RKO) and show that HNE-dependent modification in cells is long-lived, disrupts CDK2 function, and correlates with a delay of progression of the cells into S-phase. We propose that adduction of CDK2 by HNE directly alters its activity, contributing to the cell cycle delay.
Globally, maternal and fetal health is greatly impacted by extraplacental inflammation. Group B Streptococcus (GBS), a leading cause of chorioamnionitis, is thought to take advantage of the uterine environment during pregnancy in order to cause inflammation and infection. In this study, we demonstrate the metabolic changes of murine macrophages caused by GBS exposure. GBS alone prompted a delayed increase in lactate production, highlighting its ability to redirect macrophage metabolism from aerobic to anaerobic respiration. This production of lactate is thought to aid in the development and propagation of GBS throughout the surrounding tissue. Additionally, this study shows that PGE2 priming was able to exacerbate lactate production, shown by the rapid and substantial lactate increases seen upon GBS exposure. These data provide a novel model to study the role of GBS exposure to macrophages with and without PGE2 priming.
Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M1dG levels in intact DNA were increased from basal levels of 1 adduct per 10(8) nucleotides to 2 adducts per 10(6) nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M1dG (to ∼ 5 adducts per 10(7) nucleotides), and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 10(6) nucleotides) and the endogenous formation of 6-oxo-M1dG. This is the first report of the production of 6-oxo-M1dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair.
A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS(3)) method was established to characterize and measure five guanine adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its cross-link (G-NM-G), the ring-opened formamidopyrimidine (FapyG) monoadduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 10(7) DNA bases when the equivalent of 5 μg of DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G monoadduct; the FapyG-NM-G cross-link adduct; and the FapyG-NM-FapyG was below the limit of detection. The NM-FapyG adducts were formed in CT DNA at a level ∼20% that of the NM-G adduct. NM-FapyG has not been previously quanitified, and the FapyG-NM-G and FapyG-NM-FapyG adducts have not been previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 10(7) bases), followed by G-NM-G (240 adducts per 10(7) bases), NM-FapyG (180 adducts per 10(7) bases), and, last, the FapyG-NM-G cross-link adduct (6.0 adducts per 10(7) bases). These lesions are expected to contribute to NM-mediated toxicity and genotoxicity in vivo.
The conformation of an N(2)-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent food mutagen, was determined in 5'-d(C(1)T(2)C(3)X(4)G(5)C(6)G(7)C(8)C(9)A(10)T(11)C(12))-3':5'-d(G(13)A(14)T(15)G(16)G(17)C(18)G(19)C(20)C(21)G(22)A(23)G(24))-3'; X = N(2)-dG-IQ, in which the modified nucleotide X(4) corresponds to G(1) in the 5'-d(G(1)G(2)CG(3)CC)-3' NarI restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the N(2)-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the anti conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C(21) was displaced into the major groove. The processing of the N(2)-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G(3) position, but not at the G(1) position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) J. Biol. Chem., 281, 25297-25306]. The structure of the N(2)-dG-IQ adduct at the nonreiterated G(1) position was compared to that of the same adduct placed at the G(3) position [Stavros, K. M., et al. (2014) Nucleic Acids Res., 42, 3450-3463]. CD indicted minimal spectral differences between the G(1) vs G(3) N(2)-dG-IQ adducts. NMR indicated that the N(2)-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G(1) and G(3) positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G(1) but a base-displaced intercalated conformation when placed at position G(3) in the NarI sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3'-flanking sequences, perhaps modulating the ability to accommodate transient strand slippage intermediates.
Pyridoxamine (PM) is a prospective drug for the treatment of diabetic complications. In order to make zwitterionic PM more lipophilic and improve its tissue distribution, PM derivatives containing medium length alkyl groups on the hydroxymethyl side chain were prepared. The synthesis of these alkylpyridoxamines (alkyl-PMs) starting from pyridoxine offers high yields and is amenable to bulk preparations. Interestingly, alkyl-PMs were found to react with methylglyoxal (MGO), a major toxic product of glucose metabolism and autoxidation, several orders of magnitude faster than PM. This suggests the formation of nonionic pyrido-1,3-oxazine as the key step in the reaction of PM with MGO. Since the primary target of MGO in proteins is the guanidine side chain of arginine, alkyl-PMs were shown to be more effective than PM in reducing the modification of N-α-benzoylarginine by MGO. Alkyl-PMs in the presence of MGO also protected the enzymatic activity of lysozyme that contains several arginine residues next to its active site. Alkyl-PMs can be expected to trap MGO and other toxic 1,2-carbonyl compounds more effectively than PM, especially in lipophilic tissue environments, thus protecting macromolecules from functional damage. This suggests potential therapeutic uses for alkyl-PMs in diabetes and other diseases characterized by the elevated levels of toxic dicarbonyl compounds.
Extracts from the rhizome of the turmeric plant are widely consumed as anti-inflammatory dietary supplements. Turmeric extract contains the three curcuminoids, curcumin (≈80% relative abundance), demethoxycurcumin (DMC; ≈15%), and bisdemethoxycurcumin (BDMC; ≈5%). A distinct feature of pure curcumin is its instability at physiological pH, resulting in rapid autoxidation to a bicyclopentadione within 10-15 min. Here, we describe oxidative transformation of turmeric extract, DMC, and BDMC and the identification of their oxidation products using LC-MS and NMR analyses. DMC autoxidized over the course of 24 h to the expected bicyclopentadione diastereomers. BDMC was resistant to autoxidation, and oxidative transformation required catalysis by horseradish peroxidase and H2O2 or potassium ferricyanide. The product of BDMC oxidation was a stable spiroepoxide that was equivalent to a reaction intermediate in the autoxidation of curcumin. The ability of DMC and BDMC to poison recombinant human topoisomerase IIα was significantly increased in the presence of potassium ferricyanide, indicating that oxidative transformation was required to achieve full DNA cleavage activity. DMC and BDMC are less prone to autoxidation than curcumin and contribute to the enhanced stability of turmeric extract at physiological pH. Their oxidative metabolites may contribute to the biological effects of turmeric extract.
Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.
Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional pesticides and metals was assayed, and eight agents that produced a significant decrease and five that produced a significant increase in activated E1 were identified. These studies suggest that E1 is a sensitive redox sensor that can be modulated by exposure to environmental agents and can regulate downstream cellular processes.
Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ sequences (X = AFB(1)−FAPY; Y = 7-deaza-dG)demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions.Structural analysis of the adduct in the 5′-XA-3′ sequence indicates the preference of the E isomer of the formamide group,attributed to formation of a hydrogen bond between the formyl oxygen and the N(6) exocyclic amino group of the 3′-neighboradenine. While the 5′-XA-3′ sequence exhibits the E isomer, the 5′-XC-3′ sequence exhibits a 7:3 E:Z ratio at equilibrium at 283K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N(4)-dC exocyclic amino group of the 3′-neighbor cytosine. The 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl oxygen and the 3′-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and β anomers and the potential to exhibit atropisomers about the C5−N(5) bond do not depend upon sequence. In each of the four DNA sequences, the AFB(1)−FAPY adduct maintains the β deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB(1) moiety that is intercalated above the 5′-face of the damaged guanine. This enforces the Ra axialc onformation for the C5−N(5) bond.