Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs.

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.

Halogen Bonds in Protein Nucleic Acid Recognition.

Identifying new binding forces between electron donor and acceptor entities is key to properly understanding molecular recognition and aggregation phenomena, which are of inmense importance to biology. For decades, the halogenation of DNA/RNA bases has been routinely carried out to solve solid state structures of nucleic acids (NA). However, the effects of this modification might be deeper than just a simple atom substitution since halogens are also known to undergo noncovalent binding (halogen bonding). Herein we show that halogenated NAs with either Br or I atoms are able to establish halogen bonds with properly disposed protein residues. An inspection of the Protein Data Bank (PDB) reveals several examples involving 5-iodo/5-bromouracil, 8-bromoadenine, and 5-iodocytosine bases that are consistent with the halogen bond geometry features. Computations reveal the favorable and moderately strong nature of this interaction, thus confirming the ability of halogenated bases to actively participate in protein-NA binding.

Quantifying Intramolecular Halogen Bonds in Nucleic Acids: A Combined Protein Data Bank and Theoretical Study.

In this study, we report experimental (Protein Data Bank (PDB) search) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the nature, stability, and directionality properties of intramolecular halogen bonding interactions (HaBs) between 5-bromo/5-iodoracil bases and backbone phosphate groups in nucleic acids (NAs). A PDB survey revealed relevant examples where intramolecular HaBs are undertaken and serve as a structural source of stability in RNA and DNA molecules. In order to develop suitable energy predictors, we started this investigation by calculating the interaction energy values and both the potential V(r) and kinetic G(r) energy densities (using Bader's "atoms in molecules" theory) of several halogen bond complexes involving 5-bromo/5-iodoracil molecules and biologically relevant electron donors. Once the energy predictors based on V(r)/G(r) energy densities were developed, we analyzed the HaBs observed in the biological examples retrieved from the PDB search in order to estimate the strength of the interaction. As far as our knowledge extends, intramolecular halogen bonds in NAs have not been previously quantified in the literature using this methodology and may be of great importance in understanding their structural properties as well as in the construction of molecular materials with DNA and other biological macromolecules.

Insights into the deactivation of 5-bromouracil after ultraviolet excitation.

5-Bromouracil is a nucleobase analogue that can replace thymine in DNA strands and acts as a strong radiosensitizer, with potential applications in molecular biology and cancer therapy. Here, the deactivation of 5-bromouracil after ultraviolet irradiation is investigated in the singlet and triplet manifold by accurate quantum chemistry calculations and non-adiabatic dynamics simulations. It is found that, after irradiation to the bright ππ* state, three main relaxation pathways are, in principle, possible: relaxation back to the ground state, intersystem crossing (ISC) and C-Br photodissociation. Based on accurate MS-CASPT2 optimizations, we propose that ground-state relaxation should be the predominant deactivation pathway in the gas phase. We then employ different electronic structure methods to assess their suitability to carry out excited-state dynamics simulations. MRCIS (multi-reference configuration interaction including single excitations) was used in surface hopping simulations to compute the ultrafast ISC dynamics, which mostly involves the 1nOπ* and 3ππ* states.This article is part of the themed issue 'Theoretical and computational studies of non-equilibrium and non-statistical dynamics in the gas phase, in the condensed phase and at interfaces'.

Rapid and random turnover of mitochondrial DNA in rat hepatocytes of primary culture.

It is known that mitochondrial DNA (mtDNA) replication is independent of the cell cycle. Even in post-mitotic cells in which nuclear DNA replication has ceased, mtDNA is believed to still be replicating. Here, we investigated the turnover rate of mtDNA in primary rat hepatocytes, which are quiescent cells. Southwestern blot analysis using 5-bromo-2'-deoxyuridine (BrdU) was employed to estimate the activity of full-length mtDNA replication and to determine efficient doses of replication inhibitors. Southern blot analysis showed that a two-day treatment with 20mM 2',3'-dideoxycytidine and 0.2mug/ml ethidium bromide caused a 37% reduction in the amount of mtDNA, indicating that the hepatocytes had a considerably high rate of turnover of mtDNA. Further, pulse-chase analysis using Southwestern analysis showed that the amount of newly synthesized mtDNA labeled with BrdU declined to 60% of the basal level within two days. Because the rate of reduction of the new mtDNA was very similar to the overall turnover rate described above, it appears that degrading mtDNA molecules were randomly chosen. Thus, we demonstrated that there is highly active and random turnover of mtDNA in hepatocytes.

Phagocytes produce 5-chlorouracil and 5-bromouracil, two mutagenic products of myeloperoxidase, in human inflammatory tissue.

Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH approximately 5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid- or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H2O2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.

Photocatalytic degradation of pesticides and bio-molecules in water.

Two approaches are suggested for the acceleration of the photocatalytic oxidation of organic contaminants of water: acceleration by oxidants and photo-enhancement by dyes. These processes were examined with several substances: two widely applied herbicides, bromacil (a uracil) and metribuzin (a triazine), and three proteins, studied as models of biocontaminated waters. The effects of oxygen and hydrogen peroxide indicated two different reaction patterns of photo-oxidation of the herbicides. With metribuzin, oxygen had a pronounced effect on the rate of photo-oxidation, while the influence of hydrogen peroxide was quite moderate; with bromacil, oxygen had a limited effect on the rate of photo-oxidation, which however was considerably enhanced by hydrogen peroxide. Acceleration of the photo-catalytic oxidation of colourless refractory contaminants by photo-excited dye was observed. Both UV and visible light were required for the enhanced decomposition. The mechanism of the reaction seems to involve a combination of oxidation by hydroxyl radicals, via the hole-electron semiconductor route, with subsequent oxidation of photo-intermediates by singlet oxygen formed by dye sensitization. The TiO2-photocatalyzed oxidation of proteins (albumin, ovalbumin and gamma-globulin) showed the susceptibility of proteins to photocleavage and of the amino acids to photocatalytic degradation. Tyrosine was the most sensitive, while the degradation of the aliphatic amino acids Gly and Asp was slow.

Binding of 5-bromouracil-containing S/MAR DNA to the nuclear matrix.

Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis.

The eosinophil peroxidase-hydrogen peroxide-bromide system of human eosinophils generates 5-bromouracil, a mutagenic thymine analogue.

Eosinophils use eosinophil peroxidase, hydrogen peroxide (H(2)O(2)), and bromide ion (Br(-)) to generate hypobromous acid (HOBr), a brominating intermediate. This potent oxidant may play a role in host defenses against invading parasites and eosinophil-mediated tissue damage. In this study, we explore the possibility that HOBr generated by eosinophil peroxidase might oxidize nucleic acids. When we exposed uracil, uridine, or deoxyuridine to reagent HOBr, each reaction mixture yielded a single major oxidation product that comigrated on reversed-phase HPLC with the corresponding authentic brominated pyrimidine. The eosinophil peroxidase-H(2)O(2)-Br(-) system also converted uracil into a single major oxidation product, and the yield was near-quantitative. Mass spectrometry, HPLC, UV--visible spectroscopy, and NMR spectroscopy identified the product as 5-bromouracil. Eosinophil peroxidase required H(2)O(2) and Br(-) to produce 5-bromouracil, implicating HOBr as an intermediate in the reaction. Primary and secondary bromamines also brominated uracil, suggesting that long-lived bromamines also might be physiologically relevant brominating intermediates. Human eosinophils used the eosinophil peroxidase-H(2)O(2)-Br(-) system to oxidize uracil. The product was identified as 5-bromouracil by mass spectrometry, HPLC, and UV--visible spectroscopy. Collectively, these results indicate that HOBr generated by eosinophil peroxidase oxidizes uracil to 5-bromouracil. Thymidine phosphorylase, a pyrimidine salvage enzyme, transforms 5-bromouracil to 5-bromodeoxyridine, a mutagenic analogue of thymidine. These findings raise the possibility that halogenated nucleobases generated by eosinophil peroxidase exert cytotoxic and mutagenic effects at eosinophil-rich sites of inflammation.

Production of brominating intermediates by myeloperoxidase. A transhalogenation pathway for generating mutagenic nucleobases during inflammation.

The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl(-) to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br(-) to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br(-) (100 microM) and Cl(-) (100 mM). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br(-) and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br(-) and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl(-)- Br(-) system but not by the eosinophil peroxidase-H2O2-Cl(-)-Br(-) system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br(-) and the myeloperoxidase-H2O2-Cl(-)-Br(-) system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br(-) to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.

Biochemical modulation of the catabolism and tissue uptake of the anticancer drug 5-fluorouracil by 5-bromovinyluracil: assessment with metabolic (19)F MR imaging.

Using chemical shift-selective (19)F magnetic resonance (MR) imaging, we investigated the biomodulating action of 5-bromovinyluracil (BVU) on the degradation of the anticancer drug 5-fluorouracil (5-FU) to its major catabolite alpha-fluoro-beta-alanine (FBAL) and the tissue uptake of 5-FU in ACI rats with transplanted Morris hepatoma. Rats in the control group (n = 7) received 200 mg/kg body weight of 5-FU intravenously, whereas the rats in the BVU group (n = 7) additionally received 30 mg/kg body weight of BVU intraperitoneally about 45 min before 5-FU injection. In each animal examination, three selective (19)F MR images were acquired sequentially after 5-FU administration with an acquisition time of 32 min each: an early 5-FU image (dominant Fourier line, 8 min p.i.) that characterized the early uptake of the drug into the various tissues, an FBAL image (dominant Fourier line, 56 min p.i.) that reflected the catabolism of the drug, and a late 5-FU image (dominant Fourier line, 78 min p.i.) that assessed the retention ("trapping") of unmetabolized 5-FU and its MR-visible anabolites. Pretreatment with BVU resulted in a highly statistical significant decrease (P < 0.001) of the FBAL signal in the liver. The marked effect of BVU on 5-FU degradation, however, improved neither the early uptake nor the retention of 5-FU in skeletal muscle and tumor tissue (P > 0.7). Moreover, our results indicate that 5-FU tumor uptake is not only dependent on the plasma concentration of unmetabolized 5-FU but is also determined by tumor-specific factors, these showing considerable variations between individual neoplasms. Magn Reson Med 42:936-943, 1999.

A possible mechanism of eighteen patient deaths caused by interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs.

A toxicokinetic study was performed using rats to investigate the possible mechanism of 18 acute deaths in Japanese patients with cancer and herpes zoster by interactions of the new oral antiviral drug, sorivudine (SRV), with one of the oral 5-fluorouracil (5-FU) prodrugs within 40 days after approval of the use of SRV. Tegafur, an anticancer 5-FU prodrug suggested to be used by most of the patients who died, and SRV were orally administered to rats simultaneously once daily. All of these rats died within 10 days, whereas rats given SRV or tegafur alone under the same dosage conditions showed no appreciable change over 20 days compared with controls. In the rats given both drugs, bone marrow and intestinal membrane mucosa were greatly damaged at an early stage of the coadministration, and before death, the animals showed marked decreases in white blood cell and platelet counts, diarrhea with bloody flux, and severe anorexia, as was also manifested by the patients who subsequently died. In the rats given both drugs for 6 days, extremely enhanced 5-FU levels were observed from the first day of administration in plasma and in all tissues examined, including bone marrow and intestines. The extreme enhancement of the tissue 5-FU levels was attributable to the facile inactivation by (E)-5-(2-bromovinyl)uracil (BVU) of hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the systemic 5-FU level in the rat and human. BVU, a major metabolite formed from SRV by gut flora, was found at considerable levels in the liver of rats orally administered SRV alone or SRV and tegafur, and there was a marked decrease in hepatic DPD activity. In the presence of NADPH, DPD purified from rat liver cytosol was rapidly and irreversibly inactivated by [14C]BVU as a suicide inhibitor with concomitant incorporation of the radioactivity into the enzyme protein, although SRV showed no inhibitory effect on DPD under the same conditions. Human liver DPD was recently demonstrated by us to be inactivated with BVU in a manner very similar to rat DPD.

A novel and simple model of the uptake of organic chemicals by vegetation from air and soil.

A novel and simple three-compartment fugacity model has been developed to predict the kinetics and equilibria of the uptake of organic chemicals in herbaceous agricultural plants at various times, including the time of harvest using only readily available input data. The chemical concentration in each of the three plant compartments leaf, stem which includes fruits and seeds, and root) is expressed as a function of both time and chemical concentrations in soil and air. The model was developed using the fugacity concept; however, the final expressions are presented in terms of concentrations in soil and air, equilibrium partition coefficients and a set of transport and transformation half-lives. An illustrative application of the model is presented which describes the uptake of bromacil by a soybean plant under hydroponic conditions. The model, which is believed to give acceptably accurate prediction of the distribution of chemicals among plant tissues, air and soil, may be used for the assessment of exposure to, and risk from contaminants consumed either directly from vegetation or indirectly in natural and agricultural food chains.

Radiolytic products of bromodeoxyuridine in solids by 60Co gamma-rays and monoenergetic soft x-rays at the K-absorption edge of bromine.

In order to investigate DNA damage due to Auger cascades in bromodeoxyuridine (BrdU), BrdU mixed with other nucleosides, as a model of DNA, was irradiated in solids by gamma-rays and monoenergetic x-rays at around the K-absorption edge of bromine (13.47 keV). The main products of BrdU were deoxyuridine produced through debromination, and bromouracil produced through the decomposition of a sugar group. The rates of the debromination and the nucleobases release of additives were markedly increased in the mixed sample. This observation indicated that the additives surrounding BrdU efficiently supplied protons and then decomposed. The major products by x-rays were the same as those by gamma-rays, indicating that Auger cascades in bromine atoms did not produce specific products. The production rates for all products from the mixed sample were about 2.5 times higher at 13.51 (above the K-absorption edge) keV than at 13.43-keV x-rays.

Production of double-stranded RNA during synthesis of bromouracil-substituted RNA by transcription with T7 RNA polymerase.

Using T7 RNA polymerase we synthesized a short oligoribonucleotide containing bromouracil by in vitro transcription of a synthetic DNA template. Whereas the major transcript obtained had the expected size and was apparently homogeneous on a sequencing gel, additional analysis revealed the presence of double-stranded RNA in this preparation. As this was not observed when the same template was transcribed in the presence of uracil, we hypothesize that bromouracil promoted the apparition of double-stranded 'parasitic' RNA presumably by favouring priming for the RNA-dependent RNA synthesis of the T7 RNA polymerase or by facilitating an end-to-end copy mechanism.

Effect of 5-fluoro- and 5-bromouracil substitution on the translation of human thymidylate synthase mRNA.

The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of incorporation of uracil (Ura) analogs on translation. Human TS cDNA was transcribed in the presence of Ura-, FUra-, or BrUTP to obtain 100% substituted mRNA. The mRNAs were translated in a rabbit reticulocyte lysate system. The TS protein that was formed from each of the templates reacted identically with TS antibody in Western blots. Time courses of TS formation revealed a characteristic peak which occurred at 45 min for the Ura- and FUra-RNAs and at 2 h for the BrUra-RNA. Substitution of Ura with FUra did not alter the rate of translation, while substitution of BrU for Ura decreased the rate of translation. Substitution of Ura with FUra or BrUra enhanced the stability of the mRNAs in the rabbit reticulocyte lysate by 3- and 10-fold, respectively. Incorporation of BrUra influenced the binding and catalysis on the ribosome, resulting in a 3.5-fold greater rate of activation (Kact) and 6-fold lower Vmax than the equivalent values for the Ura- and FUra-substituted mRNAs. Nondenaturing gel electrophoresis revealed that different conformations exist among the mRNAs. These data show that translation can be influenced by the incorporation of fraudulent bases into mRNA and those bases that stabilize RNA secondary structure will have the greatest inhibitory effect on translation.

Photo-cross-linking of CRP to nonspecific DNA in the absence of cAMP. DNA interacts with both the N- and C-terminal parts of the protein.

Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli. cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra). The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced. We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP. This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein.

Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking.

The bZIP DNA-binding proteins are characterized by a 50-amino-acid DNA binding and dimerization motif, consisting of a highly basic DNA-binding region ('b') followed by a leucine zipper dimerization region ('ZIP'). The best characterized bZIP DNA-binding protein is GCN4, a yeast transcriptional activator. GCN4 binds to a 9-base-pair two-fold-symmetric DNA site, 5'-A-4T-3G-2A-1C0T+1C+2A+3T+4-3' (refs 7-10). A detailed model known as the 'induced helical fork' model has been proposed for the structure of the GCN4-DNA complex. Using a site-specific bromouracil-mediated photocrosslinking method, we show here that the alanine at position 238 of GCN4 contacts, or is close to, the thymine 5-methyl of A.T at position +3 of the DNA site in the GCN4-DNA complex. Our results strongly support the induced helical fork model. Our site-specific bromouracil-mediated photocrosslinking method requires no prior information regarding the structure of the protein or the structure of the protein-DNA complex and should be generalizable to DNA-binding proteins that interact with the DNA major groove.