A copper-catalyzed click reaction inside living mammalian cells is demonstrated with the use of a cell-penetrating peptide-tethered CuI ligand.
The apurinic/apyrimidinic (AP) site is a common lesion of DNA damage. The levels of AP sites reported in the literature range widely, which is primarily due to the artefactual generation or loss of AP sites during processing of the DNA. Herein, we have developed a method to quantitate AP sites with a largely reduced level of artifacts by derivatizing AP sites before DNA isolation. A rapid digestion of nuclear protein was performed to minimize enzymatic DNA repair, followed by direct derivatization of AP sites in the nuclear lysate with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA), yielding an oxime derivative that is stable through subsequent DNA processing steps. Quantitation was done using highly selective and sensitive liquid chromatography-tandem mass spectrometry, with a limit of quantitation at 2.2 lesions per 10 8 nucleotides (nts, 0.9 fmol on column). The method was applied in vivo to measure AP sites in rats undergoing oxidative stress [liver: 3.31 ± 0.47/10 7 nts (dosed) vs 0.91 ± 0.06/10 7 nts (control), kidney: 1.60 ± 0.07/10 7 nts (dosed) vs 1.13 ± 0.12/10 7 nts (control)]. The basal AP level was significantly lower than literature values. The method was also used to measure AP sites induced by the chemotherapeutic nitrogen mustard in vitro.
The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a powerful tool for bioconjugation of biomolecules, particularly proteins and peptides. The major drawback limiting the use of the CuAAC reaction in biological systems is the copper-mediated formation of reactive oxygen species (ROS), leading to the oxidative degradation of proteins or peptides. From the studies on a limited number of proteins and peptides, it is known that, in general, the copper mediated oxidative damage is associated with the copper coordination environment and solvent accessibility. However, there is a lack of data to help estimate the extent of copper-mediated oxidation on a wide range of proteins and peptides. To begin to address this need, we quantitatively measured the degree of copper-mediated oxidation on libraries of 1200 tetrapeptides and a model protein (bovine serum albumin, BSA) using liquid chromatography mass spectrometry (LC-MS). The collected data will be useful to researchers planning to use the CuAAC reaction for bioconjugaton on peptides or proteins.
Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2–6 and 2–17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.
Gut bacteria face a key problem in how they capture enough energy needed to sustain their growth and physiology. The gut bacterium Clostridium sporogenes obtains its energy by utilizing amino acids in pairs, coupling the oxidation of one to the reduction of another – the Stickland reaction. Oxidative pathways produce ATP via substrate level phosphorylation, whereas reductive pathways are thought to balance redox. Here, we investigated whether these reductive pathways are also linked to energy generation and the production of microbial metabolites that may circulate and impact host physiology. Using metabolomics, we find that during growth in vitro, C. sporogenes produces 15 metabolites, 13 of which are present in the gut of C. sporogenes colonized mice. Four of these compounds are reductive Stickland metabolites that circulate in the blood of gnotobiotic mice and are also detected in plasma from healthy humans. Gene clusters for reductive Stickland pathways suggest involvement of electron transfer proteins, and experiments in vitro demonstrate that reductive metabolism is coupled to ATP formation and not just redox balance. Genetic analysis points to the broadly conserved Rnf complex as a key coupling site for energy transduction. Rnf complex mutants show aberrant amino acid metabolism in defined medium and are attenuated for growth in the mouse gut, demonstrating a role of the Rnf complex in Stickland metabolism and gut colonization. Our findings reveal that the production of circulating metabolites by a commensal bacterium within the host gut is linked to an ATP-yielding redox process.
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