2014, Müller, Nathalie

Mitochondrial DNA (mtDNA) is organized in nucleoids, structures comprising an association of multiple DNA molecules and proteins with various functions. Its vicinity to the electron transport chain, the main production site of superoxide, makes it highly vulnerable to oxidative damage. Recent findings by our group have revealed the presence of the major superoxide-detoxifying enzyme Manganese Superoxide Dismutase (MnSOD) within the nucleoid structure of tissues from different species and of several cell lines. The binding of MnSOD to DNA was shown to be direct and salt-sensitive, suggesting the implication of ionic forces.
This led to the investigation in the present work of the nature of this binding and the possible involvement of positively charged lysine residues of the enzyme with the negatively charged backbone of the mtDNA. After mutation of three specific lysines of human MnSOD by sitedirected mutagenesis, expression in E. coli and purification, binding of MnSOD mutants to oligonucleotides was measured by a Surface Plasmon Resonance based method. Wild-type and mutant MnSODs all exhibited an association with DNA, suggesting that the binding does not rely on these specific lysines or at least not exclusively.
The isolation of nucleoids on sucrose density gradients revealed the lack of an association of MnSOD to mtDNA in the Hela cell line and in the Parkinson model cell line LUHMES. MnSOD was present in nucleoids of Xenopus laevis oocytes of stages 1 and 6 while absent in oocytes of stage 3.
An Fpg-based version of the automated Fluorimetric Analysis of DNA Unwinding assay for the detection of 8-oxo-,8-dihydroguanine (8-oxodG) was developed by our group. The method was validated by the concurrent detection of 8-oxodG levels in the same plasmid DNA samples by HPLC coupled with LC/MS which displayed a high correlation in the values measured. The method allowed the detection of 8-oxodG formation induced by the peroxynitrite donor 3-Morpholinosyndnomine (Sin-1) in a dose-dependent manner which was prevented by addition of MnSOD, as well as uric acid and minocycline.
The damaging effects of peroxynitrite on mitochondrial biomolecules were further investigated by the detection of tyrosine nitration of mitochondrial proteins in peroxynitritetreated human platelets by Western Blot. Peroxynitrite induced tyrosine nitration of recombinant human MnSOD which led to its inactivation. RAW264.7 macrophage cells treated with Sin-1 exhibited increased 8-nitroguanine levels detected by immunofluorescence
and increased mitochondrial 8-oxodG levels measured by HPLC LC/MS.

2013-08-09, Müller, Nathalie, Moreno-Villanueva, Maria, Fischbach, Arthur, Kienhöfer, Joachim, Martello, Rita, Dedon, Peter C., Ullrich, Volker, Bürkle, Alexander, Mangerich, Aswin

The oxidation of guanine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks (Moreno-Villanueva et al., 2009. BMC Biotechnol. 9, 39). The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC-MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e., uric acid, and a protein, i.e., manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.

2011-02-18, Schildknecht, Stefan, Pape, Regina, Müller, Nathalie, Robotta, Marta, Marquardt, Andreas, Bürkle, Alexander, Drescher, Malte, Leist, Marcel

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or •NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.