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The functional significance of manganese superoxide dismutase binding to mitochondrial DNA

The functional significance of manganese superoxide dismutase binding to mitochondrial DNA

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MÜLLER, Nathalie, 2014. The functional significance of manganese superoxide dismutase binding to mitochondrial DNA

@phdthesis{Muller2014funct-28843, title={The functional significance of manganese superoxide dismutase binding to mitochondrial DNA}, year={2014}, author={Müller, Nathalie}, address={Konstanz}, school={Universität Konstanz} }

2014-08-21T07:13:58Z deposit-license eng Müller, Nathalie Müller, Nathalie Die funktionelle Wahrscheinlichkeit der Mangan-Superoxide-Dismutase an mitochondriale DNA zu binden The functional significance of manganese superoxide dismutase binding to mitochondrial DNA 2014 2014-08-21T07:13:58Z 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.<br />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.<br />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.<br />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.<br />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<br />and increased mitochondrial 8-oxodG levels measured by HPLC LC/MS.

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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