04 Fakultät Energie-, Verfahrens- und Biotechnik

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1990 - 199912010 - 20172

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    Biochemical characterisation of TET DNA hydroxylases
    (2017) Ravichandran, Mirunalini; Jurkowski, Tomasz (Jun.-Prof. Dr.)
    Methylation of DNA in CpG dinucleotide plays an important role in mammalian development. The recent discovery of TET enzymes showed that DNA demethylation can occur through stepwise oxidation of 5-methylcytosine (5mC) to 5-hyrdoxymethylcytosine (5hmC), 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5-caC) followed by the removal of the higher oxidised bases by Thymine DNA glycosylase (TDG) and base excision repair mechanism. Genetic studies revealed that the TET enzymes are involved in numerous biological processes such as transcriptional regulation, hematopoietic stem cell differentiation, embryonic, primodial germ cells (PGCs) development and are commonly misregulated in cancer. While the biological functions of TET enzymes have been studied extensively, very little is known about their biochemical properties. In this body of work, the biochemistry of TET enzymes was investigated in detail with the focus on their catalytic and kinetic behaviour, which would allow us to understand the molecular mechanisms of TET enzymes. First of all, an in vitro system including a novel plate assay to quantify the oxidation products catalysed by TET enzymes, was established. As a proof of principle, several analogues of α-Ketoglutarate, the intermediates of citric acid cycle (oncometabolites) were tested. Moreover, the effect of divalent metal ions was tested both in vitro and in vivo and it was demonstrated that the addition of nickel ions to mammalian cells decreased the level of 5hmC through inhibition of TET enzymes by displacing the Fe2+ from the catalytic centre. Furthermore, using detailed biochemical studies, it was demonstrated that ascorbic acid (AscA) modulates the activity of TET enzymes through efficient recycling of Fe2+, which challenges the existing view of AscA as a bound cofactor of TET enzymes. Finally, using biochemical approach followed by next generation sequencing and bioinformatics analysis, the catalytic behaviour of TET enzymes on single molecule level was elucidated. Using linear double stranded DNA containing multiple 5hmC-substrates in different flanking sequence context, it was shown that mammalian TET enzymes oxidize 5hmC substrates in both CG and non-CG context. Importantly, both mammalian TET enzymes and Naegleria gruberi Tet1 like dioxygenase (nTet) showed a strong and distinct flanking sequence preference. In addition, it was shown that TET enzymes (both mammalian and nTet) might catalyse the substrates on DNA in distributive manner.
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    ItemOpen Access
    Dioxygenolytic cleavage of aryl ether bonds: 1,2-Dihydro-1,2-dihydroxy-4-carboxybenzophenone as evidence for initial 1,2-dioxygenation in 3- and 4-carboxy biphenyl ether degradation
    (1990) Engesser, Karl-Heinrich; Fietz, Walter H.; Fischer, Peter; Schulte, P.; Knackmuss, Hans-Joachim
    A bacterial strain, Pseudomonas sp. POB 310, was enriched with 4-carboxy biphenyl ether as sole source of carbon and energy. Resting cells of POB 310 co-oxidize a substrate analogue, 4-carboxybenzophenone, yielding 1,2-dihydro-1,2-dihydroxy-4-carboxy-benzophenone. The ether bond of 3- and 4-carboxy biphenyl ether is cleaved analogously by initial 1,2-dioxygenation, yielding a hemiacetal which is hydrolysed to proto-catechuate and phenol. These intermediates are degraded via an ortho and meta pathway, respectively. Alternative 2,3- and 3,4-dioxygenation can be ruled out as triggering steps in carboxy biphenyl ether degradation.
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    Enzymatic characterization of protein lysine methyltransferases
    (2017) Weirich, Sara; Jeltsch, Albert (Prof. Dr.)
    Histone lysine methylation is an epigenetic mechanism that is involved in the regulation of many biological processes. Over the last decade, the global interest in protein lysine methylation events increased drastically, because several protein lysine methyltransferases (PKMTs) and lysine methylation sites were identified in the genomes and proteomes of many organisms also including non-histone proteins functioning as substrates for PKMTs. The fast development of this field has moved the understanding of the biological outcome of lysine methylation into the center of research. Most urgently, it is necessary to improve our knowledge about lysine methylation by connecting specific target sites with the responsible PKMT and identifying the full substrate spectrum of PKMTs. In this thesis substrate specificity analysis was performed to tackle this challenge. It was shown that methylation of substrate specificity arrays is a good approach to analyze the substrate preference of PKMTs and identify subtle differences between related enzymes with same overall specificity. Furthermore, substrate specificity analysis was shown to be useful for the identification of novel substrates, which was successfully demonstrated for SUV4-20H1, SUV4-20H2, MLL1 and MLL3 in the present study. In vitro methylation experiments indicated that SUV4-20H1 and SUV4-20H2 introduce dimethylation on H4K20 using monomethylated H4K20 as substrate. SUV4-20H1 and SUV4-20H2 have an overlapping sequence motif, but SUV4-20H2 is less specific. This result was supported by the identification of one novel non-histone substrate for SUV4-20H1 and three non-histone targets for SUV4-20H2. MLL1 and MLL3 are H3K4 methyltransferases, but they belong to different MLL subfamilies. MLL1 catalyzes H3K4 trimethylation at promotors of developmental genes, whereas MLL3 introduces H3K4 monomethylation at enhancers. MLL1 and MLL3 are parts of related multi protein complexes also containing WDR5, RBBP5 and ASH2L. Substrate specificity analysis of MLL1 showed that it accepts several other residues at many positions of the target sequence, in addition to the residues in the original sequences of H3. At the protein level two novel substrates (TICRR and ZNF862) were methylated by MLL1. Comparison of the relative activity showed that the H3 protein was the best target in the absence of complex partners, but ZNF862 was preferred in presence of WRA. Finally, my data indicate that the substrate specificity of MLL3-WRA differed slightly from MLL1, suggesting that they may have different non-histone substrates. In several publications, assignments between PKMTs and methylated histone or non-histone target sites have been reported, but in some cases the data are questionable. This could lead to wrong interpretation of biological processes and misleading of follow-up studies. It has been shown for two examples in this study, that substrate specificity analysis can be used to identify problematic assignments between PKMT and methylation events, which need to be studied experimentally to confirm the published findings. Vougiouklakis et al. (2015) reported that SUV4-20H1 methylates ERK1 at K302 and K361, but these target sites do not fit to the specificity profile of SUV4-20H1. Indeed, I could not detect methylation of ERK1 by SUV4-20H1 or SUV4-20H2 at peptide and protein level although positive controls showed the expected methylation. Dhami et al. (2013) reported that Numb protein is methylated by SET8 at K158 and K163, which was not in agreement with the specificity data of SET8. In this thesis, Numb peptide and protein methylation was studied using recombinant SET8 purified from E.coli or HEK293 cells. In both cases, no methylation of Numb could be observed. These data suggest that these assignments of methylation substrates and PKMTs are likely not correct. Whole genome and whole transcriptome sequencing projects have frequently found somatic mutations in epigenetic enzymes in cancers. Somatic cancer mutations can have loss-of-function or gain-of-function effects on the enzymatic properties of PKMTs. Especially gain-of-function effects are a challenge in understanding their role in carcinogenesis. In this study, the effects of somatic cancer mutations found in the SET domain of MLL1 and MLL3 were analyzed. Four somatic cancer mutations of MLL1 and three of MLL3 were selected for analysis on the basis of their location close to binding sites of AdoMet, peptide or the interaction partners. The investigation of somatic cancer mutations in MLL1 and MLL3 indicated that each specific mutation has its unique effect on the enzymatic activity, product or substrate specificity and principle regulatory mechanism indicating that each mutant needs specific in depth experimental investigation in order to understand its carcinogenic effect. Moreover, inhibitor studies demonstrated that each mutant needs to be experimentally studied to allow for the development of mutation specific therapeutic strategies.