Browsing by Author "Brockmeyer, Jens"

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Open Access
Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns
(2022) Adam, Sabrina; Bräcker, Julia; Klingel, Viviane; Osteresch, Bernd; Radde, Nicole E.; Brockmeyer, Jens; Bashtrykov, Pavel; Jeltsch, Albert
TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.
Open Access
Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression
(2023) Broche, Julian; Köhler, Anja R.; Kühnel, Fiona; Osteresch, Bernd; Chandrasekaran, Thyagarajan T.; Adam, Sabrina; Brockmeyer, Jens; Jeltsch, Albert
While cytosine-C5 methylation of DNA is an essential regulatory system in higher eukaryotes, the presence and relevance of 6-methyladenine (m6dA) in human cells is controversial. To study the role of m6dA in human DNA, we introduced it in human cells at a genome-wide scale at GANTC and GATC sites by expression of bacterial DNA methyltransferases and observed concomitant reductions in cell viability, in particular after global GANTC methylation. We identified several genes that are directly regulated by m6dA in a GANTC context. Upregulated genes showed m6dA-dependent reduction of H3K27me3 suggesting that the PRC2 complex is inhibited by m6dA. Genes downregulated by m6dA showed enrichment of JUN family transcription factor binding sites. JUN binds m6dA containing DNA with reduced affinity suggesting that m6dA can reduce the recruitment of JUN transcription factors to target genes. Our study documents that global introduction of m6dA in human DNA has physiological effects. Furthermore, we identified a set of target genes which are directly regulated by m6dA in human cells, and we defined two molecular pathways with opposing effects by which artificially introduced m6dA in GANTC motifs can directly control gene expression and phenotypes of human cells.
Open Access
Identification and characterization of IgE‐reactive proteins and a new allergen (Cic a 1.01) from chickpea (Cicer arietinum)
(2020) Wangorsch, Andrea; Kulkarni, Anuja; Jamin, Annette; Spiric, Jelena; Bräcker, Julia; Brockmeyer, Jens; Mahler, Vera; Blanca‐López, Natalia; Ferrer, Marta; Blanca, Miguel; Torres, Maria; Gomez, Paqui; Bartra, Joan; García‐Moral, Alba; Goikoetxea, María J.; Vieths, Stefan; Toda, Masako; Zoccatelli, Gianni; Scheurer, Stephan
Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.
Open Access
Inter-laboratory validation of an HPLC-MS/MS method for the detection of microbial transglutaminase in meat and meat products
(2022) Jira, Wolfgang; Behnke, Thomas; Brockmeyer, Jens; Frost, Kirstin; Hiller, Ekkehard; Möllers, Manfred; Niedzwiecka, Alicia; Pöpping, Bert; Uhlig, Steffen; Weidner, Markus; Wittke, Stefan; Becker, René
Microbial transglutaminase (TG) is an enzyme isolated on an industrial scale from Streptomyces mobaraensis. Technical TG, a formulated powder, is primarily used to restructure meat in the meat-processing industry, typically at a 1% concentration and is often referred to as “meat glue.” In the European Union, meat restructured with TG requires the indication “formed meat” on the label according to Regulation (EU) No 1169/2011. In order to detect food fraud like the undeclared TG usage in meat and meat products, a qualitative mass spectrometric method using specific tryptic marker peptides has been published in 2017. Here the successful inter-laboratory validation and first-time standardization of a proteomics method for food control is described, which was subsequently included into the Official Collection of Analysis Methods according to the German Food and Feed Code (§ 64 LFGB). Thirteen laboratories from governmental, academic, and private institutions participated in the study, whereas four laboratories did not meet the minimal quality criteria and therefore their results had to be excluded. Three different test materials containing between 0.2 and 2% technical TG as well as blank samples were produced and tested. The laboratories used triple-quadrupole mass spectrometers from several vendors as well as quadrupole time-of-flight instruments. The detection of TG was considered to be positive, if three mass transitions for the marker peptides VTPPAEPLDR (TG-1) and SPFYSALR (TG-2), each, showed a signal-to-noise ratio of at least 3. The level of detection LOD95% for the median laboratory with intermediate performance was 0.31%, the false-positive rate was 0% and the false-negative rate was 2.1%.
Open Access
The lipidome of an omnivorous insect responds to diet composition and social environment
(2022) Gutiérrez, Yeisson; Fresch, Marion; Scherber, Christoph; Brockmeyer, Jens
Lipids are biomolecules with essential roles in metabolic processes, signaling, and cellular architecture. In this study, we investigated changes in the lipidome of the house cricket Acheta domesticus subjected to diets of different nutritional composition (i.e., protein to carbohydrate ratio) and two distinct social environments (i.e., solitary or in groups). We measured relative abundances of 811 lipid species in whole‐body cricket samples using flow injection analysis coupled to tandem mass spectrometry. We assessed differences in the relative abundances of lipid species induced by diet composition and social environment in female and male A. domesticus. Additionally, we performed a functional analysis of the lipids with significant differences using a recently developed database. We found that most differences in the relative abundances of lipid species were explained by sex alone. Furthermore, the lipidome of female A. domesticus was responsive to diet composition. Females fed with the balanced diet had an even higher abundance of lipids involved in lipid storage than their counterparts fed with a protein‐rich diet. Interestingly, the male cricket lipidome was not responsive to diet composition. In addition, the social environment did not induce significant changes in the lipid profile neither in female nor in male crickets.