Markoutsa S.,Goethe University Frankfurt |
Surun D.,University of Frankfurt Medical School |
Karas M.,Goethe University Frankfurt |
Hofmann B.,Goethe University Frankfurt |
And 2 more authors.
FEBS Journal | Year: 2014
The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites. © 2014 FEBS.
Brendel C.,Paul Ehrlich Institute |
Muller-Kuller U.,Paul Ehrlich Institute |
Muller-Kuller U.,University of Frankfurt Medical School |
Schultze-Strasser S.,Paul Ehrlich Institute |
And 7 more authors.
Gene Therapy | Year: 2012
Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site. © 2012 Macmillan Publishers Limited All rights reserved.
Heidler J.,University of Frankfurt Medical School |
Strecker V.,Goethe University Frankfurt |
Csintalan F.,Goethe University Frankfurt |
Bleier L.,Goethe University Frankfurt |
Wittig I.,Goethe University Frankfurt
Methods in Molecular Biology | Year: 2013
Blue native electrophoresis (BNE) is a long established method for the analysis of native protein complexes. Applications of BNE range from investigating subunit composition, stoichiometry, and assembly of single protein complexes to profiling of whole complexomes. BNE is an indispensible tool to diagnostically analyze cells and tissues from patients with mitochondrial disorders or model organisms. Since functional proteomic studies often require quantification of protein complexes, we describe here different quantification methods subsequent to protein complex separation by BNE. © 2013 Springer Science+Business Media, LLC.
Schneider K.,Albert Ludwigs University of Freiburg |
Weyerbrock A.,Albert Ludwigs University of Freiburg |
Doostkam S.,Albert Ludwigs University of Freiburg |
Plate K.,University of Frankfurt Medical School |
Machein M.R.,Albert Ludwigs University of Freiburg
Journal of Neuro-Oncology | Year: 2015
Placenta growth factor (PlGF) is a member of vascular endothelial growth factor family which can promote cancer growth by various mechanisms. Placenta growth factor is upregulated in many neoplastic diseases and serum levels of PlGF are increased in cancer patients following anti-angiogenic therapy. However, its role in glioma growth is yet not fully elucidated. In this study we analyzed the expression of PlGF mRNA using real time PCR in human gliomas of different WHO grades. Placenta growth factor mRNA levels were highly variable and did not correlate with WHO grades, arguing against a significant role in glioma progression. The highest PlGF expression was observed in anaplastic astrocytomas whereas grade II astrocytomas and glioblastomas displayed lower levels of expression. Immunohistochemical analysis showed that PlGF was expressed by inflammatory and endothelial cells in addition to tumor cells. Placenta growth factor mRNA expression in 12 matched glioblastoma samples before and after therapy, including bevacizumab and cilengitide treatment was largely unaffected by the aforementioned treatment modalities. In vitro, the exposure of VEGFR-1 expressing glioma cells to bevacizumab did not increase the expression levels of PlGF mRNA. In summary, our results do not support the hypothesis that PlGF plays a major role in the resistance of gliomas after anti-angiogenic therapy. © 2014, Springer Science+Business Media New York.
University Of Frankfurt Medical School | Date: 2010-11-24
The present invention relates to a method of producing a cell comprising a conditionally active transgene in its genome, the method comprising (a) introducing into the cell a targeting vector, wherein the targeting vector comprises (i) a 5 recombinase recognition site specifically recognised by a first recombinase, wherein the first recombinase is endogenously present in the cell or wherein the first recombinase or a nucleic acid molecule encoding said first recombinase in expressible form is introduced into the cell; followed by (ii) a 5 recombinase recognition site specifically recognised by a second recombinase, wherein the second recombinase is not endogenously present or is not active in the cell; followed by (iii) a selection cassette comprising a positively selectable marker gene; followed by (iv) a 3 recombinase recognition site specifically recognised by a third recombinase, wherein the third recombinase is not endogenously present or is not active in the cell; followed by (v) the transgene; followed by (vi) a 3 recombinase recognition site specifically recognised by a fourth recombinase, wherein the fourth recombinase is endogenously present in the cell or wherein the fourth recombinase or a nucleic acid molecule encoding said fourth recombinase in expressible form is introduced into the cell; wherein the genome of the cell comprises a 5 recombinase recognition site and a 3 recombinase recognition site that are identical to the recombinase recognition sites of (i) and (vi), and wherein said recombinase recognition sites comprised in the genome of the cell are located 3 of an endogenous cellular promoter such that introduction of the targeting vector into the genome by site specific recombination results in the promoter being operatively linked to the selectable marker gene; and (b) culturing the cell in the presence of a selection medium specific for the selectable marker encoded by the selectable marker gene of (iii). The present invention further relates to a method of producing a conditional transgenic non-human mammalian animal as well as to a conditional transgenic non-human mammalian animal obtainable by said method. The present invention also relates to a transgenic TDP-43 mouse, comprising a transgenic cassette in intron 1 of the mouse Tardbp gene.