Flierl U.,University of Melbourne |
Flierl U.,Hannover Medical School |
Nero T.L.,St. Vincents Institute |
Nero T.L.,University of Melbourne |
And 15 more authors.
Journal of Experimental Medicine | Year: 2015
Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics. © 2015 Flierl et al. Source
Roth H.M.,Rudolf Virchow Center for Experimental Biomedicine |
Romer J.,Rudolf Virchow Center for Experimental Biomedicine |
Grundler V.,Rudolf Virchow Center for Experimental Biomedicine |
Van Houten B.,University of Pittsburgh |
And 2 more authors.
DNA Repair | Year: 2012
Bax1 has recently been identified as a novel binding partner for the archaeal helicase XPB. We previously characterized Bax1 from Thermoplasma acidophilum as a Mg 2+-dependent structure-specific endonuclease. Here we directly compare the endonuclease activity of Bax1 alone or in combination with XPB. Using several biochemical and biophysical approaches, we demonstrate regulation of Bax1 endonuclease activity by XPB. Interestingly, incision assays with Bax1 and XPB/Bax1 clearly demonstrate that Bax1 produces different incision patterns depending on the presence or absence of XPB. Using atomic force microscopy (AFM), we directly visualize and compare binding of Bax1 and XPB/Bax1 to different DNA substrates. Our AFM data support enhanced DNA binding affinity of Bax1 in the presence of XPB. Taken together, the DNA incision and binding results suggest that XPB is able to load and position Bax1 on the scissile DNA substrate, thus increasing the DNA substrate range of Bax1. © 2011 Elsevier B.V.. Source
Elbashir R.,Rudolf Virchow Center for Experimental Biomedicine |
Vanselow J.T.,Rudolf Virchow Center for Experimental Biomedicine |
Kraus A.,University of Wurzburg |
Janzen C.J.,Biocenter University of Wuerzburg |
And 2 more authors.
Analytical Chemistry | Year: 2015
We introduce fragment ion patchwork quantification as a new mass spectrometry-based approach for the highly accurate quantification of site-specific acetylation degrees. This method combines 13C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Acetylation degrees are determined from the isotope patterns of acetylated b and y ions. We show that this approach allows to determine site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei. In addition, we demonstrate how this approach can be used to identify substrate sites of histone acetyltransferases. © 2015 American Chemical Society. Source
Atak S.,University of Wurzburg |
Langlhofer G.,University of Wurzburg |
Schaefer N.,University of Wurzburg |
Kessler D.,University of Wurzburg |
And 4 more authors.
Frontiers in Molecular Neuroscience | Year: 2015
Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-n interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR a1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 a1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. © 2015 Atak, Langlhofer, Schaefer, Kessler, Meiselbach, Delto, Schindelin and Villmann. Source