Rudolf Virchow Center for Experimental Biomedicine

Würzburg, Germany

Rudolf Virchow Center for Experimental Biomedicine

Würzburg, Germany
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News Article | February 15, 2017
Site: www.eurekalert.org

The small protein ubiquitin regulates a plethora of physiological and pathophysiological processes in the human body. It lives up to its name quite literally by being ubiquitous, both in terms of its abundance and its far-reaching regulatory impact. How ubiquitin exerts its diverse functions is intensely studied all over the world. Finding answers to this question is essential to exploit the ubiquitin system efficiently for therapeutic purposes. Researchers from Würzburg have taken a key step towards this goal. Their results reveal new ways of regulating a ubiquitin ligase. "Ubiquitin ligases are enzymes that decorate cellular target proteins with ubiquitin and thus determine the fate of these target proteins," says Dr. Sonja Lorenz, senior author on the study. Ubiquitin can act as a "molecular postal code" that can guide target proteins to specific locations in the cell, lead them to serve distinct functions, carry molecular signals, integrate into large complexes, or even be destroyed. Sonja Lorenz heads a research group at the Rudolf Virchow Center for Experimental Biomedicine at the University of Würzburg. Her team and colleagues study a particular ubiquitin ligase, HUWE1, that has been ascribed key roles in tumor formation and is considered a promising, yet unexploited cancer-therapeutic target. Their new results on the molecular mechanism of HUWE1 are reported in the journal eLife. With almost 4.400 amino acids HUWE1 is an extremely large protein. Its three-dimensional structure, for the most part, is unknown. "The enormous size of HUWE1 and its flexibility present a considerable challenge for structural biologists," says Sonja Lorenz. To get a handle on the protein giant, her research team followed the ancient Roman principle "divide et impera - divide and rule" and has initially determined the atomic structure of a portion of HUWE1 using X-ray crystallography. This structure reveals a new and intriguing feature of HUWE1: Two HUWE1 molecules can pair up to form a complex known as a "dimer", thereby shutting down their enzymatic activities. How does the cell prevent HUWE1 from forming dimers when the enzyme needs to be active? The Würzburg researchers also provide an answer to this question: HUWE1 exists in a fine-tuned balance of inactive dimers and single, active molecules. "Various cellular factors can regulate this balance," says Sonja Lorenz. The tumor suppressor protein p14ARF is one such factor. It inhibits HUWE1, but is frequently lost in cancer cells. The new study provides the first mechanistic explanation of how p14ARF inhibits HUWE1. "The effects of p14ARF on the structure and activity of HUWE1 are extremely exciting," says Sonja Lorenz. "They open up a range of possibilities to manipulate HUWE1 activity that we are following up on." Dr. Sonja Lorenz holds an Emmy Noether grant from the German Research Foundation with which she established her lab at the Rudolf Virchow Center of the University of Würzburg in April 2014. She is the deputy speaker of the new Research Training Group 2243, "Understanding Ubiquitylation: From Molecular Mechanisms to Disease", that will start in April 2017. Her studies on the interplay of HUWE1 and p14ARF are supported by the Wilhelm Sander-Foundation for medical research. The human ubiquitin ligase HUWE1 is regulated by a conformational switch. Bodo Sander, Wenshan Xu, Martin Eilers, Nikita Popov, Sonja Lorenz. DOI: 10.7554/eLife.21036


Flierl U.,University of Melbourne | Flierl U.,Hannover Medical School | Nero T.L.,St. Vincent's 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.


PubMed | Hannover Medical School, Monash University, French Institute of Health and Medical Research, Rudolf Virchow Center for Experimental Biomedicine and 3 more.
Type: Journal Article | Journal: The 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.


Atak S.,University of Würzburg | Langlhofer G.,University of Würzburg | Schaefer N.,University of Würzburg | Kessler D.,University of Würzburg | 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.


Elbashir R.,Rudolf Virchow Center for Experimental Biomedicine | Vanselow J.T.,Rudolf Virchow Center for Experimental Biomedicine | Kraus A.,University of Würzburg | 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.


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..


PubMed | Rudolf Virchow Center for Experimental Biomedicine
Type: Journal Article | Journal: 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-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.


News Article | February 15, 2017
Site: phys.org

The small protein ubiquitin regulates many physiological and pathophysiological processes in the human body. It lives up to its name quite literally by being ubiquitous, both in terms of its abundance and its far-reaching regulatory impact. How ubiquitin exerts its diverse functions is intensely studied all over the world. Finding answers to this question is essential to exploit the ubiquitin system efficiently for therapeutic purposes. Researchers from Würzburg have taken a key step toward this goal. Their results reveal new ways of regulating a ubiquitin ligase. "Ubiquitin ligases are enzymes that decorate cellular target proteins with ubiquitin and thus determine the fate of these target proteins," says Dr. Sonja Lorenz, senior author on the study. Ubiquitin can act as a "molecular postal code" to guide target proteins to specific locations in the cell, lead them to serve distinct functions, carry molecular signals, integrate into large complexes, or even be destroyed. Sonja Lorenz heads a research group at the Rudolf Virchow Center for Experimental Biomedicine at the University of Würzburg. Her team and colleagues study a particular ubiquitin ligase, HUWE1, that has been ascribed key roles in tumor formation and is considered a promising, yet unexploited cancer-therapeutic target. Their new results on the molecular mechanism of HUWE1 are reported in the journal eLife. With almost 4,400 amino acids, HUWE1 is an extremely large protein. Its three-dimensional structure, for the most part, is unknown. "The enormous size of HUWE1 and its flexibility present a considerable challenge for structural biologists," says Sonja Lorenz. To get a handle on the protein giant, her research team followed the ancient Roman principle "Divide and rule," and has initially determined the atomic structure of a portion of HUWE1 using X-ray crystallography. This structure reveals a new and intriguing feature of HUWE1: Two HUWE1 molecules can pair up to form a complex known as a "dimer," thereby shutting down their enzymatic activities. How does the cell prevent HUWE1 from forming dimers when the enzyme needs to be active? The Würzburg researchers also provide an answer to this question: HUWE1 exists in a fine-tuned balance of inactive dimers and single, active molecules. "Various cellular factors can regulate this balance," says Sonja Lorenz. The tumor suppressor protein p14ARF is one such factor. It inhibits HUWE1, but is frequently lost in cancer cells. The new study provides the first mechanistic explanation of how p14ARF inhibits HUWE1. "The effects of p14ARF on the structure and activity of HUWE1 are extremely exciting," says Sonja Lorenz. "They open up a range of possibilities to manipulate HUWE1 activity that we are following up on." Explore further: Too much protein HUWE1 causes intellectual disability More information: Bodo Sander et al, A conformational switch regulates the ubiquitin ligase HUWE1, eLife (2017). DOI: 10.7554/eLife.21036

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