TY  - JOUR
A1  - Gassen, Alwine
A1  - Brechtefeld, Doris
A1  - Schandry, Niklas
A1  - Arteaga-Salas, J. Manuel
A1  - Israel, Lars
A1  - Imhof, Axel
A1  - Janzen, Christian J.
T1  - DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei
JF  - Nucleic Acids Research
N2  - Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.
KW  - variants
KW  - cell-cycle regulation
KW  - blood-stream forms
KW  - african trypanosomes
KW  - mammalian cells
KW  - DNA replication
KW  - DOT1
KW  - protein
KW  - transcription
KW  - cultivation
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131449
VL  - 40
IS  - 20
ER  - 
TY  - JOUR
A1  - Dmochewitz, Lydia
A1  - Förtsch, Christina
A1  - Zwerger, Christian
A1  - Vaeth, Martin
A1  - Felder, Edward
A1  - Huber-Lang, Markus
A1  - Barth, Holger
T1  - A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages
JF  - PLoS ONE
N2  - Background: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. 
Methodology/Principal Findings: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (~50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.
Conclusions/Significance: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.
KW  - C3
KW  - mammalian cells
KW  - clostridium botulinum
KW  - ADP-ribosylation toxins
KW  - botulinum C2 toxin
KW  - membrane translocation
KW  - bacterial toxins
KW  - actin filaments
KW  - cellular uptake
KW  - ribosyltransferase
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131189
VL  - 8
IS  - 1
ER  -