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  - 
TY  - JOUR
A1  - Mueller, Kerstin
A1  - Quandt, Jasmin
A1  - Marienfeld, Ralf B.
A1  - Weihrich, Petra
A1  - Fiedler, Katja
A1  - Claussnitzer, Melina
A1  - Laumen, Helmut
A1  - Vaeth, Martin
A1  - Berberich-Siebelt, Frederike
A1  - Serfling, Edgar
A1  - Wirth, Thomas
A1  - Brunner, Cornelia
T1  - Octamer-dependent transcription in T cells is mediated by NFAT and \(NF-\kappa B\)
JF  - Nucleic Acids Research
N2  - The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and \(NF-\kappa B\)-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/\(NF-\kappa B\) sites. An array of genetic and biochemical analyses illustrates the involvement of the \(Ca^{2+}\)/calmodulin-dependent phosphatase calcineurin as well as NFAT and \(NF-\kappa B\) transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or \(NF-\kappa B\) transcription factors.
KW  - germinal center formation
KW  - OBF-1 OCA-B
KW  - coactivator OBF-1
KW  - gene expression
KW  - functional characterization
KW  - immunoglobulin promoters
KW  - OCT-1-deficient mice
KW  - embryonic lethality
KW  - endothelial cells
KW  - murine homolog
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123280
SN  - 1362-4962
VL  - 41
IS  - 4
ER  -