O changes in expression of the ribosomal proteins, hnRNPA1, and PTBP1 were observed. Thus, the down-regulation of these proteins in cytosol is terminated as an early effect of the clostridial cytotoxins. Protein glucosidase 2 (alpha and beta subunit, GANAB and PRKCSH), the chaperons endoplasmin (HSP90B1), protein disulfide-isomerases (P4HB and PDIA4) were induced after 5 h treatment with rTcdA wt. (Figure 4B, 5A, B). Endoplasmin is a glycoprotein [34] that has been linked to the Toll-like receptor pathway [35], response to hypoxia [36], and this protein is required for innate immunity but not cell viability [37]. It forms a large chaperone multiprotein complex with protein disulfide-isomerase A4 and other chaperonins and is essential for translocation of C. botulinum C2 toxin and uptake of C. perfringens iota toxin into eukaryotic cells [38,39]. Indeed, endoplasmin has been reported to bind to TcdA at the cell surface and is translocated together with TcdA into the cytoplasm [40]. We propose that toxin-dependent endoplasmin translocation from membrane into the cytoplasma led to an increased cytoplasmic concentration of this protein that was not observed after treatment with mutant rTcdA. Glucosidase 2 beta catalyzes the removal of three glucose residues from the peptide-bound Glc3-Man9-GlcNAc2 oligosaccharide remodelling the asparagine-linked precursor [41] and UDP-glucose:glycoprotein glycosyltransferase. The latter plays a possible role in control of protein glycosylation [42] and regulation of N-glycoprotein transport or protein degradation [43,44]. These data indicate that posttranslational modifications like glycosylation are influenced by large clostridial toxins. However, after 24 h treatment with rTcdA wt glucosidase 2 beta decreased by 2-fold.Jochim et al. Proteome Science 2011, 9:48 http://www.proteomesci.com/content/9/1/Page 9 ofAfter the long-term treatment of cells with rTcdA wt several cytoskeletal proteins (beta-, gamma-actin, filamine A and B, villin 1) were down regulated. This is in good accordance to the cytopathic cell rounding of rTcdA wt treated cells. Interestingly, filamine A was also down-regulated by rTcdA mutant. Thus, a Rho-independent signaling should be involved in filamine regulation. Peroxiredoxins 1, 2, 6 were found down-regulated after treatment with rTcdA wt for 24 h; peroxiredoxins 1 and 2 were up-regulated after short time incubation with mutant rTcdA (Figure 4A, C). Kim et.al. reported that C. difficile Toxin A induces the release of reactive oxygen species (ROS) and influences the regulation of cyclooxigenase-2 and prostaglandin E2 synthesis by ROS [9,45]. The expression changes of peroxiredoxins are involved in redox control and might be induced after 5 h to protect cells from ROS. Down regulation of these protecting enzymes after 24 h of toxin treatment might be a hint that the cells have become more apoptotic. The presented data indicate that large clostridial cytotoxins like TcdA act not only on Rho proteins and induce Rho-dependent pathways. Rho-independent effects evoked by a catalytic inactive toxin mutant point to new cellular target structures Vorinostat in eukaryotic cells and so far unknown ways of signaling. For the first PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 time evidence has been provided that TcdA influences more cellular functions than cytoskeleton homeostasis and apoptosis. The response of colonic cells to TcdA is time-dependent and different protein patterns are evident after short (5 h) and long (24 h) incubation times. Activit.