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In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that β-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase. Originally included in thesis in manuscript form.

PMT is a protein toxin produced by Pasteurella multocida serotypes A and D. As causative agent of atrophic rhinitis in swine, it leads to rapid degradation of the nasal turbinate bone. The toxin acts as a deamidase to modify a crucial glutamine in heterotrimeric G proteins, which results in constitutive activation of the G proteins and permanent stimulation of numerous downstream signaling pathways.Using a lentiviral based genome wide CRISPR knockout screen in combination with a lethal toxin chimera, consisting of full length inactive PMT and the catalytic domain of diphtheria toxin, we identified the LRP1 gene encoding the Low-Density Lipoprotein Receptor-related protein 1 as a critical host factor for PMT function. Loss of LRP1 reduced PMT binding and abolished the cellular response and deamidation of heterotrimeric G proteins, confirming LRP1 to be crucial for PMT uptake. Expression of LRP1 or cluster 4 of LRP1, respectively, restored intoxication of the knockout cells. In summary our data demonstrate secretory cells as entry site of PMT into airway epithelia and present LRP1 as crucial host entry factor for PMT intoxication by acting as its primary cell surface receptor.

Prostate cancer represents one of the leading causes of morbidity and mortality of men worldwide. In precision medicine, tumors are screened for specific genetic alterations known as predictive markers for targeted therapy. In androgen-independent prostate cancer cells and in tissue samples of a prostate cancer patient treated with Goserelin, we identified the self-activating splice variant Rac1b. Importantly, the expression of Rac1b was sufficient to induce AR-dependent gene synthesis. We hypothesized that Rac1b antagonizes androgen depletion induced cancer cell death by blocking pro-apoptotic signalling pathways. In line with that selective knockdown of Rac1b or inhibition of Rac-dependent signalling pathways reinduced apoptosis in androgen-independent prostate cancer cells suggesting Rac1b inhibition as a potential novel therapeutic add on strategy against prostate cancer.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.

Photorhabdus luminescens Tc toxins are large tripartite ABC-type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-acetylglucosamine transferase I (Mgat1 gene product)-deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N-glycans. Restoring Mgat1 gene activity, and hence complex N-glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild-type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan-binding and uptake into target cells. The data suggest that TcdA1-binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogenYersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author SummaryThe RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) fromYersinia pseudotuberculosisis a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.

Clostridium perfringens toxin TpeL belongs to the family of large clostridial glycosylating toxins. The toxin causes N-acetylglucosaminylation of Ras proteins at threonine35 thereby inactivating the small GTPases. Here, we show that all main types of oncogenic Ras proteins (H-Ras, K-Ras and N-Ras) are modified by the toxin in vitro and in vivo. Toxin-catalyzed modification of Ras was accompanied by inhibition of the MAP kinase pathway. Importantly, TpeL inhibited the paradoxical activation of the MAP kinase pathway induced by the BRAF inhibitor Vemurafenib in the human melanoma cell line SBCL2. The toxin also blocked Ras signaling in a zebrafish embryo model expressing oncogenic H-Ras$^{G12V}$, resulting in a reduction of melanocyte number. By using the binding and translocation component of anthrax toxin (protective antigen), the glucosyltransferase domain of TpeL was effectively introduced into target cells that were not sensitive to native TpeL toxin. To reach a higher specificity towards cancer cells, a chimeric TpeL toxin was engineered that possessed the knob region of adenovirus serotype 35 fiber, which interacts with CD46 of target cells frequently overexpressed in cancer cells. The chimeric TpeL fusion toxin efficiently inhibited Ras and MAP kinases in human pancreatic cancer Capan-2 cells, which were insensitive to the wild-type toxin. The data reveal that TpeL and TpeL-related immunotoxins provide a new toolset as Ras-inactivating agents.

// Sarah Lang 1 , Hauke Busch 2, 3 , Melanie Boerries 3, 4 , Tilman Brummer 3, 4, 5 , Sylvia Timme 6 , Silke Lassmann 4, 5, 6 , Klaus Aktories 1 and Gudula Schmidt 1 1 Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany 2 Lubeck Institute of Experimental Dermatology, Institute for Cardiogenetics, University of Lubeck, Lubeck, Germany 3 Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 German Cancer Consortium (DKTK), Freiburg, Germany, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Center for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany 6 Institute for Surgical Pathology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany Correspondence to: Gudula Schmidt, email: Gudula.Schmidt@pharmakol.uni-freiburg.de Keywords: Rho GTPase, invasion, cyclooxygenase, breast cancer Received: June 14, 2017 Accepted: August 26, 2017 Published: September 16, 2017 ABSTRACT Rho GTPases are regulators of many cellular functions and are often dysregulated in cancer. However, the precise role of Rho proteins for tumor development is not well understood. In breast cancer, overexpression of RhoC is linked with poor prognosis. Here, we aim to compare the function of RhoC and its homolog family member RhoA in breast cancer progression. We established stable breast epithelial cell lines with inducible expression of RhoA and RhoC, respectively. Moreover, we made use of Rho-activating bacterial toxins (Cytotoxic Necrotizing Factors) to stimulate the endogenous pool of Rho GTPases in benign breast epithelial cells and simultaneously knocked down specific Rho proteins. Whereas activation of Rho GTPases was sufficient to induce an invasive phenotype in three-dimensional culture systems, overexpression of RhoA or RhoC were not. However, RhoC but not RhoA was required for invasion, whereas RhoA and RhoC equally regulated proliferation. We further identified downstream target genes of RhoC involved in invasion and identified PTGS2 (COX-2) being preferentially upregulated by RhoC. Consistently, the COX-2 inhibitor Celecoxib blocked the invasive phenotype induced by the Rho-activating toxins.

// Kristine Ostevold 1, 2 , Ana V. Melendez 1 , Friederike Lehmann 1, 3, 4 , Gudula Schmidt 1 , Klaus Aktories 1, 5 and Carsten Schwan 1 1 Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, University of Freiburg, 79104 Freiburg, Germany 2 Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany 3 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104 Freiburg, Germany 4 Faculty of Chemistry and Pharmacy, University of Freiburg, 79104 Freiburg, Germany 5 Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany Correspondence to: Carsten Schwan, email: carsten.schwan@pharmakol.uni-freiburg.de Klaus Aktories, email: klaus.aktories@pharmakol.uni-freiburg.de Keywords: septin, microtentacles, microtubules, Clostridium difficile toxin, actin ADP-ribosylation Received: June 21, 2017 Accepted: August 17, 2017 Published: September 11, 2017 ABSTRACT Microtentacles are mostly microtubule-based cell protrusions that are formed by detached tumor cells. Here, we report that the formation of tumor cell microtentacles depends on the presence and dynamics of guanine nucleotide-binding proteins of the septin family, which are part of the cytoskeleton. In matrix-attached breast, lung, prostate and pancreas cancer cells, septins are associated with the cytosolic actin cytoskeleton. Detachment of cells causes redistribution of septins to the membrane, where microtentacle formation occurs. Forchlorfenuron, which inhibits septin functions, blocks microtentacle formation. The small GTPase Cdc42 and its effector proteins Borgs regulate septins and are essential for microtentacle formation. Dominant active and inactive Cdc42 inhibit microtentacle formation indicating that the free cycling of Cdc42 between its active and inactive state is essential for septin regulation and microtentacle formation. Cell attachment and aggregation models suggest that septins play an essential role in the metastatic behavior of tumor cells.

The article shows successful engineering of the syringe-like injection apparatus of Photorhabdus luminescens as standalone nanomachine for delivery of protein toxins and enzymes from different species into mammalian cells., Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches.Photorhabdus luminescenstoxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe like nano-machine for delivery of protein toxins from different species. Additionally, we loaded the highly active copepod luciferaseMetridia longaM-Luc7 for accurate quantification of injected molecules. We suggest that besides the size also the charge of the cargo defines the efficiency of packing and transport into mammalian cells. Our data show that thePhotorhabdus luminescenstoxin complex constitutes a powerful system to inject recombinant proteins, peptides and potentially other molecules like aptamers into mammalian cells. In contrast to other protein transporters based on pore formation, the cargo is protected from degradation. The system opens new perspectives for cell research and pharmacology.

Background Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. Methods We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Results Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Conclusions Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies.

Bacterial pathogens developed several strategies to overcome defense systems of eukaryotic hosts. Within the infection process they need to attach to and cross through epithelial layers, escape from the innate and adaptive immune response, and find a physiological niche to survive. For this purpose bacteria developed toxins that specifically target central eukaryotic proteins, for example actin or Rho GTPases as regulators of the actin cytoskeleton. Some bacterial toxins catalyze a covalent modification of Rho GTPases to keep these molecular switches in a constitutive active or inactive state. This leads to rearrangement of the actin cytoskeleton. Toxin-treated cells show typical morphological changes depending on substrate specificity and action of the toxins. In this chapter I describe methods to illustrate how bacterial toxins may help to study the involvement of Rho GTPases in physiological and pathophysiological processes.

Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, pro-inflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients.

Bacterial pathogens developed several strategies to overcome defense systems of eukaryotic hosts. Within the infection process they need to attach to and cross through epithelial layers, escape from the innate and adaptive immune response, and find a physiological niche to survive. For this purpose bacteria developed toxins that specifically target central eukaryotic proteins, for example actin or Rho GTPases as regulators of the actin cytoskeleton. Some bacterial toxins catalyze a covalent modification of Rho GTPases to keep these molecular switches in a constitutive active or inactive state. This leads to rearrangement of the actin cytoskeleton. Toxin-treated cells show typical morphological changes depending on substrate specificity and action of the toxins. In this chapter I describe methods to illustrate how bacterial toxins may help to study the involvement of Rho GTPases in physiological and pathophysiological processes.

Summary PTC3 and PTC5 are tripartite Tc (toxin complex) toxins from Photorhabdus luminescens, which consist of the binding component TcdA1, the linker component TcdB2 and the enzyme components TccC3 and TccC5 respectively. While PTC5 adenosine diphosphate (ADP)-ribosylates Rho proteins at Gln61/63 resulting in constitutive activation of the GTPases, PTC3 ADP-ribosylates actin at Thr148 thereby inducing actin polymerization. Here, we identified amino acids involved in ADP-ribosyltransferase activity of TccC3 and TccC5 and analysed the substrate specificity of Rho-activating TccC5. We compared the time dependency of Rho protein activation by PTC5 in HeLa cells with the effects of Escherichia coli cytotoxic necrotizing factor 1, which activates Rho GTPases by deamidation of Gln61/63. Using a luciferase reporter assay, we show that PTC5 and PTC3 stimulated gene transcription via myocardin-related transcription factor A (also called MAL) and AP1. MAL activation by PTC5 involved Rho kinase and formins. Activation of AP1 by PTC5 occurred via two MAP kinase pathways involving extracellular signal-regulated kinase and Jun kinase respectively.

ErbB family receptor tyrosine kinases (ErbBs) play a role in cell adhesion and migration and are frequently overexpressed in esophageal squamous cell carcinomas (ESCCs) or esophageal adenocarcinomas (EACs). Targeting ErbBs by tyrosine kinase inhibitors (TKIs) may therefore limit esophageal cancer cell migration. Here, we studied the impact of TKIs on ErbB dimerization, cell signaling pathways, and cell migration in three esophageal cell lines: OE21 (ESCC), OE33 (EAC), and Het-1A (non-neoplastic esophageal epithelium). In OE21 cells, the TKIs erlotinib, gefitinib, and lapatinib slightly affected epidermal growth factor receptor EGFR/EGFR, but not EGFR/HER2 dimerization as detected by in situ proximity ligation assay (in situ PLA). Still, TKIs inhibited ERK1/2, Akt, STAT3, and RhoA activity in OE21 cells, as assessed by Western blot, antibody arrays, and Rho GTPase effector pull-down assays. This was accompanied by reduced OE21 cell migration, induction of focal adhesions, and actin cytoskeleton reorganization, as shown by Oris™ migration assay and focal adhesion kinase (FAK)/phalloidin staining. In contrast, in OE33 cells, only lapatinib decreased STAT5, Src family kinase (SFK), and FAK activity as well as β-catenin expression. This impeded cell migration and induced morphological changes in OE33 cells. No alterations were seen for the non-neoplastic Het-1A cells. Thus, we identified the ErbB signaling network as regulator of esophageal cancer cell’s actin cytoskeleton, focal adhesions, and cell migration. ErbB targeted TKIs therefore also limit ESCC and EAC cell motility and migration. • Clinical tyrosine kinase inhibitors (TKIs) reduce esophageal cancer cell migration. • Loss of cell migration is linked to reduced Akt, ERK1/2, STAT (3 or 5), FAK, SFKs, and RhoA activity. • Clinical TKIs act via distinct signaling in the two main histotypes of esophageal cancer.

Kindlins are a family of integrin adapter and cell–matrix adhesion proteins causally linked to human genetic disorders. Kindlin-2 is a ubiquitously expressed protein with manifold functions and interactions. The contribution of kindlin-2 to integrin-based cell–matrix adhesions has been extensively explored, while other integrin-independent roles emerge. Because of the early involvement of kindlin-2 in development, no viable animal models with its constitutional knockout are available to study its physiological functions in adult skin. Here, we uncovered a critical physiological role of kindlin-2 in the epidermis by using a skin-equivalent model with shRNA-mediated knock-down of kindlin-2 in keratinocytes. Kindlin-2-deficient keratinocytes built stratified epidermal layers, but displayed impaired dermal–epidermal and intra-epidermal adhesion and barrier function. Co-immunoprecipitation studies demonstrated that kindlin-2 interacts with both integrin- and cadherin-based adhesions. In kindlin-2-deficient keratinocytes, reduced cell–cell adhesion was associated with abnormal cytoplasmic distribution of adherens junctions and desmosomal proteins, which was dependent on RhoA activation. Direct activation of RhoA with recombinant bacterial cytotoxic necrotizing factor y (CNFy) reverted the abnormal phenotype and barrier function of kindlin-2-deficient keratinocytes and skin equivalents. These findings have physiological and pathological significance, since kindlin-2 expression modulates the phenotype in Kindler syndrome, a skin fragility disorder caused by kindlin-1 deficiency. Our results suggest that pharmacological regulation of RhoGTPase activity may represent a therapeutic option for skin fragility. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

RhoA is reportedly involved in signal transducers and activators of transcription (STAT)-dependent transcription. However, the pathway connecting the GTPase and STAT signaling has not been characterized. Here, we made use of bacterial toxins, which directly activate Rho GTPases to analyze this pathway. Cytotoxic necrotizing factors (CNFs) are produced by pathogenic Escherichia coli strains and by Yersinia pseudotuberculosis. They activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. We show that RhoA activation leads to phosphorylation and activation of STAT3 and identify signal proteins involved in this pathway. RhoA-dependent STAT3 stimulation requires ROCK and Jun kinase activation as well as AP1-induced protein synthesis. The secretion of one or more factors activates the JAK-STAT pathway in an auto/paracrine manner. We identify CCL1/I-309 as an essential cytokine, which is produced and secreted upon RhoA activation and which is able to activate STAT3-dependent signaling pathways.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

The Escherichia coli Cytotoxic Necrotizing Factors, CNF1, CNF2, CNF3 and CNFY from Yersinia pseudotuberculosis belong to a family of deamidating toxins. CNFs deamidate glutamine 63/61 in the switch II region of Rho GTPases that is essential for GTP hydrolysing activity. Deamidation leads to constitutive activation of Rho GTPases. However, cellular mechanisms like proteasomal degradation of the activated Rho proteins restrict the action of the GTPases. This review describes the differences between the toxin family members concerning expression, cellular entry and substrate specificity.

RhoA is a small GTPase shown to be crucial for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. Analyzing mice with a keratinocyte-restricted deletion of the RhoA gene, we find that RhoA is not required for skin development and maintenance but has specific functions in vitro., RhoA is a small guanosine-5’-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell–cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC–mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

Photorhabdus luminescens produces several types of protein toxins, which are essential for participation in a trilateral symbiosis with nematodes and insects. The nematodes, carrying the bacteria, invade insect larvae and release the bacteria, which kill the insects with their toxins. Recently, the molecular mechanisms of the toxin complexes PTC3 and PTC5 have been elucidated. The biologically active components of the toxin complexes are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively. The actions of the toxins are described and compared with other bacterial protein toxins acting on the cytoskeleton.

To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation.

The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin alpha2 (KPNA2) as a direct interaction partner of Rac1. The C-terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2-dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.

The Escherichia coli Cytotoxic Necrotizing Factors, CNF1, CNF2 and CNFY from Yersinia pseudotuberculosis belong to a family of deamidating toxins. CNFs deamidate glutamine 63/61 in the switch II region of Rho GTPases that is essential for GTPase activity. Recently, a novel member of the CNF family has been described in Necrotoxigenic E. coli (NTEC) from sheep and goats which shares about 70% sequence identity with CNF1. Here we cloned, expressed, purified and characterized the toxin with respect to substrate specificity and uptake into different cell lines. As reported for CNF1 and CNF2, CNF3 deamidates RhoA, Rac and Cdc42. However, the toxin activates RhoA much stronger as compared to CNF1. The uptake of the toxin is restricted to the same cell lines, which also respond to CNFY.

Low molecular weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. RhoA governs the formation of actin stress fibers and is responsible for the formation of the contractile ring in cytokinesis. Cytokinesis completion requires RhoA inactivation resulting in disassembly of the contractile ring. Cytokinesis thus requires switching of RhoA activity. This switch of RhoA activity is blocked by Rho-modifying bacterial protein toxins that either activate or inactivate RhoA by covalent modifications. Exoenzyme C3 from Clostridium limosum (C3-lim) and Clostridium difficile toxin B (TcdB) inactivate RhoA by mono-ADP-ribosylation and mono-glucosylation, respectively. Cytotoxic necrotizing factors (CNF), produced by either Yersinia pseudotuberculosis (CNFY) or uropathogenic strains of E. coli (CNF1), deamidate and thereby activate RhoA. This study provides evidence that RhoA-activating as well as RhoA-inactivating toxins cause inhibition of cytokinesis and cell division. The toxins' effects on cytokinesis were analyzed in Hela cells synchronized using the thymidine double block technique. Treatment of G2-phase cells with either the RhoA-activating CNFY or CNF1 or the RhoA-inactivating C3-lim or TcdB resulted in cytokinesis inhibition, as evidenced by the formation of a 4N population on flow cytometry, the inhibition of contractile ring formation, and the formation of bi-nucleated cells. While TcdB and CNF1 modify a broad-spectrum of Rho proteins, C3-lim and CNFY specifically target RhoA. Since C3-lim and CNFY both caused cytokinesis inhibition, our study re-inforces the critical role of RhoA (not Rac1 or Cdc42) in cytokinesis and cell division.

// Bernd A. Czulkies 1 , Justin Mastroianni 2 , Lisa Lutz 3 , Sarah Lang 1 , Carsten Schwan 1 , Gudula Schmidt 1 , Silke Lassmann 3, 4, 5 , Robert Zeiser 2, 5 , Klaus Aktories 1, 5, 6 , Panagiotis Papatheodorou 1, 7, 8 1 Institute of Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs-University (ALU), Freiburg, Germany 2 Department of Hematology and Oncology, University Medical Center, ALU, Freiburg, Germany 3 Department of Pathology, University Medical Center, ALU, Freiburg, Germany 4 German Consortium for Translational Cancer Research (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Centre for Biological Signalling Studies (BIOSS), ALU, Freiburg, Germany 6 Freiburg Institute for Advanced Studies (FRIAS), ALU, Freiburg, Germany 7 Present address: Institute of Pharmaceutical Biotechnology. University of Ulm, Ulm, Germany 8 Present address: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany Correspondence to: Panagiotis Papatheodorou, email: panagiotis.papatheodorou@pharmakol.uni-freiburg.de Keywords: tumor growth, xenograft, epithelial barrier, cell morphology, cell-cell contact Received: March 21, 2016 Accepted: June 13, 2016 Published: July 06, 2016 ABSTRACT The lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor, serves as host receptor for clostridial iota-like toxins and is involved in the formation of tricellular contacts. Of particular interest is the role of LSR in progression of various cancers. Here we aimed to study the tumor growth of LSR-deficient colon carcinoma-derived cell lines HCT116 and CaCo-2 in a mouse xenograft model. Whereas knockout of LSR had no effect on tumor growth of HCT116 cells, we observed that CaCo-2 LSR knockout tumors grew to a smaller size than their wild-type counterparts. Histological analysis revealed increased apoptotic and necrotic cell death in a tumor originating from LSR-deficient CaCo-2 cells. LSR-deficient CaCo-2 cells exhibited increased cell proliferation in vitro and an altered epithelial morphology with impaired targeting of tricellulin to tricellular contacts. In addition, loss of LSR reduced the transepithelial electrical resistance of CaCo-2 cell monolayers and increased permeability for small molecules. Moreover, LSR-deficient CaCo-2 cells formed larger cysts in 3D culture than their wild-type counterparts. Our study provides evidence that LSR affects epithelial morphology and barrier formation in CaCo-2 cells and examines for the first time the effects of LSR deficiency on the tumor growth properties of colon carcinoma-derived cell lines.

The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNF Y of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNF Y exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNF Y does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNF Y into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNF Y , suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes.

Cytotoxic necrotizing factors CNF1 and CNF2 are produced by pathogenic Escherichia coli strains. They constitutively activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. Recently, a novel CNF (CNFY) encompassing 65% identity to CNF1 has been identified in Yersinia pseudotuberculosis. In contrast to the E. coli toxins, which activate several isoforms of Rho family GTPases, CNFY is a strong and selective activator of RhoA in vivo. By constructing chimeras between CNF1 and CNFY, we show that this substrate specificity is based on differences in the catalytic domains, whereas the receptor binding and translocation domains have no influence. We further define a loop element (L8) on the surface of the catalytic domains as important for substrate recognition. A single amino acid exchange in L8 is sufficient to shift substrate specificity of CNF1. Moreover, it is shown that RhoA activation by CNF1 is transient, which may be the consequence of the broader substrate specificity of the E. coli toxin, leading to cross-talk between the activated GTPases.

This article reviews the Escherichia coli toxins called cytotoxic necrotizing factors (CNFs), which cause activation of Rho GTPases. It describes their modes of action, structure-function relationships, and roles in disease. Rho GTPases, the targets of CNFs, belong to the Ras superfamily of low molecular mass GTPases and act as molecular switches in various signaling pathways. Low molecular mass GTPases of the Rho family are known as master regulators of the actin cytoskeleton. Moreover, they are involved in various signal transduction processes, from transcriptional activation, cell cycle progression, and cell transformation to apoptosis. CNFs are cytotoxic for a wide variety of cells, including 3T3 fibroblasts, Chinese hamster ovary cells, Vero cells, HeLa cells, and cell lines of neuronal origin. This implies that a commonly expressed receptor is responsible for the uptake of CNF1. Cultured mammalian cells treated with CNFs are characterized by dramatic changes in actin-containing structures, including stress fibers, lamellipodia, and filopodia. Most striking is the formation of multinucleation in these cells. Rho GTPases are increasingly recognized as essential factors in the development of cancer and metastasis. This fact has initiated a discussion as to whether activation of Rho proteins by CNFs might be involved in tumorigenesis. Moreover, CNF1 increases the expression of the cyclooxygenase 2 (Cox2) gene in fibroblasts. Increased expression of Cox2 is observed in some types of tumors, e.g., colon carcinoma. Lipid-mediators produced by the enzyme are suggested to be responsible for tumor progression.

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica , is glucosylated by clostridial cytotoxins. Recombinant glutathione S -transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi , which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[ 14 C]glucose.

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV–immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)–mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG–triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G(1) phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G(1) phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.