Gudula Schmidt

Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1

The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases,, which act as molecular switches in diverse signal-transduction processes. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation or glucosylation. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2),. These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure

The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid‐encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase‐activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild‐type strain. Expression of wild‐type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co‐expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.

The N-terminal Domain of Pseudomonas aeruginosaExoenzyme S Is a GTPase-activating Protein for Rho GTPases

Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin. The ADP-ribosyltransferase domain is located within the C terminus part of ExoS. Recent studies showed that the N terminus part of ExoS (amino acid residues 1–234, ExoS(1–234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells. Here we studied the effects of ExoS(1–234) on nucleotide binding and hydrolysis by Rho GTPases. ExoS(1–234) (100–500 nm) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis. A similar increase in GTPase activity was stimulated by full-length ExoS. Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10–11 nm ExoS(1–234), respectively. We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins. These data identify ExoS as a GTPase-activating protein for Rho GTPases.

Rho GTPases as Targets of Bacterial Protein Toxins

Abstract Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The ‘injected’ toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/ transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.

Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering

Tripartite Toxin Luminescent bacterial symbionts of nematode worms that attack insects have long stirred interest in their possibilities for biological control. The bacteria produce a family of toxins composed of at least three subunits that resemble a widely occurring class of bacterial toxins also produced by human pathogens. Lang et al. (p. 1139) have elucidated the mode of action and structural interactions of some of these tripartite protein toxins and found that they poison the cells actin cytoskeleton by catalyzing unusual reactions. One toxin mediated adenosine diphosphate (ADP)–ribosylation at threonine-148 to cause actin polymerization, another ADP-ribosylated Rho protein at glutamine-63, and both synergized to cause actin clustering and cell paralysis. A bacterial toxin targets and modifies the actin cytoskeleton in insect larvae. The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)–ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins.

The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity.

Recently, it has been reported that cytotoxic necrotizing factor 1 (CNF1) from Escherichia coliinduces formation of stress fibers by deamidation of glutamine 63 of RhoA (Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725–729); Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P. (1997) Nature 387, 729–733). By using mass spectrometric analysis, we show now that the toxin transfers ethylenediamine, putrescine, and dansylcadaverine specifically onto glutamine 63 of RhoA. RhoA was also a substrate for guinea pig liver transglutaminase, which modified not only glutamine 63, but also glutamine residues at positions 52 and 136. Treatment of the fully active N-terminal fragment of CNF1 (amino acid residues 709–1014) with iodoacetamide inhibited both deamidation and transglutamination activities. Moreover, exchange of cysteine 866 with serine blocked the enzyme activity of the N-terminal CNF1 fragment. In addition, we identified histidine 881 to be essential for the enzyme activity of CNF1. The data indicate that CNF1 shares a catalytic dyad of cysteine and histidine residues with eukaryotic transglutaminases and cysteine proteases.

Pseudomonas aeruginosa ExoT Is a Rho GTPase-Activating Protein

ABSTRACT Transient intracellular expression of ExoT in CHO cells stimulated cell rounding and actin reorganization. Biochemical studies showed that ExoT was a GTPase-activating protein for RhoA, Rac1, and Cdc42. Together, these data show that ExoT interferes with Rho signal transduction pathways, which regulate actin organization, exocytosis, cell cycle progression, and phagocytosis.

Inhibition of Rho A activity causes pemphigus skin blistering

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.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation

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.

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

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.