Ingo Just

Rho GTPases are over-expressed in human tumors.

Small GTPases of the Rho family are involved in the regulation of a variety of cellular processes, such as the organization of the microfilamental network, cell‐cell contact and malignant transformation. To address the question of whether Rho proteins are involved in carcinogenesis in man, we compared their expression in tumors from colon, breast and lung with that of the corresponding normal tissue originating from the same patient. As shown by Rho‐specific 32P‐ADP‐ribosylation, as well as Western‐blot analysis, the amount of RhoA protein was largely increased in all 3 types of tumors tested. The most dramatic differences in the expression of Rho GTPases were observed in breast tissue. All breast tumors analyzed showed high levels of RhoA, Rac and Cdc42 proteins, whereas in the corresponding normal tissue these Rho proteins were hardly or not detectable. Progression of breast tumors from WHO grade I to grade III was accompanied by a significant average increase in RhoA protein. Overall, increase in the amount of Rho GTPases, in particular RhoA, appears to be a frequent event in different types of human tumors. This supports the view that Rho GTPases are involved in human carcinogenesis. Int. J. Cancer 81:682–687, 1999.

Localization of the Glucosyltransferase Activity of Clostridium difficile Toxin B to the N-terminal Part of the Holotoxin

Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.

CLOSTRIDIUM NOVYI ALPHA -TOXIN-CATALYZED INCORPORATION OF GLCNAC INTO RHO SUBFAMILY PROTEINS

The lethal and edema-inducing α-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. α-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the α-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for α-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi α-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.

Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress.

Abstract. Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-α-actin (ADP-r-α-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15–100xa0ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5xa0ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-α-actin. Intercellular gap formation was mediated by increased α-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of α-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.

A Novel C3-like ADP-ribosyltransferase fromStaphylococcus aureus Modifying RhoE and Rnd3

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3Stau was cloned from the genomic DNA. At the amino acid level, C3Stau revealed an identity of 35% to C3 from C. botulinum and Clostridium limosumexoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3Stau modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3Stau but not by C3 from C. botulinum. The poor cell accessibility of C3Stau was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3Stau caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3Stauis a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.

Gene expression of the small GTP-binding proteins RhoA, RhoB, Rac1 and Cdc42 in adult rat brain

GTPases of the Rho subfamily, i.e. Rho, Rac and Cdc42, are molecular switches in various signaling pathways. Best characterized are their functions in the regulation of the actin cytoskeleton. In neuronal cell lines they are involved in the mechanisms leading to synapse formation and plasticity. It is still unknown whether they have respective functions in the mammalian CNS. In this case, they should be present in the adult brain, especially in areas known for their synaptic remodeling. We have studied the expression of the Rho GTPases in adult rat brain with in situ hybridization and Western blot analysis. High amounts of RhoA, RhoB, Rac1 and Cdc42 mRNAs were detected in neurons of the hippocampus, i.e. in pyramidal cells of the CA1-CA4 regions as well as in granule cells of the dentate gyrus and in hilar cells. Also in cerebellum, Purkinje and granular cells expressed the four mRNAs. Strong gene expression was also found in brainstem, thalamus and neocortex. Using Western blot analysis, RhoA and Cdc42 proteins were detected in hippocampus, cerebellum, thalamus and neocortex. It is concluded that GTPases of the Rho family play a role in the regulation of cellular functions in the adult brain.

Interaction of mastoparan with the low molecular mass GTP‐binding proteins rho/rac

Mastoparan, which has been shown to active G proteins [1], inhibits the ADP‐ribosylation of 20 kDa human platelet membrane proteins catalyzed by Clostridium botulinum exoenzyme C3 half‐maximally and maximally (90%) at 20 and 100 μM concentrations, respectively. Inhibition of ADP‐ribosylation was enhanced by GTP‐γS. Mastoparan increased GTP hydrolysis by porcine brain rho protein and stimulated GTP binding in a concentration dependent manner. The data suggest that mastoparan not only interacts with heterotrimeric G proteins but also with low molecular mass GTP‐binding proteins of the rho/rac family.

Molecular mode of action of the large clostridial cytotoxins.

The large clostridial cytotoxins are a family of functionally and structurally related toxins produced by clostridia comprising Clostridium difficile toxin A and toxin B, Clostridium sordellii lethal and haemorrhagic toxin and Clostridium novyi α-toxin (Table 1). These toxins are exotoxins which induce morphological changes of the cultured target cells based on the redistribution of the microfilament system. The cytotoxic activity on cultured cell lines led to their designation as cytotoxins. Despite their comparable in vitro effects, the cytotoxins — as major pathogenicity factors — are involved in different diseases and clinical outcomes. Clostridium difficile toxins A and B are of major clinical importance because both toxins are the causative agents in about 20% of antibiotic-associated diarrhoea and in almost all cases of pseudomembranous colitis (Kelly et al. 1994; Kelly and Lamont 1998; Bartlett 1994).

ADP-ribosylation of Rho proteins inhibits sperm motility

The highly homologous Rho proteins RhoA, RhoB and RhoC are low‐molecular‐mass GTP‐binding proteins. They are selectively ADP‐ribosylated by Clostridium botulinum ADP‐ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme‐like toxins ADP‐ribosylate small GTP‐binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

Clostridial Actin-ADP-Ribosylating Toxins

Several ADP-ribosylating toxins, such as cholera toxin, pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A have been the focus of intensive research for many years. Studies on these toxins, which are described in other chapters of this volume, have brought about insights into the pathogenetic mechanisms of diseases related to the toxin-producing bacteria. Furthermore, especially cholera and pertussis toxins, which ADP-ribosylate GTP-binding proteins, have been proved to be excellent instruments for elucidation of the physiological functions of their target proteins (for recent reviews see also Moss and Vaughan 1990)