Proteasomal Degradation of Cytotoxic Necrotizing Factor 1-Activated Rac

Lerm, Maria; Pop, Marius; Fritz, Gerhard; Aktories, Klaus; Schmidt, Gudula

doi:10.1128/iai.70.8.4053-4058.2002

Cited by 83 publications

(75 citation statements)

References 29 publications

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“…In PAE cells, CNF1 seems to deaminate Rho, Rac, and Cdc42 to the same extent. However, GTPase degradation, which follows covalent modification (10,22,24), was more rapid for Rho than for Rac and Cdc42. In PAE cells, major CNF1-induced F-actin reorganization may result from the dominant contribution of Cdc42 activity to the overall cytoskeletal remodeling in this cell type.…”

Section: Discussionmentioning

confidence: 99%

Actin Can Reorganize into Podosomes in Aortic Endothelial Cells, a Process Controlled by Cdc42 and RhoA

Moreau

Tatin

Varon

et al. 2003

Molecular and Cellular Biology

179

162

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Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

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Section: Discussionmentioning

confidence: 99%

Actin Can Reorganize into Podosomes in Aortic Endothelial Cells, a Process Controlled by Cdc42 and RhoA

Moreau

Tatin

Varon

et al. 2003

Molecular and Cellular Biology

179

162

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“…It consists of a permanent activation of Rho proteins, which results in their sensitization to ubiquitin-mediated proteasomal degradation (12,13,15,16). We have previously established that this dual mechanism of action confers higher invasive properties to pathogenic bacteria (15).…”

Section: Activation Of Rho Gtpases By Cnf1 Interferes With Host Cellmentioning

confidence: 99%

Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

Munro

Flatau

Doye

et al. 2004

Journal of Biological Chemistry

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The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3␣ (MIP-3␣) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3␣, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitinmediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.

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“…phox and Rac1 are of interest as both of them have wide tissue distribution and both of them were reported to be expressed in HEK293 cells (25,26).…”

Section: Duced As Aliases For P47mentioning

confidence: 99%

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

Bánfi¹,

Clark²,

Steger³

et al. 2003

Journal of Biological Chemistry

441

369

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NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47 phox and p67 phox subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47 phox and p67 phox are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47 phox homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67 phox homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47 phox was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).Superoxide generation by phagocytes plays a crucial role in the elimination of invading microorganisms. It is catalyzed by the phagocyte NADPH oxidase, an enzyme consisting of two transmembrane subunits, p22 phox and gp91 phox , and at least three cytosolic subunits, p47 phox , p67 phox , and Rac2 (1). Upon activation, the NADPH oxidase subunits assemble, and electrons are transported from intracellular NADPH to extracellular oxygen by the flavo-heme gp91 phox subunit (2). Recently six gp91 phox homologues have been described in mammals: NOX1 1 (3, 4), NOX3 (5, 6), and NOX4 (7, 8) with an overall structure similar to gp91 phox (alias NOX2), NOX5 with an N-terminal EF hand-containing extension (9), and DUOX1 and DUOX2 with an additional peroxidase homology domain (10 -12). NOX1 is found mainly in colon epithelium (3, 4); NOX3 in embryonic kidney (5, 6), NOX4 in the kidney cortex (7, 8), NOX5 in lymphoid organs and testis (9), DUOX1 in thyroid and lung, and DUOX2 in thyroid and colon (10 -12).Based on their primary structure all members of the NOX/ DUOX family should be flavo-heme electron transporters. However, it is not established whether all NOX enzymes transfer electrons to oxygen or whether some of them may use other electron acceptors as has been shown for a yeast homologue of gp91phox that functions as a ferric reductase (13). Among NOX enzymes, only gp91 phox and NOX5 have appeared capable of generating large amounts of superoxide, both of them in a stimulus-dependent manner (1, 9).Based on data gained with NOX1-transfected NIH 3T3 cell clones NOX1 has been suggested to be a subunit-independent, low capacity superoxide-generating enzyme involved in the regulation of mitogenesis (4, 14). However, we have not been able to measure any superoxide generat...

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Proteasomal Degradation of Cytotoxic Necrotizing Factor 1-Activated Rac

Cited by 83 publications

References 29 publications

Actin Can Reorganize into Podosomes in Aortic Endothelial Cells, a Process Controlled by Cdc42 and RhoA

Actin Can Reorganize into Podosomes in Aortic Endothelial Cells, a Process Controlled by Cdc42 and RhoA

Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

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