Bacterial Toxins Activating Rho GTPases

Munro, Patrick; Lemichez, Emmanuel

doi:10.1007/3-540-27511-8_10

Cited by 12 publications

(8 citation statements)

References 54 publications

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“…GTP-bound GTPases act on effectors that then induce changes to cytoskeletal dynamics and gene regulation (41). In the endothelium, RhoGTPases are activated in response to inflammatory stimuli (such as TNF-␣, IL-1␤, and bacterial toxins), hypoxia, shear stress (33,(42)(43)(44)(45)(46), or endothelial-leukocyte interactions (19,31). RhoA and Rac1 were activated in BMVEC after TNF-␣ treatment and further enhanced by monocyte-BMVEC engagement or simulation of this process by ICAM-1 Ab crosslinking.…”

Section: Discussionmentioning

confidence: 99%

Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes

Ramirez

Heilman

Morsey

et al. 2008

The Journal of Immunology

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Under inflammatory conditions (including HIV-1 encephalitis and multiple sclerosis), activated brain endothelium enhances the adhesion and transmigration of monocytes across the blood-brain barrier (BBB). Synthetic ligands that activate the peroxisome proliferator-activated receptors (PPARs) have anti-inflammatory properties, and PPAR stimulation prevents the interaction of leukocytes with cytokine stimulated-endothelium. However, the mechanism underlying these effects of PPAR ligands and their ability to intervene with leukocyte adhesion and migration across brain endothelial cells has yet to be explored. For the first time, using primary human brain endothelial cells (BMVEC), we demonstrated that monocyte adhesion and transendothelial migration across inflamed endothelium were markedly reduced by PPARγ activation. In contrast to non-brain-derived endothelial cells, PPARα activation in the BMVEC had no significant effect on monocyte-endothelial interaction. Previously, our work indicated a critical role of Rho GTPases (like RhoA) in BMVEC to control migration of HIV-1 infected monocytes across BBB. In this study, we show that in the BMVEC PPARγ stimulation prevented activation of two GTPases, Rac1 and RhoA, which correlated with decreased monocyte adhesion to and migration across brain endothelium. Relevant to HIV-1 neuropathogenesis, enhanced adhesion and migration of HIV-1 infected monocytes across the BBB were significantly reduced when BMVEC were treated with PPARγ agonist. These findings indicate that Rac1 and RhoA inhibition by PPARγ agonists could be a new approach for treatment of neuroinflammation by preventing monocyte migration across the BBB.

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Section: Discussionmentioning

confidence: 99%

Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes

Ramirez

Heilman

Morsey

et al. 2008

The Journal of Immunology

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“…The DNA can be amplified and visualized, which enables unbiased quantification of endogenous protein complexes at single-molecule resolution. The GTP-loading state of endogenous Rho GTPases can be manipulated with the bacterial toxins CNF Y and CNF1 (37,38). These toxins deamidate a single glutamine residue that is required for GTPase activity.…”

Section: Log 2 Cdc42mentioning

confidence: 99%

Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners

Paul

Zauber

Berg³

et al. 2017

Molecular & Cellular Proteomics

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Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissuederived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases. The Ras superfamily of small guanosine triphosphatases (Ras GTPases) consists of more than 150 members in mammals and conserved orthologs in all eukaryotes (1). As molecular switches, they cycle between an active GTP-and an inactive GDP-bound state. GTPase-activating proteins (GAPs) 1 stimulate the slow intrinsic GTPase activity, which inactivates the GTPase. Conversely, guanine nucleotide exchange factors (GEFs) promote release of GDP, which is replaced by GTP, thereby transforming the GTPase into the active state. The GTP-bound form binds to downstream effector proteins to initiate their specific cellular function. In this manner, GTPases control numerous biological processes, including cytoskeletal rearrangements, membrane dynamics, and gene expression. Rho GTPases form a subfamily of the Ras superfamily (2). Of its 22 mammalian members, RhoA, Rac1, and Cdc42 have been studied most intensively and are best known for their role in regulating the actin cytoskeleton (3).Identifying effector proteins is key to understanding Rho GTPase function. More than 70 effector proteins have already been identified for each of the three prototypical family members, RhoA, Rac1, and Cdc42 (4). However, new effector proteins are still being discovered, and little is known about effectors of less well studied family members. Systematic screens for GTPase effectors employed the yeast two-hybrid approach (5) or immobilized GTPases for affinity purification (6, 7). However, these approaches are semiquantitative at best, which makes it difficult to distinguish loading-statespecific binders from constitutive interactors and nonspecific contaminants. Due to these challenges, an unbiased interactor screen for multiple Rho GTPases has not yet been reported.We sought to systematically identify proteins that interact with Rho GTPases in a loading-state-specific manner. Quantitative affinity purification combined with mass spectrometry is a powerful technology that can be used to identify proteinprotein interactions (PPIs) in an unbiased way (8 -11). Here, we develop quantitative GTPase affinity purification (qGAP) as a novel variant of quantitative affinity purification combined with mass spectro...

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“…Activation of Rac1 and RhoA by bacterial toxins such as cytotoxic necrotizing factor 1 (CNF1) leads, after 4-6 hours, to proteasome-mediated degradation (Munro and Lemichez, 2005;Pop et al, 2004). Thus, cells possess endogenous mechanisms to degrade activated GTPases, representing a third level of regulation, in addition to membrane translocation and GTP loading and hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway

Nethe

Anthony

Fernandez‐Borja

et al. 2010

Journal of Cell Science

109

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SummaryDirectional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling.

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Bacterial Toxins Activating Rho GTPases

Cited by 12 publications

References 54 publications

Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes

Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes

Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners

Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway

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