High catalytic efficiency and resistance to denaturing in bacterial Rho GTPase-activating proteins

Several major bacterial pathogens use the type III secretion system (TTSS) to deliver virulence factors into host cells. Bacterial Rho GTPase activating proteins (RhoGAPs) comprise a remarkable family of type III secreted toxins that modulate cytoskeletal dynamics and manipulate cellular signaling pathways. We show that the RhoGAP activity of Salmonella SptP and Pseudomonas ExoS toxins is resistant to variations in the concentration of NaCl or MgCl(2), unlike the known salt dependant nature of the activity of some eukaryotic GAPs such as p190, RanGAP and p120GAP. Furthermore, SptP-GAP and ExoS-GAP display full activity after treatment at 80°C or with 6 m urea, which suggests that these protein domains are capable of spontaneous folding into an active state following denaturing such as what might occur upon transit through the TTSS needle. We determined the catalytic activity of bacterial GAPs for Rac1, CDC42 and RhoA GTPases and found that ExoS, in addition to Yersinia YopE and Aeromonas AexT toxins, display higher catalytic efficiencies for Rac1 and CDC42 than the known eukaryotic GAPs, making them the most catalytically efficient RhoGAPs known. This study expands our knowledge of the mechanism of action of GAPs and of the ways bacteria mimic host activities and promote catalysis of eukaryotic signaling proteins.     

ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor

ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains. Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear. The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP. A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases. Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol. A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression. HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP. This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation. A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.     

Phospholipids can switch the GTPase substrate preference of a GTPase-activating protein

The major cellular inhibitors of the small GTPases of the Ras superfamily are the GTPase-activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of GTPases, thereby inactivating them. The catalytic activity of several GAPs is reportedly inhibited or stimulated by various phospholipids and fatty acids in vitro, indicating a likely physiological role for lipids in regulating small GTPases. We find that the p190 RhoGAP, a potent GAP for the Rho and Rac GTPases, is similarly sensitive to phospholipids. Interestingly, however, several of the tested phospholipids were found to effectively inhibit the RhoGAP activity of p190 but stimulate its RacGAP activity. Thus, phospholipids have the ability to "switch" the GTPase substrate preference of a GAP, thereby providing a novel regulatory mechanism for the small GTPases.     

The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence

A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

Rho GTPases as targets of bacterial protein toxins

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.     

How the Pseudomonas aeruginosa ExoS toxin downregulates Rac

Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.     

Burkholderia cenocepacia infection: Disruption of phagocyte immune functions through Rho GTPase inactivation

Phagocytosis is an important component of innate immunity that contributes to the eradication of infectious microorganisms; however, successful bacterial pathogens often evade different aspects of host immune responses. A common bacterial evasion strategy entails the production of toxins and/or effectors that disrupt normal host cell processes and because of their importance Rho-family GTPases are often targeted. Burkholderia cenocepacia, an opportunistic pathogen that has a propensity to infect cystic fibrosis patients, is an example of a pathogenic bacterium that has only recently been shown to disrupt Rho GTPase function in professional phagocytes. More specifically, B. cenocepacia disrupts Rac and Cdc42 seemingly through perturbation of guanine nucleotide exchange factor function. Inactivation of Rac, Cdc42 and conceivably other Rho GTPases seriously compromises phagocyte function.     

Burkholderia cenocepacia infection

Phagocytosis is an important component of innate immunity that contributes to the eradication of infectious microorganisms; however, successful bacterial pathogens often evade different aspects of host immune responses. A common bacterial evasion strategy entails the production of toxins and/or effectors that disrupt normal host cell processes and because of their importance Rho-family GTPases are often targeted. Burkholderia cenocepacia, an opportunistic pathogen that has a propensity to infect cystic fibrosis patients, is an example of a pathogenic bacterium that has only recently been shown to disrupt Rho GTPase function in professional phagocytes. More specifically, B. cenocepacia disrupts Rac and Cdc42 seemingly through perturbation of guanine nucleotide exchange factor function. Inactivation of Rac, Cdc42 and conceivably other Rho GTPases seriously compromises phagocyte function.     

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.     

The GAP Activity of Type III Effector YopE Triggers Killing of Yersinia in Macrophages

The mammalian immune system has the ability to discriminate between pathogens and innocuous microbes by detecting conserved molecular patterns. In addition to conserved microbial patterns, the mammalian immune system may recognize distinct pathogen-induced processes through a mechanism which is poorly understood. Previous studies have shown that a type III secretion system (T3SS) in Yersinia pseudotuberculosis leads to decreased survival of this bacterium in primary murine macrophages by unknown mechanisms. Here, we use colony forming unit assays and fluorescence microscopy to investigate how the T3SS triggers killing of Yersinia in macrophages. We present evidence that Yersinia outer protein E (YopE) delivered by the T3SS triggers intracellular killing response against Yersinia. YopE mimics eukaryotic GTPase activating proteins (GAPs) and inactivates Rho GTPases in host cells. Unlike wild-type YopE, catalytically dead YopER144A is impaired in restricting Yersinia intracellular survival, highlighting that the GAP activity of YopE is detected as a danger signal. Additionally, a second translocated effector, YopT, counteracts the YopE triggered killing effect by decreasing the translocation level of YopE and possibly by competing for the same pool of Rho GTPase targets. Moreover, inactivation of Rho GTPases by Clostridium difficile Toxin B mimics the effect of YopE and promotes increased killing of Yersinia in macrophages. Using a Rac inhibitor NSC23766 and a Rho inhibitor TAT-C3, we show that macrophages restrict Yersinia intracellular survival in response to Rac1 inhibition, but not Rho inhibition. In summary, our findings reveal that primary macrophages sense manipulation of Rho GTPases by Yersinia YopE and actively counteract pathogenic infection by restricting intracellular bacterial survival. Our results uncover a new mode of innate immune recognition in response to pathogenic infection.     

Genome-wide analysis reveals the functional and expressional correlation between RhoGAP and RhoGEF in mouse

Rho GTPases play essential roles in various life activities. Rho GTPase-activating protein (RhoGAP) and Rho guanine nucleotide exchange factor (RhoGEF) are the main regulators of Rho GTPases. RhoGAP, RhoGEF and Rho make up a molecular switch and exert crucial roles in signaling pathways. The genome-wide studies can provide us a comprehensive information of special protein family, but the genome-wide information of RhoGAP and RhoGEF families are still lacking in the mammal lineage. Here, we report the correlations between mouse RhoGAPs and RhoGEFs in gene quantities, evolution, molecular function, and their expression levels in heart embryonic development and cardiovascular medicine treatment at genome-wide scale. Besides, we find that the 3D structures of RhoGAP domains between different species are highly conserved, but that of RhoGEF domains are variable between species. Our present study contributes to a better understanding of the complex regulation mechanisms of RhoGAP and RhoGEF families.     

Bacterial mimics of eukaryotic GTPase-activating proteins (GAPs)

Bacterial GTPase-activating proteins (GAPs) subvert their host's eukaryotic Rho GTPases to their own advantage. Studies of bacterial GAPs extend our understanding of the action of eukaryotic GAPs, provide new tools for studies of cytoskeletal dynamics and offer new targets for anti-bacterial drugs.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.     

Current knowledge of the large RhoGAP family of proteins

The Rho GTPases are implicated in almost every fundamental cellular process. They act as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state. Their slow intrinsic GTPase activity is greatly enhanced by RhoGAPs (Rho GTPase-activating proteins), thus causing their inactivation. To date, more than 70 RhoGAPs have been identified in eukaryotes, ranging from yeast to human, and based on sequence homology of their RhoGAP domain, we have grouped them into subfamilies. In the present Review, we discuss their regulation, biological functions and implication in human diseases.     

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.