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1.
ExoS (453 amino acids) is a bi-functional type-III cytotoxin of Pseudomonas aeruginosa. Residues 96-233 comprise the Rho GTPase-activating protein (Rho GAP) domain, while residues 234-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Residues 51-72 represent a membrane localization domain (MLD), which targets ExoS to perinuclear vesicles within mammalian cells. YopE (219 amino acids) is a type-III cytotoxin of Yersinia that is also a Rho GAP. Residues 96-219 comprise the YopE Rho GAP domain. While the Rho GAP domains of ExoS and YopE share structural homology, unlike ExoS, the intracellular localization of YopE within mammalian cells has not been resolved and is the subject of this investigation. Deletion mapping showed that the N terminus of YopE was required for intracellular membrane localization of YopE in CHO cells. A fusion protein containing the N-terminal 84 amino acids of YopE localized to a punctate-perinuclear region in mammalian cells and co-localized with a fusion protein containing the MLD of ExoS. Residues 54-75 of YopE (termed YopE-MLD) were necessary and sufficient for intracellular localization in mammalian cells. The YopE-MLD localized ExoS to intracellular membranes and targeted ExoS to ADP-ribosylate small molecular weight membrane proteins as observed for native type-III delivered ExoS. These data indicate that the YopE MLD functionally complements the ExoS MLD for intracellular targeting in mammalian cells.  相似文献   

2.
Maresso AW  Riese MJ  Barbieri JT 《Biochemistry》2003,42(48):14249-14257
Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin. The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases. A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells. Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells. Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity. Bacterial-associated ExoS and ExoS that had been secreted by P. aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9. The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers. Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis. ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS. Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS. This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS. ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.  相似文献   

3.
ExoS is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-232 comprise the Rho GTPase activating protein (Rho GAP) domain, whereas residues 233-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies showed that the N-terminus targeted ExoS to intracellular membranes within eukaryotic cells. This N-terminal targeting region is now characterized for cellular and biological contributions to intoxications by ExoS. An ExoS(1-107)-green fluorescent protein (GFP) fusion protein co-localized with alpha-mannosidase, which indicated that the fusion protein localized near the Golgi. Residues 51-72 of ExoS (termed the membrane localization domain, MLD) were necessary and sufficient for membrane localization within eukaryotic cells. Deletion of the MLD did not inhibit type III secretion of ExoS from P. aeruginosa or type III delivery of ExoS into eukaryotic cells. Type III-delivered ExoS(DeltaMLD) localized within the cytosol of eukaryotic cells, whereas type III-delivered ExoS was membrane associated. Although type III-delivered ExoS(DeltaMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP-ribosylate Ras. Type III-delivered ExoS(DeltaMLD) and ExoS showed similar capacities for eliciting a cytotoxic response in CHO cells, which uncoupled the ADP-ribosylation of Ras from the cytotoxicity elicited by ExoS.  相似文献   

4.
ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.  相似文献   

5.
Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS   总被引:4,自引:0,他引:4  
Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [(32)P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro. To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [(32)P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.  相似文献   

6.
ExoS (453 amino acids) is a bi-functional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-219 include the Rho GTPase-activating protein (RhoGAP) domain, and residues 234-453 include the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies also identified an N-terminal domain (termed the membrane localization domain) that comprises residues 51-77 and includes a novel leucine-rich motif that targets ExoS to the perinuclear region of cultured cells. There is limited information on how ExoS or other type III cytotoxins enter and target intracellular host proteins. Type III-delivered ExoS localized to both plasma membrane and perinuclear region, whereas ExoS(DeltaMLD) was localized to the cytosol. Plasma membrane localization of ExoS was transient and had a half-life of approximately 20 min. Type III-delivered ExoS co-immunoprecipitated 14-3-3 proteins and Rab9, Rab6, and Rab5. Immunofluorescence experiments showed that ExoS colocalized with Rab9, Rab6, and Rab5. Fluorescent energy transfer was detected between ExoS and 14-3-3 proteins but not between ExoS and Rabs proteins. Together, these results indicate that type III-delivered ExoS localizes on the host endosomes and utilizes multiple pathways to traffic from the plasma membrane to the perinuclear region of intoxicated host cells.  相似文献   

7.
Pseudomonas aeruginosa ExoS (453 amino acids) is a bifunctional type III cytotoxin, comprising a Rho GTPase-activating protein domain (RhoGAP), and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain (termed MLD, residues 51-77) which localizes and traffics ExoS within intoxicated host cells. While membrane localization has been shown to be essential for ExoS to ADP-ribosylate Ras, the relationship between intracellular localization and expression of RhoGAP activity has not been addressed. In this study, loss of MLD function was observed to abolish expression of ExoS RhoGAP activity in HeLa cells. One mutation within the MLD (R56, R63, D70 mutated to N, RRD-->N) diminished plasma membrane localization and altered the cell rounding phenotype elicited by ExoS RhoGAP. In addition, cell rounding caused by ExoS-MLD(RRD-->N) was reversed by dominant active Rac1, but not dominant active Cdc42, indicating a switch in ExoS RhoGAP substrate specificity. Mutation of the C-terminal polybasic region abolished the ability of dominant active Rac1 to protect HeLa cells from expression of the RhoGAP activity of ExoS-MLD(RRD-->N). This study shows the importance of membrane localization in the targeting of Rho GTPases by ExoS RhoGAP.  相似文献   

8.
Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.  相似文献   

9.
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.  相似文献   

10.
ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains. The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42. Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis. Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity. Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates. While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins. Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.  相似文献   

11.
Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin. The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity. The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation. Infection of HeLa cells with P. aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes. Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation. Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins. ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras. Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS. Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells. The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.  相似文献   

12.
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.  相似文献   

13.
Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.  相似文献   

14.
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.  相似文献   

15.
Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway. Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (DeltaC234). Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234. Cytoskeleton rearrangement elicited by DeltaC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1). CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation. This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by DeltaC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.  相似文献   

16.
14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells, which play an important role in a multitude of signaling pathways. 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 200 14-3-3 binding partners have been found that are involved in cell cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. A phosphorylation-independent interaction has been reported to occur between 14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study, we have investigated the effect of amino acid mutations in this C-terminal domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interaction. Our results suggest that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity, as cytotoxicity analysis reveals that substitution of this leucine significantly weakens the ability of ExoS to mediate cell death. Leucine-428 is also required for the ability of ExoS to modify the eukaryotic endogenous target Ras. Finally, single amino acid substitutions of positions 426-428 reduce the interaction potential of 14-3-3 with ExoS in vitro.  相似文献   

17.
L Zhang  H Wang  S C Masters  B Wang  J T Barbieri  H Fu 《Biochemistry》1999,38(37):12159-12164
Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.  相似文献   

18.
We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.  相似文献   

19.
14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.  相似文献   

20.
Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).  相似文献   

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