Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn5Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenotype (388::Tn5Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzyme S trans-regulatory operon. 388::Tn5Tc 469 and 550 mapped to a region downstream of the trans-regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB-L proteins of the yersiniae Yop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB-L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co-ordinately regulated proteins.
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PMID:Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. 897 19

Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts. Previous studies indicated that P. aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells. In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P. aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype. In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology. Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy. Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain. In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay. The expression of cytotoxicity requires a functional exsA allele. In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression. Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53). Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT. These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants. In the absence of a functional cytotoxicity pathway (exsA::omega strains), invasion of eukaryotic cells is detectable.
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PMID:Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. 900 16

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.
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PMID:Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa. 904 20

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.
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PMID:Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49. 915 24

Pseudomonas aeruginosa produces two ADP-ribosyltransferases, exotoxin A and exoenzyme S (ExoS). Although the physiological target protein remains to be defined, ExoS has been shown to ADP-ribosylate several eukaryotic proteins in vitro, including vimentin and members of the family of low-molecular-weight GTP-binding proteins. Recently, ExoS ADP-ribosyltransferase activity has been detected in the pleural fluid of rabbits infected with P. aeruginosa. This observation prompted an examination of the potential for ExoS to function as an ecto-ADP-ribosyltransferase. We have observed that ExoS preferentially ADP-ribosylated two extracellular serum proteins with molecular masses of 150 and 27 kDa. The ADP-ribosylation of these serum proteins by ExoS was stimulated by, but not dependent upon, exogenous FAS (for factor activating exoenzyme S), which indicated that serum contained endogenous FAS activity. Biochemical analysis showed that the 150-kDa ADP-ribosylated protein was immunoglobulin of the immunoglobulin G (IgG) and IgA classes. Subtyping showed that ExoS preferentially ADP-ribosylated human IgG3 and that ADP-ribosylation occurred within its Fc region. The 27-kDa protein ADP-ribosylated by ExoS was determined to be apolipoprotein A1. These data demonstrate ecto-ADP-ribosyltransferase activity by ExoS. This may extend the potential physiological consequences of ExoS during infection by P. aeruginosa beyond the implicated type III secretion-mediated intracellular delivery of ExoS into sensitive eukaryotic cells.
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PMID:Ecto-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exoenzyme S. 923 91

Exoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria-cell contact-dependent mechanism involving the ysc-encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.
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PMID:Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. 935 Aug 68

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.
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PMID:Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A. 957 4

Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa 388 and an isogenic mutant strain, 388DeltaexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388DeltaexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388DeltaexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.
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PMID:Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S. 959 23

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.
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PMID:ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. 981 98

This study was initiated to characterize the regulation and secretion of ExoS by Pseudomonas aeruginosa during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when P. aeruginosa was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when P. aeruginosa was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of P. aeruginosa were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.
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PMID:Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. 991 8


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