Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.
Mol Biol Cell 1996 Sep
PMID:Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton. 888 36

ADP-ribosylation factors (ARFs) are approximately 20-kDa, guanine nucleotide-binding proteins, initially discovered as stimulators of cholera toxin ADP-ribosyltransferase activity and subsequently shown to participate in vesicular trafficking. Five of the six mammalian ARFs have been identified in human tissues by molecular cloning. They fall into three classes (class I: ARFs 1-3; class II: ARFs 4, 5; class III: ARF 6) based on deduced amino acid sequence, size, phylogenetic analysis, and gene structure. Similar to the rab family of approximately 20 kDa guanine nucleotide-binding proteins, the ARFs appear to function in specific trafficking pathways. The presence of a specific ARF might serve as a marker for that pathway. To verify expression of ARF mRNA and protein in human umbilical vein endothelial cells, immunoreactivity using antibodies specific for each ARF class, quantitative polymerase chain reaction (PCR) using ARF-specific, internal cRNA standards containing unique restriction enzyme cleavage sites introduced by point mutations, and Northern analysis with probes specific for ARFs 1, and 3-6, were utilized. PCR and Northern analysis were in agreement in showing that amounts of mRNA for ARF 1 and ARF 4 were similar and higher than those of ARF 3 and ARF 5 which were greater than ARF 6. Primarily, Class 1 ARF proteins were detected by immunoreactivity, with the majority in the supernatant fraction. The relative expression of ARFs in endothelial cells thus differs from that in neuronal tissues where it had been found that ARF3 is the predominant species.
J Mol Cell Cardiol 1996 Sep
PMID:Expression in human endothelial cells of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding proteins involved in the initiation of vesicular transport. 889 50

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.
Mol Microbiol 1996 Dec
PMID:Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. 897 19

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.
Mol Microbiol 1997 Sep
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

Exoenzyme S of Pseudomonas aeruginosa is an ADP-ribosyltransferase, which is secreted via a type III-dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino-terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS-dependent ADP-ribosyltransferase activity to the carboxyl-terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP-ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl-terminal 222 amino acids of exoenzyme S, which represent the FAS-dependent ADP-ribosyltransferase domain (termed deltaN222), and a point mutant, deltaN222-E381A, which possesses a 2000-fold reduction in the capacity to ADP-ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with deltaN222 expressed ADP-ribosyltransferase activity. Co-transfection of deltaN222, but not deltaN222-E381A, resulted in a decrease in the steady-state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with deltaN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either deltaN222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.
Mol Microbiol 1998 Nov
PMID:Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells. 1009 23

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.
Mol Microbiol 1999 Apr
PMID:The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. 1023 94

We reported previously that the arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes specifically modified actin, thereby inhibiting actin polymerization in vitro. In the present study, we investigated the effect of ADP-ribosylation on actin polymerization in situ. In the leukocytes, the introduction of NAD inhibited the increase in filamentous actin contents induced by a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, while introduction of NAD together with novobiocin, a specific inhibitor for ADP-ribosyltransferase, did not. These results suggest that ADP-ribosylation regulates the formation of filamentous actin by the covalent modification of the protein in vivo.
Biochem Mol Biol Int 1999 Apr
PMID:Introduction of NAD decreases fMLP-induced actin polymerization in chicken polymorphonuclear leukocytes--the role of intracellular ADP-ribosylation of actin for cytoskeletal organization. 1031 13

Recent evidence suggests that a new member of the mono-ADP-ribosyltransferase/NAD glycohydrolase family, RT6, may be important in immune regulation. RT6 is expressed in two allelic forms and is present on post-thymic T cells in the rat. RT6-expressing T cells in the rat may have a regulatory role, a conclusion based on their ability to prevent autoimmune diabetes in the BB rat model of insulin-dependent diabetes mellitus. This observation led to investigation of RT6 at a molecular and biochemical level resulting in the determination that RT6 protein exists as both glycosylated and non-glycosylated glycosylphosphatidylinositol (GPI)-linked cell surface molecules. RT6, like many GPI-linked proteins, can mediate cell signal transduction events associated with T cell activation, and is also present in a soluble form in the circulation. The discovery that RT6 is an NAD glycohydrolase and auto-ADP-ribosyltransferase led to the ongoing investigations into the role that enzymatic activity may have in the immunoregulatory function of rat RT6+ T cells. A homologue of rat RT6, termed Rt6, has been identified in the mouse. Rt6 is predominately an ADP-ribosyltransferase enzyme as determined using simple guanidino compounds (e.g. arginine) as ribose acceptors. Abnormalities in mouse Rt6 mRNA are associated with the expression of autoimmunity. In the present manuscript, we review recent data on RT6/Rt6, and discuss the potential mechanisms by which RT6-expressing cells, and perhaps RT6 protein itself, may mediate immune regulation.
Mol Cell Biochem 1999 Mar
PMID:The RT6 (Art2) family of ADP-ribosyltransferases in rat and mouse. 1033 39

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.
Mol Cell Biochem 1999 Mar
PMID:Characterization of NAD:arginine ADP-ribosyltransferases. 1033 46

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Exp Mol Med 1999 Jun 30
PMID:Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin. 1041 Mar 10


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