Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.
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PMID:Obscurin is a ligand for small ankyrin 1 in skeletal muscle. 1263 29

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.
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PMID:Fibrous sheath of mammalian spermatozoa. 1267 26

The p21-activated protein kinases (Paks) are serine/threonine protein kinases activated by binding to Rho family small GTPases, Rac and Cdc42. Recently, Pak family members have been subdivided into two groups, I and II. Group II Paks, including Pak4, Pak5, and Pak6, does not contain the highly conserved autoinhibitory domain that is found in the group I Paks members, i.e. Pak1, Pak2, and Pak3. In the present study, we have purified the glutathione S-transferase fusion form of Pak5 and shown for the first time that Pak5 autophosphorylation can be activated by GTP bound form of Cdc42. Mutation of histidine residues 19 and 22 to leucine on the p21-binding domain of Pak5 completely abolished the binding of Cdc42 and the Cdc42-mediated autophosphorylation. On the other hand, mutation of tyrosine 40 to cysteine of Cdc42 did not knockout the binding of Pak5. Analysis of C-terminal deletion mutants has identified an autoinhibitory fragment of Pak5 that is absent from other group II Pak family members. Taken together, these results suggest that Pak5, like Pak1, contains an autoinhibitory domain and its activity is regulated by Cdc42.
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PMID:Identification of an autoinhibitory domain of p21-activated protein kinase 5. 1286 Sep 98

Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.
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PMID:The C terminus of YopT is crucial for activity and the N terminus is crucial for substrate binding. 1287 42

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.
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PMID:Direct activation of phospholipase C-epsilon by Rho. 1290 Apr 2

Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.
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PMID:Characterization of Pseudomonas aeruginosa exoenzyme S as a bifunctional enzyme in J774A.1 macrophages. 1293 77

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.
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PMID:Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. 1294 36

We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12. The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. We have previously observed the absence of RhoGAP transcript in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.
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PMID:Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins. 1498 79

A cDNA clone encoding a rac-like small GTP binding protein was isolated from a cDNA library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds and named Brac1. The Brac1 cDNA contains an open reading frame encoding 198 amino acid residues with an estimated molecular mass of 21,690 Da and this coding region has conserved residues and motifs unique to the Rho subfamily of proteins. The deduced amino acid sequence of the Brac1 protein is closely related to that of Arabidopsis thaliana Arac3 (91%), but it shares relatively little homology with other members of the Ras superfamily (about 30% identity). To further characterize Brac1, a pGBrac1 expression vector consisting of PCR-amplified Brac1 cDNA plus glutathione S-transferase (GST) and pBKS(+)II was used to purify the protein. Using a PEI-cellulose/TLC plate, GTPase activity of this protein was confirmed and competition binding studies, using the guanine nucleotides, ATP, UTP and CTP, revealed that the di- and triphosphate forms of guanine nucleotides strongly bind Brac1. Membrane-bound PLD activity was synergistically enhanced by Brac1 in the presence of protein kinase C, but not in the presence of ARF (ADP-ribosylation factor). Genomic analysis indicated that Brac1 belongs to a multigene family. Brac1 transcripts were expressed in all the organs of Brassica, but were especially prevalent in flower buds.
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PMID:A rac-like small G-protein from Brassica campestris activates a PKC-dependent phospholipase D. 1469 72

The cytotoxic necrotizing factors (CNF)1 and CNF2 from pathogenic Escherichia coli strains activate RhoA, Rac1, and Cdc42 by deamidation of Gln63 (RhoA) or Gln61 (Rac and Cdc42). Recently, a novel cytotoxic necrotizing factor termed CNFY was identified in Yersinia pseudotuberculosis strains (Lockman, H. A., Gillespie, R. A., Baker, B. D., and Shakhnovich, E. (2002) Infect. Immun. 70, 2708-2714). We amplified the cnfy gene from genomic DNA of Y. pseudotuberculosis, cloned and expressed the recombinant protein, and studied its activity. Recombinant GST-CNFY induced morphological changes in HeLa cells and caused an upward shift of RhoA in SDS-PAGE, as is known for GST-CNF1 and GST-CNF2. Mass spectrometric analysis of GST-CNFY-treated RhoA confirmed deamidation at Glu63. Treatment of RhoA, Rac1, and Cdc42 with GST-CNFY decreased their GTPase activities, indicating that all of these Rho proteins could serve as substrates for GST-CNFY in vitro. In contrast, RhoA, but not Rac or Cdc42, was the substrate of GST-CNFY in culture cells. GST-CNFY caused marked stress fiber formation in HeLa cells after 2 h. In contrast to GST-CNF1, formation of filopodia or lamellipodia was not induced with GST-CNFY. Accordingly, effector pull-down experiments with lysates of toxin-treated cells revealed strong activation of RhoA but no activation of Rac1 or Cdc42 after 6 h of GST-CNFY-treatment. Moreover, in rat hippocampal neurons, GST-CNFY results in the retraction of neurites, indicating RhoA activation. In contrast, no activation of Rac or Cdc42 was found. Altogether, our data suggest that CNFY from Y. pseudotuberculosis is a strong, selective activator of RhoA, which can be used as a powerful tool for constitutive RhoA activation without concomitant activation of Rac1 or Cdc42.
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PMID:The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA. 1476 41


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