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)

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.
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PMID:Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis. 1265 53

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.
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PMID:Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane. 1281 30

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.
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PMID:The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. 1286 Sep 94

Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes. The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin. Here we have revealed functions of Arp5 and Arp8 by analysis of mutants. arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype. Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization. The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps. Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain. GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function. These findings show direct involvement of Arps in the chromatin-remodeling process.
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PMID:Involvement of actin-related proteins in ATP-dependent chromatin remodeling. 1288

The study investigates the effect of aqueous extract of fenugreek seeds (Trigonella foenum graecum) on lipid peroxidation and antioxidant status in experimental ethanol toxicity in rats. The ability of the seed extract to prevent iron-induced lipid peroxidation in vitro was also investigated. Ethanol feeding for 60 days resulted in significant increases in the activities of serum aspartate transaminase, alanine transaminase and alkaline phosphatase. The levels of serum lipid hydroperoxides and thiobarbituric acid reactive substances in liver and brain were also significantly elevated. Significantly lower activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione reductase were observed in liver and brain accompanied by depletion in glutathione, ascorbic acid and alpha-tocopherol concentrations. Activity of Ca(2+) ATPase in brain was significantly lowered. Simultaneous administration of aqueous extract of fenugreek seeds with ethanol prevented the enzymatic leakage and the rise in lipid peroxidation and enhanced the antioxidant potential. The seeds exhibited appreciable antioxidant property in vitro which was comparable with that of reduced glutathione and alpha-tocopherol. Further, histopathological examination of liver and brain revealed that, aqueous extract of fenugreek seeds could offer a significant protection against ethanol toxicity.
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PMID:Protective effect of fenugreek (Trigonella foenum graecum) seeds in experimental ethanol toxicity. 1291 70

The biological functions of the myosin light chain 1 (LC1) have not been clearly elucidated yet. In this work we cloned and expressed N- and C- terminal fragments of human ventricular LC1 (HVLC1) containing amino acid residues 1-98 and 99-195 and two parts, NN and NC of N fragment in GST-fusion forms, respectively. Using GST pull-down assay, the direct binding experiments of LC1 with rat cardiac G-actin, F-actin and thin filaments, as well as rat cardiac myosin heavy chain (RCMHC) have been performed. Furthermore, the recombinant complexes of rat myosin S1 with N- and C-fragments, as well as the whole molecular of HVLC1 were generated. The results suggested that both binding sites of HVLC1 for actin and myosin heavy chain are positioned in its N-terminal fragment, which may contain several actin-binding sites in tandem. The polymerization of G-actin, the tropomyosin and troponin molecules located in the thin filaments do not hinder the binding of N-terminal fragment of HVLC1 with actin and thin filaments in vitro. The recombinant complex of rat cardiac myosin S1 (RCMS1) with N fragment of HVLC1 greatly decreased actin-activated Mg(2+)-ATPase activity for lack of C fragment. We conclude that the N-fragment is the binding domain of human ventricular LC1, whereas the C-fragment serves as a functional domain, which may be more involved in the modulation of the actin-activated ATPase activity of myosin.
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PMID:The functional domains of human ventricular myosin light chain 1. 1451 12

Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein-protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.
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PMID:Structure and function of human Vps20 and Snf7 proteins. 1458 93

Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.
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PMID:Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain. 1460 47

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 Delta strain. These mutations define >60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G(1) cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a two-hybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1.Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G(1) Pcl1.Pho85 complex regulates mitotic exit machinery.
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PMID:The identification of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit. 1508 39

The non-random pattern of genome-wide DNA methylation in mammalian cells is established and maintained by DNA methyltransferases DNMT1, 3A, and 3B. De novo DNA methyltransferase DNMT3B is critical for embryonic development and is mutated in ICF syndrome. Despite its importance in normal cellular functioning, little is known about how DNMT3B operates in the context of chromatin. Here we demonstrate that DNMT3B associates with four chromatin-associated enzymatic activities common to transcriptionally repressed, heterochromatic regions of the genome: DNA methyltransferase, histone deacetylase, ATPase, and histone methylase activities. By immunoprecipitation and GST pull-down, we show that DNMT3B interacts with HDAC1, HDAC2, HP1 proteins, Suv39h1, and the ATP-dependent chromatin remodeling enzyme hSNF2H. Endogenous hSNF2H is also associated with DNA methyltransferase activity. These proteins co-localize extensively with DNMT3B in heterochromatic regions. Our results therefore link DNMT3B to three other components of the epigenetic machinery and provide important insights into how DNA methylation patterns may be established within the chromatin environment.
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PMID:DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system. 1512 Jun 35


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