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Biomedical subjects

S G Rhee

Publications and source records attributed to S G Rhee.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity.

Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys(59)/Cys(64) adjacent to the flavin ring of FAD and another center consisting of Cys(497)/selenocysteine (SeCys)(498) near the C terminus. We now show that the C-terminal Cys(497)-SH/SeCys(498)-Se(-) of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS(2)) or H(2)O(2). The Cys(59)-SH/Cys(64)-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS(2), the Cys(59)-SH/Cys(64)-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys(497)-SH/SeCys(498)-Se(-). Specific alkylation of SeCys(498) completely blocked the TrxS(2)-induced oxidation of Cys(59)-SH/Cys(64)-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys(498) with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys(497)/Cys(498) mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5, 5'-dithiobis(2-nitrobenzoic acid) or TrxS(2), respectively, as substrate. Disulfide formation induced by excess TrxS(2) in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.

Animals↗
PubMed2000

Differential roles of the Src homology 2 domains of phospholipase C-gamma1 (PLC-gamma1) in platelet-derived growth factor-induced activation of PLC-gamma1 in intact cells.

Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-gamma1 (PLC-gamma1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4, 5-bisphosphate. Association of PLC-gamma1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-gamma1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-gamma1 proteins were transiently expressed in a PLC-gamma1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH(2)-terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH(2)-terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-gamma1 is required for PDGF-induced activation of PLC-gamma1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-gamma1, suggesting that recruitment of PLC-gamma1 to the particulate fraction does not involve the SH2 domains.

Animals↗
PubMed1999

Rapid degradation of PrxI and PrxII induced by silica in Rat2 cells.

Peroxidases of the peroxiredoxin (Prx) family catalyze the reduction of H(2)O(2) and lipid peroxides. The effects of H(2)O(2), 12-O-tetradecanoylphorbol 13-acetate (TPA), and silica on the abundance of two cytosolic isoforms of Prx (PrxI and PrxII) were examined in Rat2 cells. TPA induces the production of reactive oxygen species (ROS) in various mammalian cell types, and silica induces the production of ROS in Rat2 cells. Whereas H(2)O(2) and TPA did not affect the concentration of PrxI or Prx II, silica triggered a rapid degradation of both Prx enzymes. Silica also induced degradation of the NF-kappaB inhibitor IkappaB-alpha. N-Acetylcysteine and diphenyleneiodonium, both of which inhibit the accumulation of intracellular ROS, each blocked silica-induced degradation of IkappaB-alpha but had no effect on that of the Prx enzymes, suggesting that ROS do not contribute to Prx proteolysis. The silica-induced degradation of Prx enzymes was also insensitive to the proteasome inhibitors MG132 and lactacystin, whereas IkappaB-alpha proteolysis was completely blocked by these inhibitors. Experiments with the Ca(2+) ionophore A23187 indicated that a Ca(2+)-dependent protease such as calpain might contribute substantially to silica-induced degradation of PrxII, but only moderately to that of PrxI. These results indicate that silica increases cellular oxidative stress not only by inducing ROS production, but also by triggering the degradation of Prx enzymes that are responsible for elimination of cellular ROS. Such aggravated oxidative stress might be important in the initial pathogenesis of silica-associated pulmonary diseases.

Animals↗
PubMed1999

Characterization of mouse peroxiredoxin I genomic DNA and its expression.

Peroxiredoxins (Prxs) are a newly defined family of anti-oxidant proteins that have been implicated, via their anti-oxidant activity, in a number of cellular functions, including cell proliferation and differentiation, protection of other proteins from oxidative damage, and intracellular signaling. We isolated genomic DNA sequences of the Prx I genes from the mouse, and characterized their molecular genetic features. Prx I was found to form a small gene family with two and three members; one functional and two pseudogenes. The Prx I-1 gene has splice donor/acceptor site sequences and five or six exons, whereas the Prx I-2 clone has several structural features characteristic of a typical retroposon found to have ORF sequences. We analyzed the expression of pseudogenes, which were not expressed on the transcription levels in the investigated organs. The functional copy of the Prx I-1 gene was expressed abundantly in liver and kidney of the adult, as well as in early developing embryos. This report, together with amino acid/nucleotide sequence similarity between human and mice, provides a basis for speculating on an even earlier event in the evolution of the Prx I gene family, i.e. the Prx I gene was well conserved in human and mice via its anti-oxidant activity.

Amino Acid Sequence↗
PubMed1999

Characterization of the murine gene encoding 1-Cys peroxiredoxin and identification of highly homologous genes.

A new type of peroxiredoxin, named 1-Cys peroxiredoxin (1-Cys Prx), reduces hydrogen peroxide with the use of electrons from unidentified electron donor(s). We have isolated the mouse gene encoding 1-Cys Prx (CP-3) and shown that it is comprised of five exons and four introns. Analysis of 5' flanking regions revealed binding sequences of several putative transcription factors such as Sp1, Pit-1a, c-Jun, c-Myc and YY1. It is noticeable that several potential Sp1 binding sites assigned the -60 through -96bp from putative transcription initiation site. The gel shift assays showed that Sp1 and Pit-1a bind specifically to each binding site in 1-Cys Prx promoter. We also isolated two highly related genes such as CP-2 and CP-5. These genes are encoded by single exons, and show 85% of nucleotide sequence homology with the CP-3. The structural features of these genes suggest that they might be intronless genes derived from the CP-3 by the mechanism involving retrotransposition. In addition, our data suggest that they are inserted to a specific site of the mouse L1 repetitive element. The 1-Cys Prx was actively transcribed in a variety of adult tissues as well as in the developing embryos. These results suggest that only the 1-Cys Prx gene might be relevant for studying the function of the 1-Cys Prx in the murine system.

Amino Acid Sequence↗
PubMed1999

Redox signaling: hydrogen peroxide as intracellular messenger.

Although superoxide anions (O2.-) and H2O2 are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. In mammalian cells, a variety of extracellular stimuli have recently been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of stimulant-dependent signaling. In the next few years, therefore, a flurry of research activity is expected in relation to the elucidation of ROS production in response to receptor stimulation, identification of ROS target molecules, and investigation of ROS elimination. The goal of this report is to review our current knowledge of ROS-regulated signal transduction and propose future directions.

Animals↗
PubMed1999

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

3T3 Cells↗
PubMed1999

AHNAK, a protein that binds and activates phospholipase C-gamma1 in the presence of arachidonic acid.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver.

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.

Amino Acid Sequence↗
PubMed1999

Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae.

A yeast peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of reducing equivalents provided by thioredoxin was identified previously and named thioredoxin peroxidase (TPx) [Chae, H. Z., Chung, S. J., and Rhee, S. G. (1994) J. Biol. Chem. 269, 27670-27678]. A second type thioredoxin-dependent peroxidase, named type II TPx, has now been purified from yeast, and several peptide sequences have been obtained. Using those sequences, the corresponding cDNA has been identified from the GenBank database. Comparison of the predicted sequence of 176 amino acids of type II TPx with that of the 195 residues of TPx, now renamed type I TPx, revealed no substantial homology except for a short segment preceding Cys62 of type II TPx. Kinetic characterization of the reactions catalyzed by type I and II TPxs revealed that type I preferentially reduces H2O2 rather than alkyl hydroperoxides, whereas type II shows the reverse specificity. Type II TPx contains three cysteine residues at positions 31, 62, and 120. Experiments with mutant proteins in which these three cysteine residues were replaced individually with serine suggest that Cys62-SH constitutes the site of oxidation by peroxides and that the oxidized Cys62 reacts with the Cys120-SH group of another type II TPx molecule to form an intermolecular disulfide linkage. The formed disulfide can then be reduced by thioredoxin, but not by glutathione. Thus, type II TPx mutants lacking Cys62 or Cys120 showed no detectable TPx activity, whereas mutation of Cys31 had no effect on TPx activity. An antioxidant function of type II TPx in intact cells was demonstrated by the observation that Escherichia coli cells overexpressing wild-type protein were less sensitive to inhibition of growth by alkyl hydroperoxides than were control cells or cells overexpressing the mutant protein lacking Cys62.

Amino Acid Sequence↗
PubMed1999

Regulation of phospholipase C isozymes: activation of phospholipase C-gamma in the absence of tyrosine-phosphorylation.

Activation of PLC-gamma isozymes in response to various agonists involves tyrosine phosphorylation of the effector enzymes. Recent evidence indicates that PLC-gamma isozymes are additionally activated by phosphatidic acid, phosphatidylinositol 3,4,5-trisphosphate and arachidonic acid in the absence of PLC-gamma tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phosphatidylinositol 3-kinase, and phospholipase A2, enzymes which are often stimulated along with PLC-gamma in response to an agonist. Furthermore, phosphatidylinositol 4,5-bisphosphate acts as a substrate for both PLC-gamma and phosphatidylinositol 3-kinase and as an activator for phospholipase D and phospholipase A2. These results reveal an elaborate mechanism of cross-talk and mutual regulation between four effector enzymes that participate in receptor signaling by acting on phospholipids.

Animals↗
PubMed1999

Inositides in the nucleus: further developments on phospholipase C beta 1 signalling during erythroid differentiation and IGF-I induced mitogenesis.

Inositol lipids originally shown to be metabolized in the cytosol have been detected also in the nucleus, where they are both synthesized and hydrolyzed. In the case of erythroid differentiation of murine erythroleukemia cells (Friend cells) it has been previously shown that PLC beta 1, which is the major nuclear PLC, undergoes down-regulation upon treatment with DMSO or tiazofurin which act as differentiative agents. On the contrary, i.e., during IGF-I induced mitogenesis, it has been shown that PLC beta 1 is rapidly activated and this event is essential for the onset of DNA synthesis. Even though its key role in cell growth has been shown, both the mechanism by which nuclear PLC beta 1 is activated and the direct relationship with erythroid differentiation are still unknown. We have addressed the question if PLC beta 1 expression and activity in the nucleus are directly related or not to the establishment of the differentiated state and we have checked the two main ways of activation, i.e., via G-protein or via phosphorylation, in order to establish whether nuclear PLC beta 1 is regulated the same way as the one at the plasma membrane or not. The data reported here show that nuclear PLC beta 1 is responsible for a continuous recycling of Friend cells, acting as a negative regulator of differentiation and that its activation is dependent on the phosphorylation state.

3T3 Cells↗
PubMed1999

Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin.

A peroxidase from yeast that reduces H2O2 with the use of electrons provided by thioredoxin (Trx) together with homologs from a wide variety of species constitute the peroxiredoxin (Prx) family of proteins. Twelve mammalian Prx members have been previously identified in association with various cellular functions apparently unrelated to peroxidase activity. These mammalian proteins have now been divided into three distinct types, Prx I, II, and III, on the basis of their deduced amino acid sequences and immunological reactivity. With the use of recombinant proteins, Prx I, II, and III have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents. None of the three proteins exhibited peroxidase activity in the presence of glutaredoxin. All three enzymes showed similar kinetic properties: the Vmax was 6-13 micromol/min per mg at 37 degrees C, the Km for Trx was 3-6 microM, and the Km for H2O2 was < 20 microM. Immunoblot analysis of various rat tissues and cultured cells indicated that most cell types contain the three Prx isoforms, the sum of which amounts to approximately 1-10 microg per milligram of soluble protein. Prx I and II are cytosolic proteins, whereas Prx IlI is localized in mitochondria. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism as well as during stimulation of cell surface receptors.

3T3 Cells↗
PubMed1998

Probing cellular protein targets of H2O2 with fluorescein-conjugated iodoacetamide and antibodies to fluorescein.

Recent studies suggest that H2O2, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H2O2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H2O2 is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H2O2 generated in response to cellular stimulation with epidermal growth factor. One of these H2O2-sensitive proteins was identified as protein tyrosine phosphatase 1B.

Antibodies↗