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Roger A. Sunde - One of the best experts on this subject based on the ideXlab platform.
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impact of glutathione peroxidase 1 GPX1 genotype on selenoenzyme and transcript expression when repleting selenium deficient mice
Biological Trace Element Research, 2018Co-Authors: Roger A. Sunde, Edward T Zemaitis, Andrew B Blink, Julia A LawingerAbstract:Glutathione peroxidase (GPX1) is the major selenoprotein in most tissues in animals. Knockout (KO) of GPX1 decreases GPX1 activity to near zero and substantially reduces liver selenium (Se) levels, but has no overt effects in otherwise healthy mice. To investigate the impact of deletion of GPX1 on Se metabolism, Se flux, and apparent Se requirements, KO, GPX1 heterozygous (Het), and GPX1 wild-type (WT) mice were fed Se-deficient diet for 17 weeks, then repleted with graded levels of Se (0–0.3 μg Se/g as Na2SeO3) for 7 days, and selenoprotein activities and transcripts were determined in blood, liver, and kidney. Se deficiency decreased the activities of plasma Gpx3, liver GPX1, liver Txnrd, and liver Gpx4 to 3, 0.3, 11, and 50% of WT Se-adequate levels, respectively, but the GPX1 genotype had no effect on growth or changes in activity or expression of selenoproteins other than GPX1. Se repletion increased selenoprotein transcripts to Se-adequate levels after 7 days; Se response curves and apparent Se requirements for selenoprotein transcripts were similar to those observed in studies starting with Se-adequate mice. With short-term Se repletion, selenoenzyme activities resulted in three Se response curve patterns: (1) liver and kidney GPX1, Gpx4, and Txnrd activities were sigmoidal or hyperbolic with breakpoints (0.08–0.19 μg Se/g) that were double those observed in studies starting with Se-adequate mice; (2) red blood cell GPX1 activity was not significantly changed; and (3) plasma Gpx3 activity only increased substantially with 0.3 μg Se/g. Plasma Gpx3 is secreted from kidney. In this short-term study, kidney Gpx3 mRNA reached plateau levels at 0.1 μg Se/g, and other kidney selenoenzyme activities reached plateau levels at ≤ 0.2 μg Se/g, so sufficient Se should have been present in kidney. Thus, the delayed increase in plasma Gpx3 activity suggests that newly synthesized and secreted kidney Gpx3 is preferentially retained in kidney or rapidly cleared by binding to basement membranes in kidney or in other tissues. This repletion study shows that loss of capacity to incorporate Se into GPX1 in GPX1 KO mice does not dramatically alter expression of other Se biomarkers, nor the short-term flux of Se from intestine to liver to kidney.
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selenium regulation of selenoprotein enzyme activity and transcripts in a pilot study with founder strains from the collaborative cross
PLOS ONE, 2018Co-Authors: Roger A. SundeAbstract:: Rodents and humans have 24-25 selenoproteins, and these proteins contain the 21st amino acid, selenocysteine, incorporated co-translationally into the peptide backbone in a series of reactions dependent on at least 6 unique gene products. In selenium (Se) deficiency, there is differential regulation of selenoprotein expression, whereby levels of some selenoproteins and their transcripts decrease dramatically in Se deficiency, but other selenoprotein transcripts are spared this decrease; the underlying mechanism, however, is not fully understood. To begin explore the genetic basis for this variation in regulation by Se status in a pilot study, we fed Se-deficient or Se-adequate diets (0.005 or 0.2 μg Se/g, respectively) for eight weeks to the eight Founder strains of the Collaborative Cross. We found rather uniform expression of selenoenzyme activity for glutathione peroxidase (Gpx) 3 in plasma, GPX1 in red blood cells, and GPX1, Gpx4, and thioredoxin reductase in liver. In Founder mice, Se deficiency decreased each of these activities to a similar extent. Regulation of selenoprotein transcript expression by Se status was also globally retained intact, with dramatic down-regulation of GPX1, Selenow, and Selenoh transcripts in all 8 strains of Founder mice. These results indicate that differential regulation of selenoprotein expression by Se status is an essential aspect of Se metabolism and selenoprotein function. A few lone differences in Se regulation were observed for individual selenoproteins in this pilot study, but these differences did not single-out one strain or one selenoprotein that consistently had unique Se regulation of selenoprotein expression. These differences should be affirmed in larger studies; use of the Diversity Outbred and Collaborative Cross strains may help to better define the functions of these selenoproteins.
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Selenium regulation of selenoprotein enzyme activity and transcripts in a pilot study with Founder strains from the Collaborative Cross - Fig 2
2018Co-Authors: Roger A. SundeAbstract:Plasma Gpx3 activity (A), RBC GPX1 activity (B), liver GPX1 activity (C), liver Gpx4 activity (D), and liver Txnrd (E) activity in male B6 mice fed 0 (-Se) or 0.2 μg Se/g (+Se) diet for 8 wk. Activities are expressed as enzyme unit/g protein. Values are mean ± SEM (n = 3/treatment); means with asterisks are significantly different from Se-adequate values (P
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Selenium regulation of selenoprotein enzyme activity and transcripts in a pilot study with Founder strains from the Collaborative Cross - Fig 3
2018Co-Authors: Roger A. SundeAbstract:Relative expression of plasma Gpx3 activity (A), RBC GPX1 activity (B), liver GPX1 activity (C), liver Gpx4 activity (D), and liver Txnrd (E) activity. Activities are expressed as a percentage of activities in Se-adequate B6 mice. Left portion of each panel shows the relative activity in B6 mice fed 0 μg Se/g (-Se) and 0.2 μg Se/g (+Se) relative to +Se B6 mice; values are mean ± SEM (n = 3/treatment). Right portion of each panel shows the relative activities in -Se and +Se Founder mice as box-and-whisker plots, where the box delineates 25 to 75%, the bar in the box the median, and the error bars 10 and 90%; also shown are individual values linked by lines for each of the 8 strains. Means with asterisks are significantly different from Se-adequate values (P
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minimum selenium requirements increase when repleting second generation selenium deficient rats but are not further altered by vitamin e deficiency
Biological Trace Element Research, 2017Co-Authors: Roger A. Sunde, Kevin M Thompson, Kevin L Fritsche, Jacqueline K EvensonAbstract:Second-generation selenium-deficient weanling rats fed graded levels of dietary Se were used (a) to study the impact of initial Se deficiency on dietary Se requirements; (b) to determine if further decreases in selenoperoxidase expression, especially glutathione peroxidase 4 (Gpx4), affect growth or gross disease; and (c) to examine the impact of vitamin E deficiency on biochemical and molecular biomarkers of Se status. Rats were fed a vitamin E-deficient and Se-deficient crystalline amino acid diet (3 ng Se/g diet) or that diet supplemented with 100 μg/g all-rac-α-tocopheryl acetate and/or 0, 0.02, 0.05, 0.075, 0.1, or 0.2 μg Se/g diet as Na2SeO3 for 28 days. Se-supplemented rats grew 6.91 g/day as compared to 2.17 and 3.87 g/day for vitamin E-deficient/Se-deficient and vitamin E-supplemented/Se-deficient groups, respectively. In Se-deficient rats, liver Se, plasma Gpx3, red blood cell GPX1, liver GPX1 and Gpx4 activities, and liver GPX1 mRNA levels decreased to 50 % higher than in previous studies that started with Se-adequate rats, demonstrating that dietary Se requirements determined using initially Se-deficient animals can result in overestimation of Se requirements.
Joseph Heitman - One of the best experts on this subject based on the ideXlab platform.
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2005. G� subunit Gpa2 recruits Kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae. Mol. Biol. Cell 16:4557–4571
2013Co-Authors: Toshiaki Harashima, Joseph Heitman, Department Of Molecular GeneticsAbstract:All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated G� subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional G � subunits, Gpa2 forms an atypical G protein complex with the kelch repeat G � mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated G � mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes. This article was published online ahead of print in MBC in Pres
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the kelch proteins gpb1 and gpb2 inhibit ras activity via association with the yeast rasgap neurofibromin homologs ira1 and ira2
Molecular Cell, 2006Co-Authors: Toshiaki Harashima, Scott Anderson, John R Yates, Joseph HeitmanAbstract:Summary The G protein-coupled receptor Gpr1 and associated Gα subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae . Gpa2 forms a protein complex with the kelch Gβ mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1.
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Gα Subunit Gpa2 Recruits Kelch Repeat Subunits That Inhibit Receptor-G Protein Coupling during cAMP-induced Dimorphic Transitions in Saccharomyces cerevisiae
Molecular Biology of the Cell, 2005Co-Authors: Toshiaki Harashima, Joseph HeitmanAbstract:All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Gα subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Gα subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gβ mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gβ mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes.
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The Gα Protein Gpa2 Controls Yeast Differentiation by Interacting with Kelch Repeat Proteins that Mimic Gβ Subunits
Molecular Cell, 2002Co-Authors: Toshiaki Harashima, Joseph HeitmanAbstract:Abstract G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Gα protein, Gpa2, which lacks any known Gβγ subunits. Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors. Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gβ subunits and instead contain seven kelch repeats implicated in protein-protein interactions. Gβ subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed β propellers. Our studies demonstrate that Gpa2 signals in conjunction with Gβ structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes.
Yoshiharu Inoue - One of the best experts on this subject based on the ideXlab platform.
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glutathione peroxidase 2 in saccharomyces cerevisiae is distributed in mitochondria and involved in sporulation
Biochemical and Biophysical Research Communications, 2011Co-Authors: Yuuta Ukai, Tomoyuki Kishimoto, Takumi Ohdate, Singo Izawa, Yoshiharu InoueAbstract:Abstract Gpx2, one of three glutathione peroxidase homologs (GPX1, Gpx2, and Gpx3) in Saccharomyces cerevisiae, is an atypical 2-Cys peroxiredoxin that prefers to use thioredoxin as a reducing agent in vitro. Despite Gpx2 being an antioxidant, no obvious phenotype of gpx2Δ mutant cells in terms of oxidative stress has yet been found. To gain a clue as to Gpx2’s physiological function in vivo, here we identify its intracellular distribution. Gpx2 was found to exist in the cytoplasm and mitochondria. In mitochondria, Gpx2 was associated with the outer membrane of the cytoplasmic-side, as well as the inner membrane of the matrix-side. The redox state of the mitochondrial Gpx2 was regulated by Trx1 and Trx2 (cytoplasmic thioredoxin), and by Trx3 (mitochondrial matrix thioredoxin). In addition, we found that the disruption of GPX2 reduced the sporulation efficiency of diploid cells.
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kinetics and redox regulation of GPX1 an atypical 2 cys peroxiredoxin in saccharomyces cerevisiae
Fems Yeast Research, 2010Co-Authors: Takumi Ohdate, Keiko Kita, Yoshiharu InoueAbstract:The budding yeast Saccharomyces cerevisiae has three homologues of glutathione peroxidase (GPX1, GPX2, and GPX3). Two structural homologues of the mammalian glutathione peroxidase, Gpx2 and Gpx3, have been proven to be atypical 2-Cys peroxiredoxins, which prefer to use thioredoxin as an electron donor. Here, we show that GPX1 is also an atypical 2-Cys peroxiredoxin, but uses glutathione and thioredoxin almost equally. We determined the redox state of GPX1 in vivo.
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regulatory mechanism for expression of GPX1 in response to glucose starvation and ca in saccharomyces cerevisiae involvement of snf1 and ras camp pathway in ca signaling
Genes to Cells, 2010Co-Authors: Takumi Ohdate, Shingo Izawa, Keiko Kita, Yoshiharu InoueAbstract:Saccharomyces cerevisiae has three homologues of the glutathione peroxidase gene, GPX1, GPX2, and GPX3. We have previously reported that the expression of GPX3 was constitutive, but that of GPX2 was induced by oxidative stress and CaCl2, and uncovered the regulatory mechanisms involved. Here, we show that the expression of GPX1 is induced by glucose starvation and treatment with CaCl2. The induction of GPX1 expression in response to glucose starvation and Ca2+ was dependent on the transcription factors Msn2 and Msn4 and cis-acting elements [stress response element (STRE)] in the GPX1 promoter. The Ras/cAMP pathway is also involved in the expression of GPX1. We found that Snf1, a Ser/Thr protein kinase, is involved in the glucose starvation- and Ca2+-induced expression of GPX1. The activation of Snf1 is accompanied by phosphorylation of Thr210. We found that the Ca2+-treatment as well as glucose starvation causes the phosphorylation of Thr210 of Snf1 in a Tos3, Sak1, and Elm1 protein kinase-dependent manner. As the timing of the initiation of Ca2+-induced expression of GPX1 was retarded in an snf1Δ mutant, the activation of Snf1 seems pivotal to the early-stage-response of GPX1 to Ca2+.
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gpx2 encoding a phospholipid hydroperoxide glutathione peroxidase homologue codes for an atypical 2 cys peroxiredoxin in saccharomyces cerevisiae
Journal of Biological Chemistry, 2005Co-Authors: Tomoaki Tanaka, Shingo Izawa, Yoshiharu InoueAbstract:Abstract We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S., and Kimura, A. (1999) J. Biol. Chem. 274, 27002–27009). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1Δtrx2Δ) under non-stressed conditions and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1Δtrx2Δ and thioredoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys37 and Cys83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H2O2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase, and NADPH (for H2O2, Km = 20 μm, kcat = 9.57 × 102 s-1; for tert-butyl hydroperoxide, Km = 62.5 μm, kcat = 3.68 × 102 s-1); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase, and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca2+, a tendency that was most obvious in gpx2Δ cells. Although the redox state of Gpx2 was not affected by Ca2+, the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca2+. Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca2+.
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genetic analysis of glutathione peroxidase in oxidative stress response of saccharomyces cerevisiae
Journal of Biological Chemistry, 1999Co-Authors: Yoshiharu Inoue, Toshifumi Matsuda, Keiichi Sugiyama, Shingo Izawa, Akira KimuraAbstract:Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Δ mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Δ and GPX1Δ/gpx2Δ/gpx3Δ mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the GPX1Δ/gpx2Δ/gpx3Δ mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Δ mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.
Toshiaki Harashima - One of the best experts on this subject based on the ideXlab platform.
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2005. G� subunit Gpa2 recruits Kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae. Mol. Biol. Cell 16:4557–4571
2013Co-Authors: Toshiaki Harashima, Joseph Heitman, Department Of Molecular GeneticsAbstract:All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated G� subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional G � subunits, Gpa2 forms an atypical G protein complex with the kelch repeat G � mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated G � mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes. This article was published online ahead of print in MBC in Pres
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the kelch proteins gpb1 and gpb2 inhibit ras activity via association with the yeast rasgap neurofibromin homologs ira1 and ira2
Molecular Cell, 2006Co-Authors: Toshiaki Harashima, Scott Anderson, John R Yates, Joseph HeitmanAbstract:Summary The G protein-coupled receptor Gpr1 and associated Gα subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae . Gpa2 forms a protein complex with the kelch Gβ mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1.
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Gα Subunit Gpa2 Recruits Kelch Repeat Subunits That Inhibit Receptor-G Protein Coupling during cAMP-induced Dimorphic Transitions in Saccharomyces cerevisiae
Molecular Biology of the Cell, 2005Co-Authors: Toshiaki Harashima, Joseph HeitmanAbstract:All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Gα subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Gα subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gβ mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gβ mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes.
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The Gα Protein Gpa2 Controls Yeast Differentiation by Interacting with Kelch Repeat Proteins that Mimic Gβ Subunits
Molecular Cell, 2002Co-Authors: Toshiaki Harashima, Joseph HeitmanAbstract:Abstract G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Gα protein, Gpa2, which lacks any known Gβγ subunits. Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors. Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gβ subunits and instead contain seven kelch repeats implicated in protein-protein interactions. Gβ subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed β propellers. Our studies demonstrate that Gpa2 signals in conjunction with Gβ structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes.
John E Hesketh - One of the best experts on this subject based on the ideXlab platform.
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influence of genetic variations in selenoprotein genes on the pattern of gene expression after supplementation with brazil nuts
Nutrients, 2017Co-Authors: Janaina L S Donadio, John E Hesketh, Marcelo Macedo Rogero, Simon Cockell, Silvia Maria Franciscato CozzolinoAbstract:Selenium (Se) is an essential micronutrient for human health. Its beneficial effects are exerted by selenoproteins, which can be quantified in blood and used as molecular biomarkers of Se status. We hypothesize that the presence of genetic polymorphisms in selenoprotein genes may: (1) influence the gene expression of specific selenoproteins and (2) influence the pattern of global gene expression after Brazil nut supplementation. The study was conducted with 130 healthy volunteers in Sao Paulo, Brazil, who consumed one Brazil nut (300 μg/Se) a day for eight weeks. Gene expression of GPX1 and SELENOP and genotyping were measured by real-time PCR using TaqMan Assays. Global gene expression was assessed by microarray using Illumina HumanHT-12 v4 BeadChips. Brazil nut supplementation significantly increased GPX1 mRNA expression only in subjects with CC genotype at rs1050450 (p < 0.05). SELENOP mRNA expression was significantly higher in A-carriers at rs7579 either before or after supplementation (p < 0.05). Genotype for rs713041 in GPX4 affected the pattern of blood cell global gene expression. Genetic variations in selenoprotein genes modulated both GPX1 and SELENOP selenoprotein gene expression and global gene expression in response to Brazil nut supplementation.
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selenium alters mirna profile in an intestinal cell line evidence that mir 185 regulates expression of gpx2 and sepsh2
Molecular Nutrition & Food Research, 2013Co-Authors: Anabel Macieldominguez, Daniel Swan, Dianne Ford, John E HeskethAbstract:Scope Intake of the essential micronutrient selenium (Se) has health implications. This work addressed whether some effects of Se on gene expression are exerted through microRNAs (miRNA). Methods and results Human colon adenocarcinoma cells (Caco-2) were grown in Se-deficient or Se-adequate medium for 72 h. RNA was extracted and subjected to analysis of 737 miRNA using microarray technology. One hundred and forty-five miRNA were found to be expressed in Caco-2 cells. Twelve miRNA showed altered expression after Se depletion: miR-625, miR-492, miR-373*, miR-22, miR-532–5p, miR-106b, miR-30b, miR-185, miR-203, miR1308, miR-28–5p, miR-10b. These changes were validated by quantitative real-time PCR (RT-qPCR). Transcriptomic analysis showed that Se depletion altered expression of 50 genes including selenoproteins GPX1, SELW, GPX3, SEPN1, SELK, SEPSH2 and GPX4. Pathway analysis identified arachidonic acid metabolism, glutathione metabolism, oxidative stress, positive acute phase response proteins and respiration of mitochondria as Se-sensitive pathways. Bioinformatic analysis identified 13 transcripts as targets for the Se-sensitive miRNA; three were predicted to be recognised by miR-185. Silencing of miR-185 increased GPX2 and SEPSH2 expression. Conclusions We propose that miR-185 plays a role in up-regulation of GPX2 and SEPHS2 expression. In the case of SEPHS2 this may contribute to maintaining selenoprotein synthesis. The data indicate that micronutrient supply can regulate the cell miRNA expression profile.
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association between polymorphisms in glutathione peroxidase and selenoprotein p genes glutathione peroxidase activity hrt use and breast cancer risk
PLOS ONE, 2013Co-Authors: Catherine Meplan, Lars O Dragsted, Gitte Ravnharen, Anne Tjonneland, Ulla Vogel, John E HeskethAbstract:Breast cancer (BC) is one of the most common cancers in women. Evidence suggests that genetic variation in antioxidant enzymes could influence BC risk, but to date the relationship between selenoproteins and BC risk remains unclear. In this report, a study population including 975 Danish cases and 975 controls matched for age and hormone replacement therapy (HRT) use was genotyped for five functional single nucleotide polymorphisms (SNPs) in SEPP1, GPX1, GPX4 and the antioxidant enzyme SOD2 genes. The influence of genetic polymorphisms on breast cancer risk was assessed using conditional logistic regression. Additionally pre-diagnosis erythrocyte GPx (eGPx) activity was measured in a sub-group of the population. A 60% reduction in risk of developing overall BC and ductal BC was observed in women who were homozygous Thr carriers for SEPP1 rs3877899. Additionally, Leu carriers for GPX1 Pro198Leu polymorphism (rs1050450) were at ,2 fold increased risk of developing a non-ductal BC. Pre-diagnosis eGPx activity was found to depend on genotype for rs713041 (GPX4), rs3877899 (SEPP1), and rs1050450 (GPX1) and on HRT use. Moreover, depending on genotype and HRT use, eGPx activity was significantly lower in women who developed BC later in life compared with controls. Furthermore, GPX1 protein levels increased in human breast adenocarcinoma MCF7 cells exposed to b-estradiol and sodium selenite.In conclusion, our data provide evidence that SNPs in SEPP1 and GPX1 modulate risk of BC and that eGPx activity is modified by SNPs in SEPP1, GPX4 and GPX1 and by estrogens. Our data thus suggest a role of selenoproteins in BC development.
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a novel single nucleotide polymorphism in the 3 untranslated region of human glutathione peroxidase 4 influences lipoxygenase metabolism
Blood Cells Molecules and Diseases, 2002Co-Authors: Stephane Villette, Janet Kyle, Katrina M Brown, Karen Pickard, John S Milne, Fergus Nicol, John R Arthur, John E HeskethAbstract:Abstract ABSTRACT Selenium (Se) is an essential micronutrient for human health. The biological roles of the essential micronutrient Se are attributed to its presence in a range of 20–30 selenoproteins including the cytosolic and phospholipid hydroperoxide glutathione peroxidases (GPX1 and GPX4). It has been suggested that GPX4 may play a role in regulation of leukotriene biosynthesis and thus inflammation. In eukaryotes Se is incorporated into selenoproteins as the amino acid selenocysteine in a process requiring a stem-loop within the 3′ untranslated region (3′UTR) of the mRNA. In this study the region of the GPX4 gene corresponding to the 3′UTR was scanned for mutations in a group of 66 volunteers. The data show a T/C variant at position 718. The distribution of this SNP in our population was 34% CC, 25% TT and 41% TC; i.e., it is in Hardy–Weinberg equilibrium. Individuals of different genotypes exhibited significant differences in the levels of lymphocyte 5-lipoxygenase total products, with C718 showing increased levels of those products compared to T718 and T/C718 (36% and 44% increases, respectively). The data suggest that the SNP718 that we have identified has functional effects and support the hypothesis that GPX4 plays a regulatory role in leukotriene biosynthesis.
