Transcriptional regulation of Saccharomyces cerevisiae CYS3 encoding cystathionine gamma-lyase.
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ABSTRACT:
SUBMITTER: Hiraishi H
PROVIDER: S-EPMC2668581 | biostudies-other | 2008 Apr
REPOSITORIES: biostudies-other
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Project description:By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) gene, encoding cystathionine gamma-lyase (EC 4.4.1.1), and isolated previously as a cycloheximide-induced gene (B. Ono, K. Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes a defect in serine O-acetyltransferase (EC 2.3.1.30), and another, designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrifugation in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as substrate. In addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) activity but could find no detectable serine O-acteyltransferease activity. In this paper, we compare the enzymatic properties of the protein with those of homologous enzymes previously reported and discuss the possibility that this enzyme has a physiological role as cystathionine Beta-lyase and cystathionine gamma-synthase in addition to its previously described role as cystathionine gamma-lyase.
| S-EPMC204932 | biostudies-other
Project description:Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/-, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.
| S-EPMC5473925 | biostudies-literature
Project description:We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine gamma-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B(6) administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects.
| S-EPMC2752209 | biostudies-literature
Project description:Background and purposel-cysteine or hydrogen sulfide (H2 S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30-100 μM. Here, we have investigated the signalling involved in the H2 S-induced contraction.Experimental approachVascular response to NaHS or l-cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ-lyase (CSE-/- ), soluble guanylyl cyclase (sGCα1 -/- ) and endothelial nitric oxide synthase (eNOS-/- ) knock-out mice. The cAMP, cGMP and inosine 3',5'-cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated.Key resultsCSE-derived H2 S-induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2 S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell-permeable analogue of cIMP elicits concentration-dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE-/- mice, confirms that H2 S-induced contraction involves cIMP.Conclusion and implicationsThe endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE-derived H2 S that is mediated by cIMP.
| S-EPMC8453910 | biostudies-literature
Project description:Hydrogen sulfide (H2 S), an endogenous signaling gaseous molecule, is involved in various physiological activities, including vessel relaxation, regulation of cellular bioenergetics, inflammation, and angiogenesis. By using xenograft orthotopic implantation of prostate cancer PC3 cells and subsequently comparing bone metastatic with primary tumor-derived cancer cells, we find that H2 S-producing enzyme cystathionine γ-lyase (CTH) is upregulated in bone-metastatic PC3 cells. Clinical data further reveal that the expression of CTH is elevated in late-stage prostate cancer patients, and higher CTH expression correlates with poor survival from The Cancer Genome Atlas (TCGA) prostate cancer RNA-seq datasets. CTH promotes NF-κB nuclear translocation through H2 S-mediated sulfhydration on cysteine-38 of the NF-κB p65 subunit, resulting in increased IL-1β expression and H2 S-induced cell invasion. Knockdown of CTH in PC3 cells results in the suppression of tumor growth and distant metastasis, while overexpression of CTH in DU145 cells promotes primary tumor growth and lymph node metastasis in the orthotopic implanted xenograft mouse model. Together, our findings provide evidence that CTH generated H2 S promotes prostate cancer progression and metastasis through IL-1β/NF-κB signaling pathways.
| S-EPMC6776913 | biostudies-literature
Project description:Saccharomyces cerevisiae myosin type II-deficient (myo1?) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1? transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1? strain may provide a simplified paradigm for cell wall stress survival.To explore the post-transcriptional regulation of the myo1? stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1? strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2?-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1? strains. Deletion of GCN2 in myo1? abolished eIF2?p phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1? strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1? and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2?-P in myo1? strains.Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1? strains. Inhibition of translation initiation factor eIF2? (eIF2?-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2?-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1? strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2?-P in myo1? strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1? stress response and possibly other stresses in yeast.
| S-EPMC3017085 | biostudies-literature
Project description:As toxic substances can enter the circulating blood and cross endothelial monolayers to reach parenchymal cells in organs, vascular endothelial cells are an important target compartment for such substances. Reactive sulfur species protect cells against oxidative stress and toxic substances, including heavy metals. Reactive sulfur species are produced by enzymes, such as cystathionine γ-lyase (CSE), cystathionine β-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase. However, little is known about the regulatory mechanisms underlying the expression of these enzymes in vascular endothelial cells. Bio-organometallics is a research field that analyzes biological systems using organic-inorganic hybrid molecules (organometallic compounds and metal coordinating compounds) as molecular probes. In the present study, we analyzed intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes in cultured bovine aortic endothelial cells, using copper diethyldithiocarbamate (Cu10). Cu10 selectively upregulated CSE gene expression in vascular endothelial cells independent of cell density. This transcriptional induction of endothelial CSE required both the diethyldithiocarbamate scaffold and the coordinated copper ion. Additionally, the present study revealed that ERK1/2, p38 MAPK, and hypoxia-inducible factor (HIF)-1α/HIF-1β pathways mediate transcriptional induction of endothelial CSE by Cu10. The transcription factors NF-κB, Sp1, and ATF4 were suggested to act in constitutive CSE expression, although the possibility that they are involved in the CSE induction by Cu10 cannot be excluded. The present study used a copper complex as a molecular probe to reveal that the transcription of CSE is regulated by multiple pathways in vascular endothelial cells, including ERK1/2, p38 MAPK, and HIF-1α/HIF-1β. Bio-organometallics appears to be an effective strategy for analyzing the functions of intracellular signaling pathways in vascular endothelial cells.
| S-EPMC7503448 | biostudies-literature
Project description:Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.
| S-EPMC2223195 | biostudies-literature
Project description:BackgroundMutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance.MethodsProteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice.ResultsWe find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice.ConclusionsWe show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.
| S-EPMC8262642 | biostudies-literature
Project description:Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 μΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.
| S-EPMC8051778 | biostudies-literature