Glutathione Biosynthesis Pathway Research Articles

GsNAC2 gene enhances saline-alkali stress tolerance by promoting plant growth and regulating glutathione metabolism in Sorghum bicolor.

The quality and yields of Sorghum bicolor plants are seriously affected by saline-alkali conditions. NAC (NAM, ATAF, and CUC) transcription factors are plant specific and have various functions in plant development and response to various stresses. To investigate how GsNAC2 functions in sorghum responses to saline-alkali treatment, the characteristics of GsNAC2 were analysed by bioinformatics methods, and NaHCO3:Na2CO3 (5:1, 75mM, pH 9.63) saline-alkali stress solution was applied when sorghum plants were 2weeks old. The research results show that GsNAC2 belongs to the NAC gene family. GsNAC2 was significantly induced by saline-alkali treatment and strongly expressed in sorghum leaves. GsNAC2-overexpressing sorghum plants had increased plant height, dry weight, moisture content, root activity, leaf length, chlorophyll content, stomatal conductance, relative root activity, relative chlorophyll content, relative stomatal conductance, and relative transpiration rate after saline-alkali treatment. Lower H2O2 and O2- levels, relative permeability of the plasma membrane, and malondialdehyde (MDA) content were found in GsNAC2-overexpressing sorghum. In transcriptome analysis, clusters of orthologous groups (COG) analysis showed that a high proportion of differentially-expressed genes (DEGs) participated in defence mechanisms at each processing time, and 18 DEGs related to synthetic glutathione were obtained. Gene expression analysis revealed that key genes in glutathione biosynthesis pathways were upregulated. GR and GSH-Px activities were increased, and GSH accumulated more with the overexpression of GsNAC2 after saline-alkali treatment. Furthermore, these results suggest that GsNAC2 acts as a potentially important regulator in response to saline-alkali stress and may be used in molecular breeding to improve crop yields under adverse environmental conditions.

Exogenous brassinolide improves the antioxidant capacity of Pinellia ternata by enhancing the enzymatic and nonenzymatic defense systems under non-stress conditions.

Brassinolide (BR) improves the antioxidant capacity of plants under various abiotic stresses. However, it is not clear about the effect of BR on the antioxidant capacity in plants under non-stress conditions. In the present study, the antioxidant defense response of Pinellia ternata was to be assessed by applying BR and propiconazole (Pcz) under non-stress conditions. BR treatment enhanced the flavonoid content, peroxidase, and ascorbate peroxidase (APX) activity by 12.31, 30.62, and 25.08% and led to an increase in 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity by 4.31% and a decrease in malondialdehyde content by 1.04%. Exogenous application of BR improved the expression levels of PAL, CHS, CHI, and DFR genes by 3. 18-, 3. 39-, 2. 21-, and 0.87-fold in flavonoid biosynthesis, PGI, PMI, and GME genes by 6. 60-, 1437. 79-, and 3.11-fold in ascorbic acid (ASA), biosynthesis, and γECs and GSHS genes by 6.08- and 2.61-fold in glutathione (GSH) biosynthesis pathway, and the expression of these genes were inhibited by Pcz treatment. In addition, BR treatment promoted the ASA–GSH cycle by enhancing the expression of APX, DHAR, and MDHAR genes, which were enhanced by 3. 33-, 157. 85-, and 154.91-fold, respectively. These results provided novel insights into the effect of BR on the antioxidant capacity in bulbil of P. ternata under non-stress conditions and useful knowledge of applying BR to enhance the antioxidant capacity of plants.

Functional prediction of environmental variables using metabolic networks

In this manuscript, we propose a novel approach to assess relationships between environment and metabolic networks. We used a comprehensive dataset of more than 5000 prokaryotic species from which we derived the metabolic networks. We compute the scope from the reconstructed graphs, which is the set of all metabolites and reactions that can potentially be synthesized when provided with external metabolites. We show using machine learning techniques that the scope is an excellent predictor of taxonomic and environmental variables, namely growth temperature, oxygen tolerance, and habitat. In the literature, metabolites and pathways are rarely used to discriminate species. We make use of the scope underlying structure—metabolites and pathways—to construct the predictive models, giving additional information on the important metabolic pathways needed to discriminate the species, which is often absent in other metabolic network properties. For example, in the particular case of growth temperature, glutathione biosynthesis pathways are specific to species growing in cold environments, whereas tungsten metabolism is specific to species in warm environments, as was hinted in current literature. From a machine learning perspective, the scope is able to reduce the dimension of our data, and can thus be considered as an interpretable graph embedding.

Intrinsic and Chemotherapeutic Stressors Modulate ABCC-Like Transport in Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6–7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.

Stress-driven dynamic regulation of multiple tolerance genes improves robustness and productive capacity of Saccharomyces cerevisiae in industrial lignocellulose fermentation

Yeast productivity in lignocellulosic ethanol fermentation is clearly impeded by stress. Enhancing the robustness of xylose-fermenting yeast is important for improving lignocellulosic ethanol production. In this study, the glutathione biosynthesis pathway and acetic acid degradation pathway were strengthened to enhance yeast tolerance to stress due to elevated reactive oxygen species (ROS) and acetic acid. Dynamic feedback regulation of the anti-stress genetic circuits was achieved using stress-driven promoters discovered from the transcriptome to maintain low intracellular ROS, relieve the metabolic burden, and ultimately improve the robustness and ethanol production of yeast. The cell growth, xylose utilization and ethanol production of the engineered strain were enhanced under both stress and nonstress conditions. The engineered strain showed 49.5% and 17.5% higher ethanol productivity in laboratory media and industrial lignocellulosic media, respectively, at 36 °C compared with the parent strain. This study provides novel insights on the rational design and construction of feedback genetic circuits for dynamically improving yeast robustness.

System χc- overexpression prevents 2-deoxy-d-ribose-induced β-cell damage

Pancreatic β-cells are vulnerable to oxidative stress, which promotes β-cell failure in type 2 diabetes. System χc- is a sodium-independent, cystine/glutamate antiporter that mediates the exchange of extracellular l-cystine and intracellular l-glutamate. The import of l-cystine through this transporter is the rate-limiting step in the glutathione (GSH) biosynthesis pathway that plays a significant role in antioxidative defense. Previously, we reported that 2-deoxy-d-ribose (dRib) induces oxidative damage through GSH depletion in pancreatic β-cells. In the current study, we elucidated the mechanism underlying the oxidative stress-induced β-cell damage. We measured the intracellular l-[14C]cystine uptake, GSH content, reactive oxygen species (ROS) levels, cytotoxicity, and apoptosis in rat insulinoma cell line, RINm5F. Treatment of dRib decreased the intracellular l-[14C]cystine uptake and GSH content and increased the intracellular ROS levels, cytotoxicity, and apoptosis in a time- and dose-dependent manner. Conversely, 2-mercaptoethanol (2-ME), a cystine uptake enhancer, recovered the dRib-induced decrease in l-[14C]cystine uptake, GSH content, and cell viability in a Na+-independent manner. In the case of isolated islets, dRib dose-dependently decreased the intracellular l-[14C]cystine uptake and cell viability; however, dRib-induced cytotoxicity was completely recovered by adding N-acetyl cysteine (NAC). To confirm that system χc- mediates the oxidative stress-induced β-cell damage, we overexpressed xCT (the substrate-specific subunit of system χc-) using a lentiviral vector in RINm5F cells. Overexpression of xCT fully recovered the dRib-induced decrease in l-[14C]cystine uptake and GSH content and prevented the dRib-induced increase in ROS levels, cytotoxicity, and apoptosis. The overexpression of xCT showed a protective effect against dRib-induced oxidative damage in RINm5F cells. Our study showed that dRib depletes intracellular GSH content through inhibition of cystine transport via system χc- in β-cells.

Genome sequence of the potential probiotic eukaryote Saccharomyces cerevisiae KCCM 51299.

Saccharomyces cerevisiae KCCM 51299, a potential probiotic yeast overproducing glutathione, has been isolated from among 272 yeast strains from the relatively safe Nuruk. The genome sequence of S. cerevisiae KCCM 51299 was analyzed and a near-complete genome (12Mb) with 19 contigs was assembled after PacBio single-molecule real-time (SMRT) sequencing. The genome of S. cerevisiae KCCM 51299 was compared to the S. cerevisiae s288c reference genome. Additionally, genes involved in glutathione biosynthesis were identified, and the glutathione biosynthesis pathway was constructed in silico based on these genes. Furthermore, S. cerevisiae KCCM 51299 genes were compared with those in other yeast genomes. Finally, genome-scale in silico flux analysis was carried out, and a metabolic engineering strategy for glutathione biosynthesis was generated. These results provide useful information to further develop eukaryotic probiotics to overproduce glutathione.

Arsenic toxicity and its mitigation in ectomycorrhizal fungus Hebeloma cylindrosporum through glutathione biosynthesis

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.

Structural biology of plant sulfur metabolism: from sulfate to glutathione.

Sulfur is an essential element for all organisms. Plants must assimilate this nutrient from the environment and convert it into metabolically useful forms for the biosynthesis of a wide range of compounds, including cysteine and glutathione. This review summarizes structural biology studies on the enzymes involved in plant sulfur assimilation [ATP sulfurylase, adenosine-5'-phosphate (APS) reductase, and sulfite reductase], cysteine biosynthesis (serine acetyltransferase and O-acetylserine sulfhydrylase), and glutathione biosynthesis (glutamate-cysteine ligase and glutathione synthetase) pathways. Overall, X-ray crystal structures of enzymes in these core pathways provide molecular-level information on the chemical events that allow plants to incorporate sulfur into essential metabolites and revealed new biochemical regulatory mechanisms, such as structural rearrangements, protein-protein interactions, and thiol-based redox switches, for controlling different steps in these pathways.

Recent advances in arsenic metabolism in plants: current status, challenges and highlighted biotechnological intervention to reduce grain arsenic in rice.

Arsenic (As), classified as a "metalloid" element, is well known for its carcinogenicity and other toxic effects to humans. Arsenic exposure in plants results in the alteration of the physiochemical and biological properties and consequently, loss of crop yield. Being a staple food for half of the world's population, the consumption of As-contaminated rice grain by humans may pose serious health issues and risks for food security. In this study, we have described the principal understanding of the molecular basis of arsenic toxicity and accumulation in plant parts. We described the measures for decreasing As accumulation in rice and understanding the mechanism and transport of As uptake, its transport from root to shoot to rice grain, its metabolism, detoxification, as well as the mechanisms lying behind its accumulation in rice grains. There are various checkpoints, such as the tuning of AsV/Pi specific Pi transporters, arsenate reductase, transporters that are involved in the efflux of As to either the vacuole or outside the cell, xylem loading, loading and unloading to the phloem, and transporters involved in the loading of As to grain, that can be targeted to reduce As accumulation in rice grain. Genes/proteins involved in As detoxification, particularly the glutathione (GSH) biosynthesis pathway, phytochelatin (PC) synthesis, and arsenic methyltransferase, also provide a great pool of pathways that can also be castellated for the low As in rice grains. Paddy rice is also used as fodder for animals, enhancing vacuolar sequestration and using constitutive promoters, which may be of concern for animal health. Therefore, using a root-specific promoter and/or converting inorganic arsenic into volatile organic arsenic might be a better strategy for low As in grain. Furthermore, in this review, the other specific approaches, such as bio-remediation, bio-augmentation practices, and molecular breeding, which have great potential to reduce As uptake from soil to rice grains, have also been highlighted.

A Pilot Data Analysis of a Metabolomic Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) Based Study of Patients with Sickle Cell/Beta Thalassemia

Background: The complex pathophysiology of Sickle Cell Disease (SCD) makes unlikely that a single therapeutic agent will prevent or reverse all SCD complications. Metabolomic analysis might help in the characterization of the endogenous and exogenous effects of potential new treatments. Metabolites are small molecules that are chemically transformed during metabolism and provide a functional readout of cellular state. Metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. The metabolome is typically defined as the collection of small molecules produced by cells and offers a window for investigating how mechanistic biochemistry relates to cellular phenotype. There are very few reports associated with SCD providing comprehensive measurements of metabolites present in blood. Low arginine bioavailability has been associated with a clinical phenotype of increased hemolytic rate, pulmonary hypertension risk and early mortality. Recently, the FDA approved the use of L-glutamine for the treatment of adults and children with SCD, on the basis of the results of randomized phase 3 clinical trials1, while L-arginine's involvement is under investigation2,3. In this context we aimed to quantify targeted metabolites' abnormalities in patients with Sickle Cell/beta thalassemia (HbS/βThal), to identify pathways that might be of interest to prevent disease complications.Patients and Methods: Thirty adult Caucasian patients with HbS/βThal aged 45.6±10.9y, (43% male), at steady-state were enrolled in the study, while 20 age-matched healthy individuals (45% male) served as controls. Along with measurements of hematologic and blood chemistry parameters, targeted metabolome analyses for 13 aminoacids and 2 aminoacid's derivatives were performed after extraction from dry blood spots (DBSs) on filter paper using LC/MS/MS, with derivatization (AB SCIEX 5500 triple quadrupole QTRAP® LC/MS/MS Systems, Framingham, MA, USA).Results: Multiple metabolite differences are identified in HbS/βThal vs. Controls (Figure1). From metabolites involved in the biosynthesis of glutathione, only L-glutamine's levels were lower in patients with HbS/βThal compared to controls, while 5-oxyproline levels, a catabolic product of glutathione metabolism, were markedly increased in patients with HbS/βThal compared to controls. Urea cycle amino acids, also involved in the production of nitric oxide, L-arginine and L-ornithine concentrations were significantly lower in patients with HbS/βThal compared to controls, with a trend towards lower L-citrulline in patients with HbS/βThal p=0.06). Finally, amino acids involved in catecholamines (dopamine, nor-epinephrine and epinephrine) biosynthesis, such as L-phenylalanine and L-tyrosine and its metabolite succinylacetone levels were significantly lower in patients with HbS/βThal compared to controls. No significant correlations were found between any metabolites and markers of hemolysis, HbF levels or iron burden.Conclusions: This study confirms prior observations concerning aberrations in multiple blood specific amino acid levels in patients with SCD compared to controls, but this is the first report on Caucasian patients with HbS/βThal and on whole blood from DBS. We also identified for the first time in Caucasian patients with HbS/βThal important metabolic abnormalities of glutathione and nitric oxide biosynthesis pathways associated with altered concentrations of the metabolites serving of substrates in these cycles. Importantly, we also demonstrate low levels of L-phenylanine and L-tyrosine, which are essential sources for multiple neurotransmitters biosynthesis and neurobehavioral health. The latter novel observation should be confirmed in larger studies, while measurements of urinary amino acid clearances are necessary to evaluate for potential etiologies of the deficiencies through urinary losses vs. low substrate availability, increased utilization or abnormal metabolism. Patients with SCD are in a precarious state with respect to many amino acid deficiencies, all of which may have clinical consequences warranting further investigation.1N Engl J Med. 2018 Jul 19;379(3):226-235.2Anesth Analg. 2017 Apr;124(4):1369-1370.3Haematologica. 2013 Sep;98(9):1375-82. DisclosuresMorris:Pfizer: Consultancy; Calithera: Consultancy; MAST Therapeutics: Research Funding; UCSF-Benioff Children's Hospital Oakland: Patents & Royalties: For nutritional supplement licensed to Lifetrients; FDA: Research Funding; NIH/NHLBI: Research Funding. Voskaridou:Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors.

Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

Abstract LB-B24: Targeting transcriptional addiction for the treatment of TSC

Abstract Introduction: Tuberous sclerosis complex (TSC) is characterized by tumor development in the brain, heart, skin, kidney and lungs. While everolimus is approved for treatment of TSC angiomyolipoma and TSC subependymal giant cell astrocytoma, responses are not complete and tumor regrowth occurs after treatment cessation. Therapies for TSC that induce a selective cytocidal response in TSC-null cells are not currently available, and are highly desirable for TSC patients. In TSC tumors loss of the TSC1/TSC2 protein complex leads to activation of mTORC1 with downstream effects on anabolism and cell growth. We hypothesized that mTORC1 activation would also enhance mRNA transcription and that this effect might create a therapeutic vulnerability. In addition, we hypothesized that rapamcyin treatment of TSC-null cells would lead to complex compensatory effects on transcription, and that synergy might be seen in co-treatment with a transcriptional inhibitor. Cyclin-dependent kinase 7 (CDK7) plays a critical role in phosphorylation of RNA polymerase II, thus regulating RNA transcription. Recent screens have identified THZ1, a selective and covalent CDK7 inhibitor, as a cytocidal compound for several cancer types. We hypothesized that THZ1 and inhibitors of CDK7 might be selectively lethal to TSC-null cells in TSC-associated tumors. Methods: Drug sensitivity assay was performed using Quant-it PicoGreen. FACS and assays of apoptosis, such as Propidium Iodide/Annexin V staining and immunoblot for cleaved caspase 3 were used to assess apoptosis. We used RNA-sequencing and metabolome profiling to assess metabolomics and transcriptional effects of THZ1, and investigate the novel hypothesis that targeting transcription triggers selective cell death in TSC-null cells. To further assess the therapeutic potential of THZ1+/-Rapamycin in TSC, In vivo treatment studies were performed using xenografts in athymic nude mice. Results: Drug sensitivity assays to THZ1+/- Rapamycin revealed selective lethality for both the TSC1-null HCV29 bladder cancer derived cell line vs. addback TSC1-HCV29 and the TSC2-null 621-101 angiomyolipoma cell line vs. TSC2 addback 621-103. The IC50 of each of TSC1/TSC2 null cells was 6-10 fold lower vs. addback lines. FACS assay of apoptosis showed 37% of HCV29 cells Annexin V+ in comparison to 3% of TSC1-HCV29 cells. IB showed cleaved Caspase 3 only in THZ1-treated TSC1-null cells. RNA-seq on samples treated with THZ1(30nm) for 6 hours identified many genes with markedly different expression: e.g. 1128 genes showed a > 5-fold lower expression in THZ1-treated HCV29 compared with THZ1-treated TSC1-HCV29 cells. Genes involved in glutathione biosynthesis pathway such as GCLC (glutamate-cysteine ligase catalytic subunit) showed an 82% reduction in mRNA levels in THZ1-treated HCV29 cells vs. TSC1-HCV 29. Metabolite analyzes confirmed that THZ1 caused a marked decrease (4 fold) in glutathione levels, and supplemental GSH-MEE rescued cell death induction in response to THZ1.THZ1(10mg/kg/twice daily) +/- Rapamycin (3mg/kg/3 days week) for 30 days markedly reduced the tumor growth rate in the in vivo TSC1-null xenograft model and prevented tumor regrowth compared to either Rapamycin or vehicle. Conclusions: These findings reveal that targeting transcriptional addiction will selectively induce cell death of TSC-null cells. We hypothesize that targeting transcription with a covalent CDK7 inhibitor has novel therapeutic benefit for patients with TSC. Citation Format: Mahsa zarei, Heng Du, Rachel E. Yan, Yubao Wang, Thingu Zhang, John M. Asara, David J. Kwiatkowski. Targeting transcriptional addiction for the treatment of TSC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B24.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017.

Metabolic Profiling Regarding Pathogenesis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, eventually fatal disease characterized by fibrosis of the lung parenchyma and loss of lung function. IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair process including uncontrolled proliferation of lung (myo) fibroblasts and excessive deposition of extracellular matrix proteins in the interstitial space; however, the pathogenic pathways involved in IPF have not been fully elucidated. In this study, we attempted to characterize metabolic changes of lung tissues involved in the pathogenesis of IPF using gas chromatography-mass spectrometry-based metabolic profiling. Partial least-squares discriminant analysis (PLS-DA) model generated from metabolite data was able to discriminate between the control subjects and IPF patients (R(2)X = 0.37, R(2)Y = 0.613 and Q(2) (cumulative) = 0.54, receiver operator characteristic AUC > 0.9). We discovered 25 metabolite signatures of IPF using both univariate and multivariate statistical analyses (FDR < 0.05 and VIP score of PLS-DA > 1). These metabolite signatures indicated alteration in metabolic pathways: adenosine triphosphate degradation pathway, glycolysis pathway, glutathione biosynthesis pathway, and ornithine aminotransferase pathway. The results could provide additional insight into understanding the disease and potential for developing biomarkers.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4.

The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPβ:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPβ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg(-/-) mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg(-/-) mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.

Genomic Profile of Fatigued Men Receiving Localized Radiation Therapy.

The purpose of this study was to explore gene expression changes in fatigued men with nonmetastatic prostate cancer receiving localized external beam radiation therapy (EBRT). Fatigue was measured in 40 men with prostate cancer (20 receiving EBRT and 20 controls on active surveillance) using the Functional Assessment of Cancer Therapy-Fatigue (FACT-F). EBRT subjects were followed from baseline to midpoint and end point of EBRT, while controls were seen at one time point. EBRT subjects were categorized into high- and low-fatigue groups based on change in FACT-F scores from baseline to EBRT completion. Full genome microarray was performed from peripheral leukocyte RNA to determine gene expression changes related to fatigue phenotypes. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay confirmed the most differentially expressed gene in the microarray experiment. At baseline, mean FACT-F scores were not different between EBRT subjects (44.3 ± 7.16) and controls (46.7 ± 4.32, p = .24). Fatigue scores of EBRT subjects decreased at treatment midpoint (38.6 ± 9.17, p = .01) and completion (37.6 ± 9.9, p = .06), indicating worsening fatigue. Differential expression of 42 genes was observed between fatigue groups when EBRT time points were controlled. Membrane-spanning four domains, subfamily A, member (MS4A1) was the most differentially expressed gene and was associated with fatigue at treatment end point (r = -.46, p = .04). Fatigue intensification was associated with MS4A1 downregulation, suggesting that fatigue during EBRT may be related to impairment in B-cell immune response. The 42 differentially expressed fatigue-related genes are associated with glutathione biosynthesis, γ-glutamyl cycle, and antigen presentation pathways.

Evaluation of Cadmium-Induced Nephrotoxicity Using Urinary Metabolomic Profiles in Sprague-Dawley Male Rats

The aim of this study was to investigate urinary metabolomic profiles associated with cadmium (Cd)-induced nephrotoxicity and their potential mechanisms. Metabolomic profiles were measured by high-resolution 1H-nuclear magnetic resonance (NMR) spectroscopy in the urine of rats after oral exposure to CdCl2 (1, 5, or 25 mg/kg) for 6 wk. The spectral data were further analyzed by a multivariate analysis to identify specific urinary metabolites. Urinary excretion levels of protein biomarkers were also measured and CdCl2 accumulated dose-dependently in the kidney. High-dose (25 mg/kg) CdCl2 exposure significantly increased serum blood urea nitrogen (BUN), but serum creatinine (sCr) levels were unchanged. High-dose CdCl2 (25 mg/kg) exposure also significantly elevated protein-based urinary biomarkers including osteopontin, monocyte chemoattractant protein-1 (MCP-1), kidney injury molecules-1 (Kim-1), and selenium-binding protein 1 (SBP1) in rat urine. Under these conditions, six urinary metabolites (citrate, serine, 3-hydroxyisovalerate, 4-hydroxyphenyllactate, dimethylamine, and betaine) were involved in mitochondrial energy metabolism. In addition, a few number of amino acids such as glycine, glutamate, tyrosine, proline, or phenylalanine and carbohydrate (glucose) were altered in urine after CdCl2 exposure. In particular, the metabolites involved in the glutathione biosynthesis pathway, including cysteine, serine, methionine, and glutamate, were markedly decreased compared to the control. Thus, these metabolites are potential biomarkers for detection of Cd-induced nephrotoxicity. Our results further indicate that redox metabolomics pathways may be associated with Cd-mediated chronic kidney injury. These findings provide a biochemical pathway for better understanding of cellular mechanism underlying Cd-induced renal injury in humans.

Genetic Variants Associated With Glycine Metabolism and Their Role in Insulin Sensitivity and Type 2 Diabetes

Circulating metabolites associated with insulin sensitivity may represent useful biomarkers, but their causal role in insulin sensitivity and diabetes is less certain. We previously identified novel metabolites correlated with insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. The top-ranking metabolites were in the glutathione and glycine biosynthesis pathways. We aimed to identify common genetic variants associated with metabolites in these pathways and test their role in insulin sensitivity and type 2 diabetes. With 1,004 nondiabetic individuals from the RISC study, we performed a genome-wide association study (GWAS) of 14 insulin sensitivity–related metabolites and one metabolite ratio. We replicated our results in the Botnia study (n = 342). We assessed the association of these variants with diabetes-related traits in GWAS meta-analyses (GENESIS [including RISC, EUGENE2, and Stanford], MAGIC, and DIAGRAM). We identified four associations with three metabolites—glycine (rs715 at CPS1), serine (rs478093 at PHGDH), and betaine (rs499368 at SLC6A12; rs17823642 at BHMT)—and one association signal with glycine-to-serine ratio (rs1107366 at ALDH1L1). There was no robust evidence for association between these variants and insulin resistance or diabetes. Genetic variants associated with genes in the glycine biosynthesis pathways do not provide consistent evidence for a role of glycine in diabetes-related traits.

A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana

To identify potentially novel and essential components of plant membrane trafficking mechanisms we performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble secretory marker. Since GSH2 is responsible for converting γ-glutamylcysteine (γ-EC) to glutathione (GSH) in the glutathione biosynthesis pathway, gsh2 mutants exhibited γ-EC hyperaccumulation and GSH deficiency. Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that γ-EC accumulation rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies. Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated. The link between glutathione biosynthesis and ER morphology and function is discussed.