Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload

Deshpande, Anup; Bhatia, Muskan; Laxman, Sunil; Bachhawat, Anand K.

doi:10.15698/mic2017.04.567

Cited by 16 publications

(15 citation statements)

References 43 publications

(66 reference statements)

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“…The involvement of threonine biosynthesis during high levels of cysteine was also reported in Escherichia coli more than three decades ago—where it was suggested that cysteine inhibits homoserine dehydrogenase (Hom6 in yeast) (44), acetohydroxyacid synthetase (Ilv6 in yeast) (45), and threonine deaminase (Ilv1 in yeast) (46). Furthermore, cysteine increases the levels of arginine and the enzymes involved in its biosynthesis, and this observation is in agreement with a recent report, suggesting that high levels of cysteine increase arginine biosynthesis for polyamine synthesis (47).…”

Section: Discussionsupporting

confidence: 93%

Ncl1-mediated metabolic rewiring critical during metabolic stress

et al. 2019

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Nutritional limitation has been vastly studied; however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in the presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine up-regulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation—events that are rescued by leucine supplementation. Through a comprehensive genetic screen, we show that leucine-mediated effect depends on a transfer RNA methyltransferase (NCL1), absence of which decouples transcription and translation in the cell, inhibits the conversion of leucine to ketoisocaproate, and leads to tricarboxylic acid cycle block. We therefore propose a role of NCL1 in regulating metabolic homeostasis through translational control.

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Section: Discussionsupporting

confidence: 93%

Ncl1-mediated metabolic rewiring critical during metabolic stress

et al. 2019

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“…Furthermore, cysteine increases the levels of arginine and the enzymes involved in its biosynthesis, and this observation is in agreement with a recent report suggesting that high levels of cysteine increase arginine biosynthesis for polyamine synthesis 47 .…”

Section: Cysteine Inhibits Translation and Rewires Amino Acid Metabolismsupporting

confidence: 93%

Ncl1 mediated metabolic rewiring critical during metabolic stress

Bhat

Chakraborty

Adlakha

et al. 2019

Preprint

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Nutritional limitation has been vastly studied, however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine upregulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation, events that are rescued by leucine supplementation. Through a comprehensive genetic screen we show that leucine mediated effect depends on a tRNA methyltransferase (Ncl1), absence of which decouples cell"s transcription and translation, inhibits the conversation of leucine to ketoisocaproate and leads to TCA cycle block. We therefore, propose a role of Ncl1 in regulating metabolic homeostasis through translational control.In this study, using Saccharomyces cerevisiae as a model, we used a combination of genetic, proteomic, transcriptomic and metabolic approach to understand cysteine induced systemic alteration. We show that cysteine inhibits protein translation and alters the amino acid metabolism, effects which are reversed by supplementation of leucine. We also show that a tRNA methyltransferase (NCL1) is involved in survival during cysteine stress, absence of which inhibited the conversation of leucine to ketoisocaproate (KIC), a necessary step for mitigating the effect of cysteine. Thus, this study not only uncovered the cellular insights during high levels cysteine, but also highlighted the novel role of NCL1 in regulating the metabolism during excess cysteine. Results Metabolic alterations due to excess amino acids induce growth defectGrowth screening of S cerevisiae (BY4741) in the presence of high concentrations of amino acids revealed that a few amino acids -cysteine, isoleucine, valine, tryptophan and phenylalanine inhibited growth (Fig. 1A). To test if the imbalance due to excess of an amino acid resulting in the growth inhibition could be abrogated in the presence of other amino acids, we did a comprehensive amino acid supplementation screening. We found that the amino acid, leucine could completely rescue the growth inhibition due to isoleucine, valine, tryptophan and phenylalanine but not cysteine, where the rescue was partial ( Fig 1B). The effect of leucine could be because the yeast strain used in this study, BY4741, is auxotrophic for leucine. To confirm this, we performed the same experiment using a prototrophic wild type strain, S288C, and found that except cysteine, none of the other amino acids inhibited growth ( Fig 1C). Further, the growth inhibitory effect of cysteine was lower compared to BY4741. This suggests that intracellular concentration of leucine might play a role in alleviating amino-acid mediated toxicity. This was further confirmed by deleting the gene invol...

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“…The protocol for RNA isolation, labeling, hybridization, scanning was followed as described previously [ 21 ]. Microarray data was analyzed and submitted to the Gene Expression Omnibus (GEO) database (GSE10859).…”

Section: Methodsmentioning

confidence: 99%

Role of phosphate limitation and pyruvate decarboxylase in rewiring of the metabolic network for increasing flux towards isoprenoid pathway in a TATA binding protein mutant of Saccharomyces cerevisiae

et al. 2018

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BackgroundProduction of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several attempts have been made to reroute metabolic flux towards isoprenoid pathway in yeast. Most approaches have focused on the core isoprenoid pathway as well as on meeting the increased precursors and cofactor requirements. To identify unexplored genetic targets that positively influence the isoprenoid pathway activity, a carotenoid based genetic screen was previously developed and three novel mutants of a global TATA binding protein SPT15 was isolated for heightened isoprenoid flux in Saccharomyces cerevisiae.ResultsIn this study, we investigated how one of the three spt15 mutants, spt15_Ala101Thr, was leading to enhanced isoprenoid pathway flux in S. cerevisiae. Metabolic flux analysis of the spt15_Ala101Thr mutant initially revealed a rerouting of the central carbon metabolism for the production of the precursor acetyl-CoA through activation of pyruvate-acetaldehyde-acetate cycle in the cytoplasm due to high flux in the reaction caused by pyruvate decarboxylase (PDC). This led to alternate routes of cytosolic NADPH generation, increased mitochondrial ATP production and phosphate demand in the mutant strain. Comparison of the transcriptomics of the spt15_Ala101Thr mutant cell with SPT15WT bearing cells shows upregulation of phosphate mobilization genes and pyruvate decarboxylase 6 (PDC6). Increasing the extracellular phosphate led to an increase in the growth rate and biomass but diverted flux away from the isoprenoid pathway. PDC6 is also shown to play a critical role in isoprenoid pathway flux under phosphate limitation conditions.ConclusionThe study not only proposes a probable mechanism as to how a spt15_Ala101Thr mutant (a global TATA binding protein mutant) could increase flux towards the isoprenoid pathway, but also PDC as a new route of metabolic manipulation for increasing the isoprenoid flux in yeast.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1000-1) contains supplementary material, which is available to authorized users.

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Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload

Cited by 16 publications

References 43 publications

Ncl1-mediated metabolic rewiring critical during metabolic stress

Ncl1-mediated metabolic rewiring critical during metabolic stress

Ncl1 mediated metabolic rewiring critical during metabolic stress

Role of phosphate limitation and pyruvate decarboxylase in rewiring of the metabolic network for increasing flux towards isoprenoid pathway in a TATA binding protein mutant of Saccharomyces cerevisiae

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