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Ncl1-mediated metabolic rewiring critical during metabolic stress

View ORCID ProfileAjay Bhat, Rahul Chakraborty, Khushboo Adlakha, View ORCID ProfileGanesh Agam, View ORCID ProfileKausik Chakraborty  Correspondence email, View ORCID ProfileShantanu Sengupta  Correspondence email
Ajay Bhat
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
2Academy of Scientific and Innovative Research, Ghaziabad, India
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  • ORCID record for Ajay Bhat
Rahul Chakraborty
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
2Academy of Scientific and Innovative Research, Ghaziabad, India
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Khushboo Adlakha
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
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Ganesh Agam
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
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  • ORCID record for Ganesh Agam
Kausik Chakraborty
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
2Academy of Scientific and Innovative Research, Ghaziabad, India
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  • For correspondence: kausik@igib.in
Shantanu Sengupta
1Council of Scientific and Industrial Research—Institute of Genomics and Integrative Biology, New Delhi, India
2Academy of Scientific and Innovative Research, Ghaziabad, India
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  • ORCID record for Shantanu Sengupta
  • For correspondence: shantanus@igib.res.in
Published 15 August 2019. DOI: 10.26508/lsa.201900360
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  • Figure 1.
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    Figure 1. Metabolic alterations because of excessive amino acids induce growth defect.

    (A) Cells of BY4741 strain were grown in higher concentration of amino acids (∼10-fold molar excess than the amount present in the media) for 12 h, and growth was monitored by measuring OD at 600 nm. Percentage growth was then calculated with respect to the untreated cells (growth in SC media containing basal concentration of amino acids). Error bar represents SD (n = 3). (B) The higher concentration of toxic amino acids (Cys, Trp, Val, Ile, and Phe) were co-supplemented with higher levels (10 times as that of in media) of other amino acids, and growth was measured at 600 nm using multimode reader. The figure represents the growth matrix of cells grown with different combinations of amino acids. The bottom most row represents the growth of the cells during normal SC media (control) and treatment with high doses of single amino acids (Val, Ile, Phe, Trp, and Cys). Growth of the cells during these conditions co-treated with other amino acids (at higher concentrations) is represented in the above matrix. (C) Prototrophic strain (S288C) was grown with higher concentration of different amino acids for 12 h and then OD (at 600 nm) was measured to calculate the percentage growth with respect to untreated cells (growth in basal SC media). Error bar represents SD (n = 3). (D) Percentage of growth during cysteine treatment (1 mM) in BY4741, S288C, and leu2Δ (LEU2 deleted in S288C background) strains. Error bar represents SD (n = 3). Statistical significance was determined by unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001). (E) Growth of S288C and leu2Δ (LEU2 deleted in S288c background) strains in the presence of toxic amino acids (Ile, Val, Phe, and Trp). (F) Overnight grown cultures of yeast were reinoculated at an OD of 0.1 in SC media and growth was monitored in the presence (0.25–5 mM) and absence of l-cysteine at different time points. The graph shows the kinetics of growth inhibition because of different concentrations of cysteine. Percentage growth inhibition was calculated by using the formula: {(OD of control cells − OD of cysteine-treated cells)/OD of control cell × 100}. Error bar represents SD (n = 3).

  • Figure S1.
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    Figure S1. Effect of aminoacids on cell size and growth.

    (A) Effect of different amino acids on cell size in Wt measured by forward scatter. Error bar represents SD (n = 3). (B) Drop dilution assay of Wt in the presence of different amino acids. (C) Effect of cysteine on the growth of prototrophic yeast strain (S288C) in leucine restrained condition. Error bar represents SD (n = 3). Statistical significance was determined by paired t test. “ns” indicates nonsignificant. (D) Effect of cysteine, leucine, and leucine + cyseine in both leucine and cysteine pretreated Wt strain. Error bar represents SD (n = 3). Statistical significance was determined by paired t test. “**” indicates P < 0.01. (E) Growth kinetics of BY4741 strain during different concentrations of cysteine. Error bar represents SD (n = 3).

  • Figure S2.
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    Figure S2. Cysteine-induced effect on protein translation.

    (A) Functional classification based on biological function of differentially expressed proteins. Protein was isolated from cells treated with cysteine for 6 and 12 h, and iTRAQ-based proteomics was performed. Differentially expressed proteins at each time interval were classified based on biological function using DAVID, and top 10 enriched pathways with P-value ≤ 0.05 are plotted. The above panels are for proteins up-regulated by cysteine at 12 h (left panel) and 6 h (right panel), respectively, and similarly the below panels are for down-regulated proteins. (B) S35-Methionine incorporation assay in Δmet6 deleted strain (cannot metabolize homocysteine to methionine). The upper panel represents the Coomassie stain of total protein, and the lower panel represents the autoradiogram of the labeled proteins.

  • Figure 2.
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    Figure 2. Amino acid metabolism and protein translation play a vital role in cysteine toxicity.

    (A) Cells were treated with cysteine for 6 and 12 h, and then iTRAQ-based relative quantification of proteins was performed. The figure represents the directionality of the expression of proteins involved in amino acid metabolism during 6 and 12 h of cysteine treatment. Left-side and right-side boxes for each protein denote its expression at 6 and 12 h of cysteine treatment, respectively. Red, green, and beige colored boxes represent the proteins down-regulated, up-regulated, and not changed, respectively, because of cysteine treatment. However, white colored box represents the proteins not detected in the proteomics experiment. (B) Heat map represents the relative expression of proteins (with respect to untreated condition) involved in amino acid metabolism during cysteine treatment and their status during co-treatment of leucine and cysteine. (C) Heat map represents the log fold change of different amino acids during cysteine (Cys) treatment and combination of leucine and cysteine (Leu + Cys). Statistical significance was determined by t test (n = 5, and n = 2 for Gln, Asn, and Trp, respectively). In the first column, the amino acids significantly altered by cysteine are labeled by “*” (P < 0.05); however, if the levels of these amino acids are significantly reverted by co-treatment of leucine and cysteine, then in the second column they are labeled by “*” (P < 0.05) or otherwise “ns” (nonsignificant). (D)35S Methionine incorporation kinetics in the presence of different concentrations (1 and 2 mM) of cysteine for different time points. In the upper panel, the upper blot is for autoradiogram, which signifies S35-Met incorporation in proteins, and the lower blot represents Coomassie stain of the same proteins, which represents equal loading of proteins. The lower panel is the quantification of 35S incorporation with respect to untreated cells. “**” indicates P < 0.01 (t test). Error bar represents SEM (n = 2). (E) Polysome profiling representing monosome and polysome fractions in the presence and absence of cysteine treatment and during co-treatment of leucine and cysteine.

  • Figure 3.
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    Figure 3. Genetic interactors of leucine-mediated rescue.

    (A) Whole genome screen in the presence of 1 mM cysteine. The upper most panel represents the schematic of the genome-wide screen. The middle panel represents scatter plot between the logarithmic ratio of growth in the presence and absence of cysteine versus growth in basal media (SC media), for each deletion strain. In this panel, the deletion strains, which show growth sensitivity and resistance for cysteine, are symbolized with red and blue dots, respectively. Strains that were very slow growing and show growth advantage with cysteine are denoted by green dots. The lower most panel represents the validation of the cysteine-sensitive strains. Strains that show growth sensitivity toward high levels of cysteine in first round of whole genome screen were again grown in the presence of cysteine and growth was monitored and compared with wild-type (Wt) strain. Three biological replicates were performed, and average percentage growth of each strain with respect to Wt was calculated. Deletion strains whose growth was significantly different from Wt (P-value ≤ 0.05) (statistical significance was determined by unpaired t test), and grows at least 20% less than Wt, were considered to be sensitive for cysteine and are denoted by red dots. (B) Screening to identify genes required by leucine for alleviating cysteine-induced toxicity. Cysteine-sensitive deletion strains were grown with and without higher concentration of cysteine (1 mM), leucine (5 mM), or combination of leucine and cysteine. The figure represents the scatter plot between percentage growth during cysteine treatment versus percentage growth during co- treatment with leucine and cysteine, with respect to wild type’s growth during cysteine treatment. Deletion strains in the left quadrant, which are denoted by red dots, represent the strains that are sensitive to cysteine but are not recovered by leucine.

  • Figure 4.
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    Figure 4. Leucine reprograms proteome through NCl1 to recover cysteine-mediated toxicity.

    (A) Percentage growth inhibition of wild-type (Wt) and ncl1Δ strain, in the presence of cysteine and combination of leucine and cysteine. (B) Box plot represents the relative expression of proteins during co-treatment of leucine and cysteine in Wt and ncl1Δ, for proteins that were up-regulated by cysteine in Wt. Each condition represents the log2-fold change of different proteins (n = 32), which is an average of three biological replicates, and statistical significance was analyzed using paired nonparametric method. (C) Spearman rank correlation between relative expression of mRNA (obtained from RNA-seq) and protein levels (from iTRAQ-based relative proteomics) in Wt (upper panel) and ncl1Δ (lower panel). (D) The bar graph represents the biological pathways enriched from proteins whose expression was reverted during co-treatment of leucine and cysteine in Wt, but not to the similar extent in Δncl1. Classification was done using Database for Annotation, Visualization & Integrated Discovery (DAVID), and pathways with P-value ≤ 0.05 are plotted in the figure.

  • Figure S3.
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    Figure S3. NCL1-mediated regulation of proteome during cysteine treatment.

    (A) The bar graph represents the biological pathways enriched from proteins differentially expressed in ncl1Δ strain during cysteine treatment, with respect to their expression in cysteine-treated Wt cells (normalized with respect to their corresponding controls). Left and right panel represents the biological pathways up-regulated and down-regulated, respectively, in ncl1Δ. (B) Box plot represents the relative expression of proteins during co-treatment of leucine and cysteine in Wt and ncl1Δ, for proteins which were down-regulated by cysteine in Wt. Each condition contains the fold change of different proteins (n = 21), which is an average of three biological replicates, and statistical significance was analyzed using a paired nonparametric method.

  • Figure 5.
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    Figure 5. Metabolism of leucine via NCL1 plays a vital role during cysteine-induced stress.

    (A) Relative amino acid (AA) levels in ncl1Δ strain during Cys treatment and combination of Leu and Cys treatment were measured and were analyzed by calculating the correlation between these two groups. (B) Bar graph represents the relative levels of leucine in Wt and ncl1Δ cells, during leucine and combination of leucine and cysteine, normalized with respect to levels of leucine in untreated Wt strain. Error bar represents SD (n = 2). Statistical significance was determined by unpaired t test. “**” indicates P < 0.01. (C) Relative expression of BAT1 in Wt and ncl1Δ during cysteine treatment, normalized with respect to its expression in cysteine-treated Wt. Error bar represents SD (n = 3). Statistical significance was determined by paired t test “*” indicates P < 0.05. (D) KIC levels were measured using MRM-based LC–MS technique. The figure represents the overlapped peak intensity of a transition of KIC (121/101 m/z), measured from Wt and ncl1Δ cells during co-treatment of leucine and cysteine. KIC was also measured in both these cells during co-treatment of KIC and cysteine. (E) Cells were co-treated with cysteine (1 mM) and different concentrations of leucine or KIC, and then growth was measured after 12 h. The graph represents the growth inhibition of Wt and ncl1Δ strain because of cysteine in the basal media and during its co-treatment with varying concentration of KIC and leucine. (F) Growth of cells in the presence of toxic amino acids (Ile, Val, Phe, and Trp) and during their respective co-treatment with KIC. (G) TCA intermediates were measured by MRM-based LC–MS technique. The bar plot represents their relative levels during cysteine treatment and its combination with KIC or leucine in ncl1Δ strain. Error bar represents SD (n = 3).

  • Figure 6.
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    Figure 6. Pyruvate and leucine co-treatment completely rescues the toxicity of cysteine.

    (A) Relative levels of pyruvate in ncl1Δ cells treated with cysteine, with respect to corresponding untreated control. Error bar represents SD (n = 3). Statistical significance was determined by paired t test. “*” indicates P < 0.05. (B) Effect of pyruvate and leucine on the growth inhibition induced by cysteine in Wt and ncl1Δ cells. Error bar represents SD (n = 3). (C) Growth inhibition of cysteine during co-treatment of leucine and pyruvate simultaneously. Error bar represents SD (n = 3). Statistical significance was determined by paired t test. “**” indicates P < 0.01.

Supplementary Materials

  • Figures
  • Table S1 List of amino acids with working concentrations.

  • Table S2 Differentially expressed proteins because of cysteine treatment for 12 h.

  • Table S3 Differentially expressed proteins because of cysteine treatment for 6 h, and their status during co-treatment of leucine and cysteine.

  • Table S4 This table includes the percentage growth of cysteine-sensitive deletion strains in comparison with wild-type strain during cysteine treatment.

  • Table S5 List of proteins differentially expressed in ncl1Δ strain during cysteine treatment with respect to wild-type strain.

  • Table S6 Proteins differentially expressed by cysteine in Wt, and their status in ncl1Δ during cysteine and leucine treatment.

  • Table S7 Relative expression of genes at RNA and proteins levels during cysteine treatment in Wt and ncl1Δ strain.

  • Table S8 Optimized parameters for targeted metabolomics.

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Ncl1-mediated metabolic homeostasis during excess cysteine
Ajay Bhat, Rahul Chakraborty, Khushboo Adlakha, Ganesh Agam, Kausik Chakraborty, Shantanu Sengupta
Life Science Alliance Aug 2019, 2 (4) e201900360; DOI: 10.26508/lsa.201900360

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Ncl1-mediated metabolic homeostasis during excess cysteine
Ajay Bhat, Rahul Chakraborty, Khushboo Adlakha, Ganesh Agam, Kausik Chakraborty, Shantanu Sengupta
Life Science Alliance Aug 2019, 2 (4) e201900360; DOI: 10.26508/lsa.201900360
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