Research ArticleHEALTH AND MEDICINE

Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span

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Science Advances  05 Aug 2020:
Vol. 6, no. 32, eaba1306
DOI: 10.1126/sciadv.aba1306
  • Fig. 1 RNA-seq and ribosome profiling revealed global transcriptional and translational regulation by GR.

    (A) Log2 of the fold change (GR versus SD) of ribosome-protected RNA is plotted against log2 fold change of the total RNA, for all the genes. The size of the dots represents the number of reads (per 1 million total reads) for the gene. Translational efficiency is measured by the deviation from the linear fit (the black line), quantified by a z score (indicated by the color; see Materials and Methods). (B) Length distributions of 5′UTR (left), coding sequence (middle), and 3′UTR (right) for genes with increased translational efficiency (z score >2) and other genes (z score ≤2). (C) Ribosome occupancy of 5′UTR and 3′UTR relative to CDS for all genes (left), genes with increased translational efficiency (z score >2, middle), and genes with decreased translational efficiency (z score <−2, right), under SD and GR conditions. (D) Transcriptional changes between SD and GR. Genes are organized into TF modules. Change of expression of a TF module is quantified by a z score, which measures the collective change of the genes in the module relative to other genes in the genome (see Materials and Methods). Top TF modules with the absolute value of z scores passing 2.5 in both experiments are shown.

  • Fig. 2 GR represses the expression of methionine biosynthetic enzymes and transporters and leads to decreased intracellular methionine level.

    (A) Fold changes of mRNA and translational efficiency for amino acid biosynthetic enzymes and transporters in two independent experiments using different GR protocols (see Materials and Methods). (B) Change of the protein expression of the major methionine transporter Mup1, measured by following the Mup1-GFP reporter in single cells in the microfluidic device. Shown is the average over 23 cells; each was normalized by the mean fluorescence before the switch. The arrow indicates the time point at which the media were switched from SD to GR. (C to F) Change of the intracellular concentration of amino acids after switching from SD to GR. (C and D) Ratios of intracellular amino acid concentrations between GR and SD in the BY4741 strain and BY4742 strain, respectively. Different colors represent different independent experiments. Box plot shows maximum, minimum, median, and top and bottom 20 percentile. (E and F) Amino acid concentration plotted against the usage in the proteome (genome frequency weighted by protein abundance). The red line is a linear fit excluding five charged amino acids (Arg, Lys, His, Glu, and Asp) and one polar amino acid (Gln). There is a log-linear correlation between the intracellular concentration and the usage.

  • Fig. 3 Adding back extra methionine (10 times the level in SD) to the GR media specifically cancels the life span extension by GR without affecting the life span in the SD media.

    (A) Survival curves of the BY4741 strain under four different conditions: GR (0.05% glucose), GR + 10xMet, SD (2% glucose), and SD + 10xMet. Numbers in parentheses indicate the average life span and the number of cells measured. The life span under GR + 10xMet and SD + 10 × Met is not significantly different from that under SD (P = 0.55 and 0.17, respectively). (B) The same survival curves under the four conditions for a different strain BY4741-MET (BY4741 with MET15 knocked in) with an intact methionine biosynthesis pathway. The life span under GR + 10xMet and SD + 10xMet is not significantly different from that under SD (P = 0.22 and 0.25, respectively). (C) Adding back valine does not abrogate the life span extension by GR. Shown are the survival curves of the BY4741 strain under three different conditions: GR (0.05% glucose), GR + 10xVal, and SD (2% glucose). Life span under GR + 10xVal is not significantly different from that under GR (P = 0.75). (D) Adding back lysine does not abrogate the life span extension by GR. Shown are the survival curves of the BY4741 strain under three different conditions: GR (0.05% glucose), GR + 10xLys, and SD (2% glucose). Life span under GR + 10xLys is not significantly different from that under GR (P = 0.1).

  • Fig. 4 Deletion of methionine biosynthesis genes extends life span.

    The methionine biosynthetic pathway is shown with enzymes in the boxes and arrows connecting metabolites. Survival curves for the single gene deletion mutants and the wide-type controls are plotted next to the enzymes. Deletion mutants were derived from the BY4742 strain.

  • Fig. 5 GR up-regulates the translation of RPN4, leading to elevated proteasome expression and activity, and RPN4 is required for the life span extension.

    (A) GR induced fold changes of footprint (left), total mRNA (middle), and translational efficiency (right) for Rpn4 and its representative targets. (B) Ribosome footprints in the coding region and the 5′UTR of RPN4 under SD (bottom track) and GR (middle track) conditions. RPN4 coding sequence and 5′uORFs are indicated on the top track. (C) Rpn11-GFP reporter level as a function of time before and after switching from SD to GR media (red curve) or SD to GR + 10xMet (purple curve). The blue curve is the control without media switch. The red, purple, and blue curves are the average over 94, 69, and 123 cells, respectively, where each single-cell curve is first normalized by the mean fluorescence intensity before switch. (D and E) Proteasome activity (chymotrypsin-like and caspase-like) in GR and GR + 10xMet versus SD in three independent experiments. (F and G) Proteasome activity in UBR2 deletion mutant and under the MR condition. (H) RPN4 deletion abolishes the life span extension by GR without affecting the life span in SD. Life spans of RPN4∆ in both SD and GR conditions are not significantly different from WT in SD (P = 0.15 and 0.21, respectively). (I) Deletion of UBR2, a ubiquitin-protein ligase that targets Rpn4 for degradation, extends life span in SD (P = 7.1 × 10−16). GR does not extend the life span of UBR2 deletion mutant further (P = 0.64). Deletion mutants were derived from BY4741.

  • Fig. 6 A graphical model summarizing the findings in this work and some of the previous studies.

    The life span extension by GR is mediated by reduced intracellular methionine (via the repression of methionine biosynthetic enzymes and transporters) and by elevated proteasome activity. MR is known to extend life span. Reducing intracellular methionine extends life span in a more general context beyond GR and MR, such as genetic perturbation of methionine pathway genes or in long-lived TF deletion mutants. Proteasome activity may be regulated by redundant pathways under GR—by intracellular methionine and by an independent pathway, via the translational up-regulation of Rpn4. Elevated proteasome activity is required for the life span extension of GR and the UBR2 deletion mutant.

Supplementary Materials

  • Supplementary Materials

    Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span

    Ke Zou, Silvia Rouskin, Kevin Dervishi, Mark A. McCormick, Arjun Sasikumar, Changhui Deng, Zhibing Chen, Matt Kaeberlein, Rachel B. Brem, Michael Polymenis, Brian K. Kennedy, Jonathan S. Weissman, Jiashun Zheng, Qi Ouyang, Hao Li

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