mTORC1-Sch9 regulates hydrogen sulfide production through the transsulfuration pathway

Endogenous hydrogen sulfide mediates anti-aging benefits of dietary restriction (DR). However, it is unclear how H2S production is regulated by pathways related to DR. Due to the importance of mTORC1 pathway in DR, we investigated the effects of Sch9, a yeast homolog of mammalian S6K1 and a major substrate of mTORC1 on H2S production in yeast Saccharomyces cerevisiae. We found that inhibition of the mTORC1-Sch9 pathway by SCH9 deletion, rapamycin or myriocin treatment resulted in a dramatic decrease in H2S production. Although deficiency of SCH9 did not alter the intracellular level of methionine, the intracellular level of cysteine increased in Δsch9 cells. The expression of CYS3 and CYS4, two transsulfuration pathway genes encoding cystathionine gamma-lyase (CGL) and cystathionine beta-synthase (CBS), were also decreased under mTORC1-Sch9 inhibition. Overexpression of CYS3 or CYS4 in Δsch9 cells or WT cells treated with rapamycin rescued the deficiency of H2S production. Finally, we also observed a reduction in H2S production and lowering of both mRNA and protein levels of CGL and CBS in cultured human cells treated with rapamycin to reduce mTORC1 pathway activity. Thus, our findings reveal a probably conserved mechanism in which H2S production by the transsulfuration pathway is regulated by mTORC1-Sch9 signaling.


INTRODUCTION
The roles of Hydrogen sulfide (H 2 S) as a gaseous signal transmitter has been well-appreciated in the last two decades [1][2][3][4]. There are many signaling pathways in a range of organisms, from yeast to human, that are regulated by H 2 S including cell death, the cell cycle, autophagy, inflammation, aging and oxidative stress. Physiologically, H 2 S plays an important role in protecting the nervous system and the cardiovascular system of animals [5,6].
Endogenous production of H 2 S is mainly catalyzed by four enzymes involved in cysteine metabolism including cystathionine gamma-lyase (CGL), cystathionine beta-synthase (CBS), cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). The production of H 2 S can be controlled by the expression of these enzymes, the bioavailability of their substrates, and enzyme activity modulating factors [3]. Therefore, the regulation of H 2 S production is complicated and more studies are required to clarify how it is controlled under physiological or pathological conditions. target of rapamycin complex 1 (mTORC1) pathway also plays a key role in the anti-aging effects of DR [9,10]. Inhibiting mTORC1 pathway by rapamycin treatment or by deletion of down-stream signaling components such as SCH9, a homologue of mammalian S6K1 in Saccharomyces cerevisiae and one of direct substrates of mTORC1, mimics DR and provides anti-aging benefits [11][12][13]. However, it is unknown if the mTORC1 pathway regulates H 2 S production even though it mediates at least some effects of DR. Since the mTORC1-Sch9 pathway in yeasts responds to DR [14,15] and is required for protein synthesis and amino acid metabolism [16][17][18], we sought to determine if mTORC1-Sch9 regulates H 2 S production via sulfide amino acids metabolism.

Inhibiting mTORC1-Sch9 inhibits H 2 S production
Sch9 is a direct substrate of yeast mTORC1 and depletion of SCH9 extends yeast lifespan through mechanisms shared with lifespan extension by calorie restriction (CR) [13,16]. Since H 2 S mediates the benefits of CR, we first compared H 2 S production in Δsch9 mutant cells to WT cells. While WT cells released measurable amounts of H 2 S, Δsch9 cells produce barely detectable amounts of H 2 S ( Figure 1A and 1B). H 2 S production was recovered if a functional SCH9 gene was added back to the mutant cells ( Figure 1A and 1B), thus, showing that Sch9 activity is required for H 2 S production. Western blotting for Sch9 was used to verify that H 2 S production correlated with the concentration of Sch9 protein present in cells (lower panels, Figure 1A).
Decreased H 2 S production by Δsch9 cells was also observed by measuring the reduction of methylene blue in different yeast strain backgrounds (BY4741 and BY4742) ( Figure 1C). Measurement of intracellular H 2 S in these two yeast strains by using WSP-1 fluorescent showed the same trends ( Figure 1D). Consistent with previous studies showing that CR enhanced H 2 S production in yeast [7], we observed a significant increase of H 2 S production in WT cell under CR ( Figure  1E). However, similar to no restriction condition, H 2 S production was still significantly impaired in Δsch9 cell even under CR ( Figure 1E).
To investigate if phosphorylation of Sch9, which correlates with its kinase activity and is required for H 2 S production, two inhibitors, Myriocin and Rapamycin, that indirectly lower Sch9 activity were used to inhibit the phosphorylation of Sch9 at the activation loop and the hydrophobic motif, respectively [19] (Figure 2A). Treatment with myriocin at 0.75 µM, but not 0.25 µM, inhibited H2S production ( Figure 2B). Similarly, Rapamycin treatments at 10 or 50 nM also resulted in decreased H2S production ( Figure 2C). These data indicate that the phosphorylation of Sch9 at the both activation loop and the hydrophobic motifs are required to the regulate H 2 S production.
Unlike mammalian cells, yeast cells convert extracellular sulfate to sulfide through the sulfur assimilatory pathway with enzymes encoded by MET14, MET16, and MET5/10 in addition to conserved the TSP pathway [7]. To verify if the TSP pathway is involved in decreased H 2 S production by mTORC1-Sch9 inhibition in yeast, we monitored the effect of rapamycin in H 2 S production by MET14, MET16 and MET5 mutants. As shown in Figure  2D, all three mutants have decreased H 2 S production upon mTORC1 inhibition by rapamycin, suggesting that interfering with the sulfur assimilatory pathway does not change the inhibitory role of rapamycin in H 2 S production and the TSP pathway is likely involved.

The role of mTORC1-Sch9 on the production of hydrogen sulfide is not caused by the alteration of methionine metabolism
The mTORC1 pathway plays an important role in regulating cell growth in response to amino acid availability [19,20]. Additionally, methionine metabolism contributes to H 2 S production through TSP pathway and methionine restriction extends lifespan from yeasts to humans [3,8]. Therefore, we asked if there is a lowered free methionine pool in Δsch9 cell which may contribute to the decreased H 2 S production. The effects of methionine on H 2 S production were investigated in BY4741, a strain with a defective MET15 gene which prevents the synthesis of methionine from sulfate in the medium, and BY4742 with functional MET15. BY4741 cells produced more H 2 S in the present of 2 mg/L methionine than in the present of 20 mg/L methionine while BY4742 cell only produced barely detectable H 2 S at both conditions ( Figure 3A). This suggests that methionine restriction indeed contributes to H 2 S production. It is worth to noting that when the methionine concentration in the medium was increased to 100 mg/L, H 2 S production increased in BY4742 cells. This may be due to the extreme abundance of substrates for H 2 S production. Similarly, in a different yeast background TB50a, decreasing the methionine concentration in the medium from 20 mg/L to 5 mg/L or 0 gave rise to significant H 2 S production while Δsch9 cell remained defective in H 2 S production under those conditions ( Figure 3B). Also, increasing methionine to 80 mg/L partially recovered H 2 S production by Δsch9 cell probably due to the extreme abundance of substrates.
To investigate if there is a high level of intracellular methionine to inhibit H 2 S production by Δsch9 cell, a AGING methionine probe plasmid pUG35-eGFP was constructed by putting eGFP expression under MET17 promoter which efficiency is inhibited by high concentration of intracellular methionine [21]. Indeed, the expression of eGFP protein by pUG35-eGFP was inhibited by increasing methionine concentration of medium ( Figure  3C upper and Figure 3D left panels), while eGFP level was not altered by exogenous methionine in the absence of the MET17 promoter ( Figure 3C lower and Figure 3D right panels) indicating that the altered eGFP levels are not due to the degradation of the protein. However, when pUG35-eGFP was transformed into Δsch9 cell the expression of eGFP was similar to that when the probe was present in WT cells ( Figure 3D left panels), indicating that the intracellular level of methionine in Δsch9 cell is not higher than that in WT cells and does not contribute to the decreased H 2 S production.

mTORC1-Sch9 regulates H 2 S production by regulating cysteine metabolism
Cysteine is another sulfur-containing amino acid whose metabolism is closely related to H 2 S production [3]. To verify that an altered intracellular level of cysteine is were transformed with pRS316-SCH9 or empty vector and inoculated into 1L of SDC medium at initial OD 600nm =0.005. H 2 S production was monitored using lead acetate strips at indicated times (Upper 3 panels) after inoculation. The level of Sch9 protein and actin loading control were determined by Western blotting as shown in the lower 2 panels. (B) Millimeters of darkening of the lead acetate strips inserted into the headspace of the culture flask shown in panel A normalized by OD 600nm . (C) Methylene blue assays of H 2 S produced by WT and Δsch9 cells in BY4741 or BY4742 background. Note that there is spontaneous oxidation of methylene blue when H 2 S is absent which gave negative readings for methylene reduction (red and blue dash lines). (D) Intracellular H 2 S production in WT and Δsch9 cells in BY4741 or BY4742 background monitored by H 2 S fluorescent with probe WSP-1. (* p˂0.05; ** p˂0.01; *** p˂0.005). (E) H 2 S production by WT and Δsch9 cells in BY4742 background assayed by using lead acetate strips which were replaced every 24 hours under caloric restriction conditions (CR, medium containing 0.5% glucose) or no restriction (NR, medium containing 2% glucose). AGING rapamycin and myriocin inhibit Sch9 through two different signaling pathways. (B) H 2 S production by BY4741 was monitored by using lead acetate strips at 24 or 48 hours after inoculation into YPD medium containing the indicated concentrations of myriocin. (C and D) H 2 S production by BY4741 or sulfur assimilatory mutants was monitored by using lead acetate strips which were replaced every 24 hours after the indicated concentrations of rapamycin were added into overnight culture of YPD (* p˂0.05 compared to control). AGING

(D) Immunoblot analysis of GFP expression in WT and
Δsch9 cells (TB50a background) with actin as loading control. Cells were transformed with either pUG35-eGFP (with MET7 promoter) or pRS316-eGFP (without MET7 promoter). The ratios of GFP to Actin are quantified by ImageJ and indicated below the lower panels. AGING involved in the regulation of H 2 S production by mTORC1-Sch9, we first investigated how H 2 S production is affected by different levels of cysteine supplementation. Adding 100 mg/L cysteine to SDC medium lacking methionine significantly decreased H 2 S production in WT TB50a cell ( Figure 4A). And increasing cysteine concentration to 500 mg/L restored H 2 S production likely due to substrate abundance ( Figure 4A). Deletion of SCH9 caused significant inhibition of H 2 S production under cysteine limited or over-supplied conditions ( Figure 4A). These data suggest that the inhibition of mTORC1-Sch9 renders H 2 S production less sensitive to exogenous cysteine, probably due to increased endogenous cysteine. Indeed, unlike the intracellular methionine level which was not changed in Δsch9 cell ( Figure 3D), the intracellular cysteine level was ~50% higher in Δsch9 cell than WT cell ( Figure 4B). And when a functional SCH9 gene was added back to mutant cells the intracellular cysteine level decreased ( Figure 4B), showing that Sch9 regulates intracellular cysteine metabolism. When the level of exogenous cysteine was 100 mg/L, the intracellular cysteine level was still higher in Δsch9 cells than WT cells until the level of exogenous cysteine reached 500 mg/L ( Figure 4C). Together, these data indicate that the decreased H 2 S production by mTORC1-Sch9 inhibition is most likely due to an increase in the level of intracellular cysteine.

mTORC1-Sch9 regulates H 2 S production via transsulfuration pathway
Increased intracellular cysteine usually inhibits the expression of enzymes in the transsulfuration pathway that required for H 2 S production [7]. To investigate if the expression of the transsulfuration enzymes is altered in Δsch9 cell, the mRNA levels of CYS3 and CYS4 which encodes Cystathionine gamma-lyase and Cystathionine beta-synthase respectively in yeast were compared in Δsch9 and WT cells. Indeed, the mRNA levels of both CYS3 and CYS4 decreased to about 50% in Δsch9 cell compared to them in WT cell. And it can be reversed by adding SCH9 back to mutant cells ( Figure 5A). Similarly, inhibiting mTORC1-Sch9 by rapamycin also decreased the expression of both CYS3 and CYS4 ( Figure 5B). These data suggest that inhibiting mTORC1-Sch9 which increases intracellular cysteine level does down-regulate transsulfuration pathway.
To verify the role of Cys3 and Cys4 in H 2 S production regulation by mTORC1-Sch9, CYS3 or CYS4 was overexpressed in Δsch9 or WT cells treated with rapamycin and H 2 S production was monitored ( Figure  5C to 5E). Overexpressing CYS3 significantly increased H 2 S production by WT cell. While Δsch9 cell with empty vector produced little H 2 S, overexpressing CYS3 restored H 2 S production to a level similar to WT cells overexpressing CYS3 (Figure 5C and 5D). Similarly, overexpressing CYS3 also restored H 2 S production in rapamycin treated WT cell ( Figure 5E). However, although overexpressing CYS4 restored H 2 S production in rapamycin treated WT cell, it did not restore H 2 S production in Δsch9 cell ( Figure 5C). These data support the hypothesis that the CYS3 and CYS4 genes in the transsulfuration pathway mediate H 2 S production and are regulated by the mTORC1-Sch9 pathway.

Inhibiting mTORC1 reduces H 2 S production and expression of transsulfuration pathway enzymes in human cells.
The anti-aging effects of H 2 S production mediated by transsulfuration pathway and regulated by mTORC1 are evolutionary conserved from yeast to mammals [7,[11][12][13]. To assess if inhibiting mTORC1 also interferes with H 2 S production in mammalian cells, we examined human 293T and HeLa cells. To measure H 2 S production in cultured 293T and HeLa cells by using the lead acetate strip assay the growth medium was supplemented with the CGL/CBS substrates Cys and cofactor pyridoxal-5′phosphate (PLP). The supplementation of Cys/PLP decreased the cell viability (Supplementary Figure 1), but significantly increased H 2 S production. With these assay conditions rapamycin treated cells have decreased the cell death induced by Cys/PLP and higher cell density (Supplementary Figure 1). However, less H 2 S is produced in both 293T and HeLa cells with rapamycin treatments ( Figure 6A and 6B), similar to what we observed in yeast cells ( Figure 2D). These data suggest that inhibiting mTORC1 in mammalian cells may also decrease H 2 S production.
To determine if reduction of H 2 S by inhibition of mTORC1 activity in mammalian cells is also accompanied by down-regulation of transsulfuration pathway enzymes, the expression of CGL and CBS was monitored by RT-qPCR in HeLa cell with or without rapamycin treatments for 1 to 4 days. Similar to what we observed in yeast cells ( Figure 5B), rapamycin treatment reduced the mRNA level of both CGL and CBS significantly ( Figure 6C and 6D). Consistent with mRNA levels, CGL and CBS protein levels were significantly decreased after 3 to 4 days of rapamycin treatments ( Figure 6E), indicating that the expression of both transsulfuration pathway enzymes is reduced upon mTORC1 inhibition in HeLa cell.

DISCUSSION
Due to the important roles of the mTORC1 and H 2 S signaling pathways in aging and longevity, we wanted to investigate if the widely studied anti-aging effect of mTORC1 is partially mediated by endogenous H 2 S production. Surprisingly, by monitoring the amount of H 2 S released from yeast cells and its intracellular level in different strain backgrounds, we found that H 2 S production was reduced when the mTORC1-Sch9 signal transduction pathway was inhibited. These observations are unexpected since both increased H 2 S production and lower mTORC1-Sch9 activity are beneficial for lifespan. The facts that DR lowers activity of the mTORC1-Sch9 pathway and enhances H 2 S production are also unexpected and suggest involvement of novel lifespan enhancing mechanisms. A plausible explanation is that the lifespan extension mediated by direct mTORC1-Sch9 inhibition under normal nutrition conditions does not require H 2 S. Conversely, H 2 S probably benefits lifespan through down-regulating mTORC1-Sch9. Thus further studies are required to investigate if H 2 S regulates mTORC1-Sch9 signaling and, if it does, then what is the mechanism.
Based on the unexpected observation of H 2 S production being down-regulated by mTORC1-Sch9 inhibition, we asked what mechanism is behind this phenomenon. Sulfur amino acid metabolism is likely involved since it is regulated by mTORC1 and contributes to endogenous H 2 S production. Indeed, H 2 S production is controlled by both methionine and cysteine levels [3], and only the cysteine level is enhanced by mTORC1-Sch9 inhibition  ( Figures 3 and 4), suggesting that an alteration of the cysteine level is involved in regulation of H 2 S production by mTORC1-Sch9. It is not surprising that inhibiting mTORC1-Sch9 increase intracellular cysteine level since many studies have shown that mTORC1-Sch9 inhibition enhances autophagy and decreases protein synthesis through its downstream factors and both processes may contribute to cysteine accumulation [22].
While cysteine is the substrate for H 2 S production and large increases in intracellular cysteine promote H 2 S production, it has been demonstrated that moderate increases in intracellular cysteine decrease H 2 S production by inhibiting expression of the transsulfuration pathway enzymes including CBS and CGL (Figure 7) [23][24][25]. In this study, we observed the downregulation of CBS and CGL accompanied with cysteine elevation upon mTORC1-Sch9 inhibition, supporting the hypothesis that decreased CBS and CGL activity in response to cysteine elevation contributes to decreased H 2 S production. The restoration of H 2 S production by restoring CBS or CGL enzyme activity during mTORC1-Sch9 inhibition is consistent with this mechanism ( Figure 5). Therefore, we established a mechanism in yeast by which mTORC1-Sch9 regulates H 2 S production through altering intracellular cysteine level and expression of CBS and CGL (Figure 7).
Additionally, our data indicate that inhibiting mTORC1-Sch9 down-regulates the transsulfuration pathway and reduces H 2 S production in ways that are conserved in yeast and human cells (Figure 7). It worth noting that an earlier study indicated that constitutively activating Figure 7. A mechanism by which mTORC1-Sch9 regulates H 2 S production via transsulfuration pathway. mTORC1-Sch9 controls the intracellular level of cysteine which is one of substrates for endogenous H 2 S production. On the other hand, cysteine regulates the expression of key transsulfuration pathway enzymes CBS and CGL which catalyze H 2 S production from homocysteine or cysteine. CBS is encoded by CBS in human and CYS4 in Saccharomyces cerevisiae while CGL is encoded by CTH in human and CYS3 in Saccharomyces cerevisiae. Dash lines indicate indirect regulations. mTORC1 in mouse hepatocytes prevents the increase of CGL expression and H 2 S production by DR [7]. However, it is not clear how the transsulfuration pathway and cysteine metabolism are altered in different types of cells at different situations. And, as indicated by our data, a moderately increased cysteine level inhibited H 2 S production while higher cysteine levels increased H 2 S production ( Figure 4A). Therefore, H 2 S production seems to be very sensitive to the extracellular or intracellular level of cysteine and published data may have been influenced by how much cysteine accumulated in the tissues under different nutrition conditions and genetic backgrounds.
Together, our study reveals crosstalk between mTORC1 and H 2 S signaling, two conserved pathways which play fundamental regulatory roles in aging of eukaryotic organisms. Further studies which elucidate how these two pathways collaborate in specific human cells and tissues will have broad implications for potential clinical applications.

Yeast strains, plasmids, and media
The S. cerevisiae strains and plasmids used in this study are listed in Table 1 and Table 2. Strains were grown at 30 °C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or synthetic dextrose complete medium (SDC) which contains no cysteine [26]. Cells transformed with plasmids carrying URA3 were grown in the SDC medium lacking uracil. For inducing the expression of DNA sequences inserted into pYES2, cells were grown in the galactose-inducing medium (2% glucose, carbon source of SDC medium, was replaced to 1% Galactose and 1% Sucrose).

Protein extraction and western blotting
For protein extraction, trichloroacetic acid yeast cell extracts were prepared according to a method described previously [17,27]. HeLa cell with indicated treatments were washed with PBS and lysed in Laemmli buffer.   [28,29]. The stripes were replaced every day or remained for entire experimental periods as described.
The methylene blue assay described previously for H 2 S detection was also performed in centrifuge tubes [30]. 36ml cells at OD600nm of 0.05 were divided in two parts. One of them was added with 2ml medium to monitor growth rate as measured by the absorbance at 600 nm and the another was the addition of 2 ml methylene blue reaction mix (1 mg/ml methylene blue, 100 mM citric acid buffer at pH 4.5), reacting with H 2 S dissolved in medium. Methylene blue decolorization by H 2 S were monitored at 663 nm and normalized to biomass.
Intracellular free H 2 S levels were also determined using the H 2 S fluorescent probe WSP-1 (Cayman, USA) [31].

H 2 S assay for cultured human cells
293T or HeLa cell was grown in DMEM (Invitrogen) supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin with or without 250 nM rapamycin. To measure H 2 S production, growth media was supplemented with or without 10 mM Cys and 10 mM pyridoxal-5′-phosphate (PLP) and a lead acetate strip was placed above the media in a 25 ml cell culture flask incubated in a CO 2 incubator at 37 °C for indicated time.

Measurement of the Cys content
The extraction and estimation of cysteine content in yeast cells were done as described previously [32]. Cells grown in SDC-Ura or medium containing different concentrations of Cys were harvested and washed with PBS twice by centrifugation. Cell pellets were dried at 50 °C until constant weight was achieved. Dried cells were lysed in liquid nitrogen and then 1ml of 10% TCA was added to 200mg cell powder. The homogenates were centrifuged at 2800×g for 60min. Acid ninhydrin were added to the extract, and reaction mixture was kept in boiled water for 10 min. After fast cooling, the A560nm absorbance of the reaction mixture was measured. The amount of cysteine in each reaction was determined using a standard curve.

Statistical analysis
All data from at least three independent experiments. Error bars are presented as averages ± SD. Statistical analysis and comparisons were performed using twotailed, unpaired Student t tests.