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Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, Gaude E, King A, Leach JD, Edrada-Ebel R, Hedley A, Morrice NA, Kalna G, Blyth K, Ruppin E, Frezza C, Gottlieb E - Nat Commun (2015)

Bottom Line: Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione.Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo.Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

No MeSH data available.


Related in: MedlinePlus

GSH succination triggers cystine uptake and GSH biosynthesis.(a–c) The loss of FH leads to the upregulation of the cystine transporter (a), increased cystine uptake (b) and accelerated GSH biosynthesis traced from 13C5-glutamine (c). (d) The steady-state pool of GSH is increased in FH-deficient cells. (e,f,g) Sulfasalazin (SSZ) inhibits GSH biosynthesis in both wt and FH-deficient cells (e) but selectively impairs cell survival and growth of FH-deficient cells (f,g). (h,i) 6-Aminonicotinamide (6-AN), an inhibitor of the PPP, inhibits pentose phosphate production in both cell lines as assessed by tracing 13C6-Glucose (h), selectively affects the proliferation of FH-deficient cells (i). The images in f are the representative images of triplicate cultures. Results were obtained from three independent experiments and represented as average±s.e.m.
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f5: GSH succination triggers cystine uptake and GSH biosynthesis.(a–c) The loss of FH leads to the upregulation of the cystine transporter (a), increased cystine uptake (b) and accelerated GSH biosynthesis traced from 13C5-glutamine (c). (d) The steady-state pool of GSH is increased in FH-deficient cells. (e,f,g) Sulfasalazin (SSZ) inhibits GSH biosynthesis in both wt and FH-deficient cells (e) but selectively impairs cell survival and growth of FH-deficient cells (f,g). (h,i) 6-Aminonicotinamide (6-AN), an inhibitor of the PPP, inhibits pentose phosphate production in both cell lines as assessed by tracing 13C6-Glucose (h), selectively affects the proliferation of FH-deficient cells (i). The images in f are the representative images of triplicate cultures. Results were obtained from three independent experiments and represented as average±s.e.m.

Mentions: We hypothesized that the chronic depletion of GSH caused by succination could perturb cell metabolism and contribute to a general deregulation of redox homeostasis. To address this question, we reconstructed the metabolic models previously generated to include GSH succination. Of note, the inclusion of this reaction improved the ability of the FH-deficient model to predict the measured flux rates (R=0.570, P=1.81E−03; Supplementary Table 2). We found that in the FH-deficient model, but not in the wt model, GSH succination significantly reshaped the generation of reducing power (Supplementary Fig. 1b). Furthermore, the model revealed that GSH succination impinged GSH metabolism and cystine uptake (Supplementary Table 3). Notably, the selective uptake of cystine (an oxidized dimer of cysteine), rather than methionine, as a precursor for cysteine and subsequently GSH biosynthesis imposes a further metabolic constraint: the reduction of cystine to cysteine requires NADPH, the supply of which becomes critical for GSH biosynthesis (see Supplementary Fig. 3b). In line with these predictions, Fh1Δ/Δ cells exhibited a significant overexpression of Slc7a11, a member of the cystine transporter xCT, which was functionally associated with increased uptake of cystine (Fig. 5a,b). The rate-limiting step in GSH biosynthesis is the synthesis of γ-glutamylcysteine from glutamate and cysteine by γ-glutamylcysteine synthetase (GCL; see Fig. 3a for a schematic representation of these reactions). GSH is then synthesized by GSH synthetase (GSS), which adds glycine to γ-glutamylcysteine. Therefore, the GSH biosynthesis rate could be assessed by the rate of incorporation of glutamine-derived glutamate after incubation with 13C5-glutamine. The incorporation of glutamate into GSH was faster in FH-deficient cells, meaning that, as predicted by the computational models, GSH production is accelerated in FH-deficient cells (Fig. 5c). Moreover, the increased biosynthesis rate of GSH caused by GSH succination led to a larger steady-state pool of GSH (Fig. 5d). In line with these findings, genes encoding for cystine transport (Slc7a11) and enzymes of the GSH biosynthetic pathway (GSS and GCL), are significantly upregulated in HLRCC patients (Supplementary Fig. 4)717. All in all, these suggest that increased GSH biosynthesis could be a compensatory mechanism that mitigates the increased oxidative stress in FH-deficient cells.


Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, Gaude E, King A, Leach JD, Edrada-Ebel R, Hedley A, Morrice NA, Kalna G, Blyth K, Ruppin E, Frezza C, Gottlieb E - Nat Commun (2015)

GSH succination triggers cystine uptake and GSH biosynthesis.(a–c) The loss of FH leads to the upregulation of the cystine transporter (a), increased cystine uptake (b) and accelerated GSH biosynthesis traced from 13C5-glutamine (c). (d) The steady-state pool of GSH is increased in FH-deficient cells. (e,f,g) Sulfasalazin (SSZ) inhibits GSH biosynthesis in both wt and FH-deficient cells (e) but selectively impairs cell survival and growth of FH-deficient cells (f,g). (h,i) 6-Aminonicotinamide (6-AN), an inhibitor of the PPP, inhibits pentose phosphate production in both cell lines as assessed by tracing 13C6-Glucose (h), selectively affects the proliferation of FH-deficient cells (i). The images in f are the representative images of triplicate cultures. Results were obtained from three independent experiments and represented as average±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340546&req=5

f5: GSH succination triggers cystine uptake and GSH biosynthesis.(a–c) The loss of FH leads to the upregulation of the cystine transporter (a), increased cystine uptake (b) and accelerated GSH biosynthesis traced from 13C5-glutamine (c). (d) The steady-state pool of GSH is increased in FH-deficient cells. (e,f,g) Sulfasalazin (SSZ) inhibits GSH biosynthesis in both wt and FH-deficient cells (e) but selectively impairs cell survival and growth of FH-deficient cells (f,g). (h,i) 6-Aminonicotinamide (6-AN), an inhibitor of the PPP, inhibits pentose phosphate production in both cell lines as assessed by tracing 13C6-Glucose (h), selectively affects the proliferation of FH-deficient cells (i). The images in f are the representative images of triplicate cultures. Results were obtained from three independent experiments and represented as average±s.e.m.
Mentions: We hypothesized that the chronic depletion of GSH caused by succination could perturb cell metabolism and contribute to a general deregulation of redox homeostasis. To address this question, we reconstructed the metabolic models previously generated to include GSH succination. Of note, the inclusion of this reaction improved the ability of the FH-deficient model to predict the measured flux rates (R=0.570, P=1.81E−03; Supplementary Table 2). We found that in the FH-deficient model, but not in the wt model, GSH succination significantly reshaped the generation of reducing power (Supplementary Fig. 1b). Furthermore, the model revealed that GSH succination impinged GSH metabolism and cystine uptake (Supplementary Table 3). Notably, the selective uptake of cystine (an oxidized dimer of cysteine), rather than methionine, as a precursor for cysteine and subsequently GSH biosynthesis imposes a further metabolic constraint: the reduction of cystine to cysteine requires NADPH, the supply of which becomes critical for GSH biosynthesis (see Supplementary Fig. 3b). In line with these predictions, Fh1Δ/Δ cells exhibited a significant overexpression of Slc7a11, a member of the cystine transporter xCT, which was functionally associated with increased uptake of cystine (Fig. 5a,b). The rate-limiting step in GSH biosynthesis is the synthesis of γ-glutamylcysteine from glutamate and cysteine by γ-glutamylcysteine synthetase (GCL; see Fig. 3a for a schematic representation of these reactions). GSH is then synthesized by GSH synthetase (GSS), which adds glycine to γ-glutamylcysteine. Therefore, the GSH biosynthesis rate could be assessed by the rate of incorporation of glutamine-derived glutamate after incubation with 13C5-glutamine. The incorporation of glutamate into GSH was faster in FH-deficient cells, meaning that, as predicted by the computational models, GSH production is accelerated in FH-deficient cells (Fig. 5c). Moreover, the increased biosynthesis rate of GSH caused by GSH succination led to a larger steady-state pool of GSH (Fig. 5d). In line with these findings, genes encoding for cystine transport (Slc7a11) and enzymes of the GSH biosynthetic pathway (GSS and GCL), are significantly upregulated in HLRCC patients (Supplementary Fig. 4)717. All in all, these suggest that increased GSH biosynthesis could be a compensatory mechanism that mitigates the increased oxidative stress in FH-deficient cells.

Bottom Line: Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione.Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo.Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

No MeSH data available.


Related in: MedlinePlus