Limits...
Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of Ferroptosis

View Article: PubMed Central - PubMed

ABSTRACT

Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

No MeSH data available.


Related in: MedlinePlus

Validating SQS as the functionally relevant target to FIN56’s lethalitya. Effects of chemical inhibitors of SQS on FIN56’s lethality. b. Effects of Farnesyl-PP on FIN56’s lethality. c. Detection of SQS using pull-down assay from HT-1080 whole cell lysate with active or inactive probes. Note that the probes are the same as what were used for chemoproteomic target identification. d. Schematics of the mevalonate pathway. Large letters are metabolites, small letters are enzymes responsible for the reactions or small molecules. Red and blue letters indicate the molecules (inhibitors or metabolites) suppressed or enhanced FIN56’s lethality. The detailed results are shown in e and f. e. Perturbation of the mevalonate pathway and their effects on FIN56’s lethality. Concentrations: cerivastatin (1 μM), metabolites (100 μM), YM-53601 (5 μM), NB-598 (25 μM), αToc (100 μM). f. Supplementation of 10 μM end-products of the MVA pathway and their effects on FIN56. g. Effect of 10 μM idebenone on FIN56 in HT-1080. h. Modulatory profiling between the modulators of the MVA pathway and various lethal compounds inducing oxidative stress. See Supplementary Fig. 9-–10 for SQS pull-down with competition and effect of statins on FIN56 lethality. a,b,g were performed in biological replicates and error-bars are s.e.m. of technical triplicates; e,f in biological duplicates and error-bars are standard errors of EC50 estimation from sigmoidal curve-fitting; h in singlicate. Full image of c is in Supplementary Figure 14.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4920070&req=5

Figure 5: Validating SQS as the functionally relevant target to FIN56’s lethalitya. Effects of chemical inhibitors of SQS on FIN56’s lethality. b. Effects of Farnesyl-PP on FIN56’s lethality. c. Detection of SQS using pull-down assay from HT-1080 whole cell lysate with active or inactive probes. Note that the probes are the same as what were used for chemoproteomic target identification. d. Schematics of the mevalonate pathway. Large letters are metabolites, small letters are enzymes responsible for the reactions or small molecules. Red and blue letters indicate the molecules (inhibitors or metabolites) suppressed or enhanced FIN56’s lethality. The detailed results are shown in e and f. e. Perturbation of the mevalonate pathway and their effects on FIN56’s lethality. Concentrations: cerivastatin (1 μM), metabolites (100 μM), YM-53601 (5 μM), NB-598 (25 μM), αToc (100 μM). f. Supplementation of 10 μM end-products of the MVA pathway and their effects on FIN56. g. Effect of 10 μM idebenone on FIN56 in HT-1080. h. Modulatory profiling between the modulators of the MVA pathway and various lethal compounds inducing oxidative stress. See Supplementary Fig. 9-–10 for SQS pull-down with competition and effect of statins on FIN56 lethality. a,b,g were performed in biological replicates and error-bars are s.e.m. of technical triplicates; e,f in biological duplicates and error-bars are standard errors of EC50 estimation from sigmoidal curve-fitting; h in singlicate. Full image of c is in Supplementary Figure 14.

Mentions: We found that four of the five shRNAs against FDFT1 mRNA (which encodes SQS protein) suppressed FIN56 consistently in all four cell lines tested, indicating that FIN56 activates, rather than inhibits, SQS (a ‘gain-of-function’ model). Therefore, we explored this possibility and studied how the FIN56-SQS interaction is relevant to FIN56’s lethality. We confirmed that not only shRNAs targeting FDFT1, but also small molecule inhibitors of the SQS activity (YM-53601 and zaragozic acid A), suppress FIN56 lethality (Fig. 5a). SQS is an enzyme acting downstream of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway. SQS couples two farnesyl pyrophosphate (FPP) molecules to form squalene. Inhibition of SQS consequently increases the pool of FPP. FPP is essential for multiple processes, including protein prenylation and metabolite synthesis (e.g., sterols, coenzyme Q10 (CoQ10), dolichol and heme) 26, some subset of which may be relevant to the modulatory effect on ferroptosis sensitivity. Supplementation with FPP indeed suppressed the lethality of FIN56 (Fig. 5b). We also examined SQS-FIN56 binding, by confirming that SQS from HT-1080 whole cell lysate binds selectively to active probes versus an inactive probe (Fig. 5c). Moreover, bacterially-expressed truncated human SQS protein27 (with the lipophilic N and C termini removed) binding to an active affinity probe was efficiently suppressed by pre-incubation of purified SQS protein with FIN56, suggesting that SQS and FIN56 directly interact (Supplementary Fig. 9).


Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of Ferroptosis
Validating SQS as the functionally relevant target to FIN56’s lethalitya. Effects of chemical inhibitors of SQS on FIN56’s lethality. b. Effects of Farnesyl-PP on FIN56’s lethality. c. Detection of SQS using pull-down assay from HT-1080 whole cell lysate with active or inactive probes. Note that the probes are the same as what were used for chemoproteomic target identification. d. Schematics of the mevalonate pathway. Large letters are metabolites, small letters are enzymes responsible for the reactions or small molecules. Red and blue letters indicate the molecules (inhibitors or metabolites) suppressed or enhanced FIN56’s lethality. The detailed results are shown in e and f. e. Perturbation of the mevalonate pathway and their effects on FIN56’s lethality. Concentrations: cerivastatin (1 μM), metabolites (100 μM), YM-53601 (5 μM), NB-598 (25 μM), αToc (100 μM). f. Supplementation of 10 μM end-products of the MVA pathway and their effects on FIN56. g. Effect of 10 μM idebenone on FIN56 in HT-1080. h. Modulatory profiling between the modulators of the MVA pathway and various lethal compounds inducing oxidative stress. See Supplementary Fig. 9-–10 for SQS pull-down with competition and effect of statins on FIN56 lethality. a,b,g were performed in biological replicates and error-bars are s.e.m. of technical triplicates; e,f in biological duplicates and error-bars are standard errors of EC50 estimation from sigmoidal curve-fitting; h in singlicate. Full image of c is in Supplementary Figure 14.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4920070&req=5

Figure 5: Validating SQS as the functionally relevant target to FIN56’s lethalitya. Effects of chemical inhibitors of SQS on FIN56’s lethality. b. Effects of Farnesyl-PP on FIN56’s lethality. c. Detection of SQS using pull-down assay from HT-1080 whole cell lysate with active or inactive probes. Note that the probes are the same as what were used for chemoproteomic target identification. d. Schematics of the mevalonate pathway. Large letters are metabolites, small letters are enzymes responsible for the reactions or small molecules. Red and blue letters indicate the molecules (inhibitors or metabolites) suppressed or enhanced FIN56’s lethality. The detailed results are shown in e and f. e. Perturbation of the mevalonate pathway and their effects on FIN56’s lethality. Concentrations: cerivastatin (1 μM), metabolites (100 μM), YM-53601 (5 μM), NB-598 (25 μM), αToc (100 μM). f. Supplementation of 10 μM end-products of the MVA pathway and their effects on FIN56. g. Effect of 10 μM idebenone on FIN56 in HT-1080. h. Modulatory profiling between the modulators of the MVA pathway and various lethal compounds inducing oxidative stress. See Supplementary Fig. 9-–10 for SQS pull-down with competition and effect of statins on FIN56 lethality. a,b,g were performed in biological replicates and error-bars are s.e.m. of technical triplicates; e,f in biological duplicates and error-bars are standard errors of EC50 estimation from sigmoidal curve-fitting; h in singlicate. Full image of c is in Supplementary Figure 14.
Mentions: We found that four of the five shRNAs against FDFT1 mRNA (which encodes SQS protein) suppressed FIN56 consistently in all four cell lines tested, indicating that FIN56 activates, rather than inhibits, SQS (a ‘gain-of-function’ model). Therefore, we explored this possibility and studied how the FIN56-SQS interaction is relevant to FIN56’s lethality. We confirmed that not only shRNAs targeting FDFT1, but also small molecule inhibitors of the SQS activity (YM-53601 and zaragozic acid A), suppress FIN56 lethality (Fig. 5a). SQS is an enzyme acting downstream of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway. SQS couples two farnesyl pyrophosphate (FPP) molecules to form squalene. Inhibition of SQS consequently increases the pool of FPP. FPP is essential for multiple processes, including protein prenylation and metabolite synthesis (e.g., sterols, coenzyme Q10 (CoQ10), dolichol and heme) 26, some subset of which may be relevant to the modulatory effect on ferroptosis sensitivity. Supplementation with FPP indeed suppressed the lethality of FIN56 (Fig. 5b). We also examined SQS-FIN56 binding, by confirming that SQS from HT-1080 whole cell lysate binds selectively to active probes versus an inactive probe (Fig. 5c). Moreover, bacterially-expressed truncated human SQS protein27 (with the lipophilic N and C termini removed) binding to an active affinity probe was efficiently suppressed by pre-incubation of purified SQS protein with FIN56, suggesting that SQS and FIN56 directly interact (Supplementary Fig. 9).

View Article: PubMed Central - PubMed

ABSTRACT

Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

No MeSH data available.


Related in: MedlinePlus