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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.


Squalene synthase (SQS) encoded by FDFT1 as FIN56’s target proteina. Active and inactive FIN56 analogs with PEG linkers. Supplementary Fig. 7 shows their potency in HT-1080 cells. b. Effects of five shRNAs against FDFT1 on FIN56. The results in two of the four cell lines were shown. Five shRNA clones targeting FDFT1 are shown in polychromatic lines. A black line indicates a shRNA which gives no effect (median AUC among tested shRNAs) in each cell line. Grey lines indicate shRNAs targeting other genes. c. Summary of proteomic target identification and shRNA screening targeting 70 identified genes on FIN56. Each dot summarizes the result of multiple shRNAs targeting a gene. Each shRNA is considered ‘consistent’ when it exerts indicated effect (enhancing or suppressing FIN56). X-axis shows the ratio: the number of consistent shRNAs inducing indicated (i.e., enhancing or suppressing FIN56) effects to the total number of shRNAs targeting the gene. Y-axis shows fold-enrichment of protein abundance on active versus inactive probe-beads in pull-down assay. See Supplementary Fig. 8 for more description. d. Effect of siRNAs against ‘loss-of-function’ candidates on BJeLR cell viability. Cells were grown under DMSO or ATOC (α-tocopherol)’s existence. shRNA screens in b,c were performed once in four cell lines. siRNA experiment in d was performed in BJeLR twice and mean of biological replicates.
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Figure 4: Squalene synthase (SQS) encoded by FDFT1 as FIN56’s target proteina. Active and inactive FIN56 analogs with PEG linkers. Supplementary Fig. 7 shows their potency in HT-1080 cells. b. Effects of five shRNAs against FDFT1 on FIN56. The results in two of the four cell lines were shown. Five shRNA clones targeting FDFT1 are shown in polychromatic lines. A black line indicates a shRNA which gives no effect (median AUC among tested shRNAs) in each cell line. Grey lines indicate shRNAs targeting other genes. c. Summary of proteomic target identification and shRNA screening targeting 70 identified genes on FIN56. Each dot summarizes the result of multiple shRNAs targeting a gene. Each shRNA is considered ‘consistent’ when it exerts indicated effect (enhancing or suppressing FIN56). X-axis shows the ratio: the number of consistent shRNAs inducing indicated (i.e., enhancing or suppressing FIN56) effects to the total number of shRNAs targeting the gene. Y-axis shows fold-enrichment of protein abundance on active versus inactive probe-beads in pull-down assay. See Supplementary Fig. 8 for more description. d. Effect of siRNAs against ‘loss-of-function’ candidates on BJeLR cell viability. Cells were grown under DMSO or ATOC (α-tocopherol)’s existence. shRNA screens in b,c were performed once in four cell lines. siRNA experiment in d was performed in BJeLR twice and mean of biological replicates.

Mentions: To better understand the mechanism of action of FIN56, we decided to seek direct binding proteins of FIN56 using a chemoproteomic approach. First, we explored structural analogs of FIN56. This resulted in creation of SRS11-31, an analog with a polyethylene glycol (PEG) moiety, which induces ferroptosis at 10-fold higher EC50 than FIN56 (Fig. 4a, Supplementary Fig. 7). On the other hand, substitution of the cyclohexyl moiety in FIN56 with a 4-tetrahydropyran (SRS8-18 (3)) or its PEG-conjugate (SRS11-66) (4) resulted in complete loss of activity. Next, both SRS11-31 (5) (active, or A) and SRS11-66 (inactive, or I) were conjugated to Profinity epoxide resin through an epoxy ring-opening reaction, and the resins were incubated with HT-1080 whole cell lysates. The pull-down proteins found with each probe were identified and quantified by mass spectrometry. 70 proteins excluding universally expressed proteins (actins, tubulins and ribosome subunits) were found to be more abundant on the resin conjugated with the active probe.


Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of Ferroptosis
Squalene synthase (SQS) encoded by FDFT1 as FIN56’s target proteina. Active and inactive FIN56 analogs with PEG linkers. Supplementary Fig. 7 shows their potency in HT-1080 cells. b. Effects of five shRNAs against FDFT1 on FIN56. The results in two of the four cell lines were shown. Five shRNA clones targeting FDFT1 are shown in polychromatic lines. A black line indicates a shRNA which gives no effect (median AUC among tested shRNAs) in each cell line. Grey lines indicate shRNAs targeting other genes. c. Summary of proteomic target identification and shRNA screening targeting 70 identified genes on FIN56. Each dot summarizes the result of multiple shRNAs targeting a gene. Each shRNA is considered ‘consistent’ when it exerts indicated effect (enhancing or suppressing FIN56). X-axis shows the ratio: the number of consistent shRNAs inducing indicated (i.e., enhancing or suppressing FIN56) effects to the total number of shRNAs targeting the gene. Y-axis shows fold-enrichment of protein abundance on active versus inactive probe-beads in pull-down assay. See Supplementary Fig. 8 for more description. d. Effect of siRNAs against ‘loss-of-function’ candidates on BJeLR cell viability. Cells were grown under DMSO or ATOC (α-tocopherol)’s existence. shRNA screens in b,c were performed once in four cell lines. siRNA experiment in d was performed in BJeLR twice and mean of biological replicates.
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Figure 4: Squalene synthase (SQS) encoded by FDFT1 as FIN56’s target proteina. Active and inactive FIN56 analogs with PEG linkers. Supplementary Fig. 7 shows their potency in HT-1080 cells. b. Effects of five shRNAs against FDFT1 on FIN56. The results in two of the four cell lines were shown. Five shRNA clones targeting FDFT1 are shown in polychromatic lines. A black line indicates a shRNA which gives no effect (median AUC among tested shRNAs) in each cell line. Grey lines indicate shRNAs targeting other genes. c. Summary of proteomic target identification and shRNA screening targeting 70 identified genes on FIN56. Each dot summarizes the result of multiple shRNAs targeting a gene. Each shRNA is considered ‘consistent’ when it exerts indicated effect (enhancing or suppressing FIN56). X-axis shows the ratio: the number of consistent shRNAs inducing indicated (i.e., enhancing or suppressing FIN56) effects to the total number of shRNAs targeting the gene. Y-axis shows fold-enrichment of protein abundance on active versus inactive probe-beads in pull-down assay. See Supplementary Fig. 8 for more description. d. Effect of siRNAs against ‘loss-of-function’ candidates on BJeLR cell viability. Cells were grown under DMSO or ATOC (α-tocopherol)’s existence. shRNA screens in b,c were performed once in four cell lines. siRNA experiment in d was performed in BJeLR twice and mean of biological replicates.
Mentions: To better understand the mechanism of action of FIN56, we decided to seek direct binding proteins of FIN56 using a chemoproteomic approach. First, we explored structural analogs of FIN56. This resulted in creation of SRS11-31, an analog with a polyethylene glycol (PEG) moiety, which induces ferroptosis at 10-fold higher EC50 than FIN56 (Fig. 4a, Supplementary Fig. 7). On the other hand, substitution of the cyclohexyl moiety in FIN56 with a 4-tetrahydropyran (SRS8-18 (3)) or its PEG-conjugate (SRS11-66) (4) resulted in complete loss of activity. Next, both SRS11-31 (5) (active, or A) and SRS11-66 (inactive, or I) were conjugated to Profinity epoxide resin through an epoxy ring-opening reaction, and the resins were incubated with HT-1080 whole cell lysates. The pull-down proteins found with each probe were identified and quantified by mass spectrometry. 70 proteins excluding universally expressed proteins (actins, tubulins and ribosome subunits) were found to be more abundant on the resin conjugated with the active probe.

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.