Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells
- Human Biology Division, Fred Hutchinson Cancer Center, United States
- Molecular Medicine & Mechanisms of Disease Program, University of Washington, United States
- Department of Chemistry and Biochemistry, New Mexico State University, United States
Figures
Figure 1 with 1 supplement
SDH inhibition blocks proliferation, which is incompletely rescued by electron acceptors but robustly restored by aspartate.
(A) Proliferation rates of 143B cells treated with vehicle (DMSO), 50 nM rotenone, or 5 µM atpenin A5 (AA5) cultured in pyruvate free DMEM with no addition, 1 mM pyruvate (PYR), 1 mM …
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Figure 1—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig1-data1-v3.zip
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Figure 1—source data 2
Uncropped western blot for Figure 1F.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig1-data2-v3.zip
Figure 1—figure supplement 1
Alternative methods of aspartate acquisition and characterization of metabolic phenotypes in SDH-impaired cells.
(A) Schematic demonstrating how the aspartate transporter SLC1A3 allows for cells to uptake aspartate, which can be measured by the incorporation of isotopically labeled extracellular aspartate …
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Figure 1—figure supplement 1—source data 1
Proliferation rates and metabolite levels by LCMS in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig1-figsupp1-data1-v3.zip
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Figure 1—figure supplement 1—source data 2
Uncropped western blot for Figure 1—figure supplement 1B and F.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig1-figsupp1-data2-v3.zip
Figure 2 with 1 supplement
CI inhibition is sufficient to induce aspartate synthesis and cell proliferation in SDH-deficient cancer cells.
(A) Proliferation rates of 143B cells cultured in DMEM with 1 mM PYR treated with vehicle (DMSO), 50 nM rotenone, 5 µM AA5, or 5 µM AA5 and 50 nM rotenone (n=3). (B) Aspartate levels of 143B cells …
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Figure 2—source data 1
Proliferation rates, relative metabolite levels by LCMS, and other data in Figure 2.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig2-data1-v3.zip
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Figure 2—source data 2
Uncropped western blots for Figure 2C and E.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig2-data2-v3.zip
Figure 2—figure supplement 1
Characterization of interactions between CI inhibition and SDH status.
(A) Mitochondrial oxygen consumption rates in WT 143B cells pretreated with vehicle (DMSO) or 5 µM AA5 for 4 hr, then injected with vehicle (DMSO) or 5 µM AA5 as indicated, followed by treatment of …
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Figure 2—figure supplement 1—source data 1
Proliferation rates, metabolite levels by LCMS, and mitochondrial oxygen consumption values in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig2-figsupp1-data1-v3.zip
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Figure 2—figure supplement 1—source data 2
Uncropped western blot for Figure 2—figure supplement 1D and I.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig2-figsupp1-data2-v3.zip
Figure 3 with 2 supplements
CI inhibition decreases mitochondrial NAD+/NADH, which is required for aspartate synthesis and proliferation in SDH-impaired cells.
(A) Whole cell NAD+/NADH measured by LCMS metabolomics of 143B cells cultured in DMEM with 1 mM PYR and treated with vehicle (DMSO), 50 nM rotenone, 5 µM AA5, or 5 µM AA5 and 50 nM rotenone for 6 hr …
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Figure 3—source data 1
Proliferation rates, relative metabolite levels by LCMS, and enzymatic assay values in Figure 3.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig3-data1-v3.zip
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Figure 3—source data 2
Uncropped western blot for Figure 3D.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig3-data2-v3.zip
Figure 3—figure supplement 1
Effects of ETC inhibition in SDH-deficient cells.
(A) Schematic showing the metabolic roles of the electron transport chain and ATP synthase and depicting the sites of action for rotenone, AA5, antimycin A, hypoxia, and oligomycin. (B) …
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Figure 3—figure supplement 1—source data 1
Proliferation rates and metabolite levels by LCMS in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig3-figsupp1-data1-v3.zip
Figure 3—figure supplement 2
Effects of PDK inhibition in SDH-deficient cells.
(A) Schematic showing the mechanism of action of AZD7545 to activate pyruvate dehydrogenase (PDH) by inhibition of the negative regulators of PDH, pyruvate dehydrogenase kinases (PDKs). (B) …
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Figure 3—figure supplement 2—source data 1
Proliferation rates, relative metabolite levels by LCMS, and mitochondrial oxygen consumption values in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig3-figsupp2-data1-v3.zip
Figure 4 with 1 supplement
Reductive carboxylation and pyruvate carboxylation drive aspartate synthesis in SDH impaired cells upon CI inhibition.
(A) Schematic depicting metabolic pathway usage for isotopologue patterns of aspartate derived from U-13C glutamine. (B) Relative ion counts for all aspartate isotopologues derived from U-13C …
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Figure 4—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 4.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig4-data1-v3.zip
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Figure 4—source data 2
Uncropped western blot for Figure 4G.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig4-data2-v3.zip
Figure 4—figure supplement 1
Effects of pyruvate versus AKB in SDH and CI impaired cells.
(A) Schematic showing how the ACLY inhibitor BMS-303141 blocks a convergent step necessary for glutamine-derived aspartate synthesis by either cytosolic or mitochondrial reductive carboxylation of …
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Figure 4—figure supplement 1—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig4-figsupp1-data1-v3.zip
Figure 5 with 1 supplement
Mitochondrial pyruvate import supports alternative aspartate synthesis in SDH-impaired cells.
(A) Western blot for MPC1 and SDHB in WT 143B cells, two MPC1 KO 143B clones, and MPC1 KO 143B clone 1 with MPC1-V5 cDNA added back (AB). SDHB is used as a loading control. (B) Proliferation rates …
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Figure 5—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 5.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig5-data1-v3.zip
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Figure 5—source data 2
Uncropped western blot for Figure 5A.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig5-data2-v3.zip
Figure 5—figure supplement 1
Mitochondrial AKG production is dependent on GPT2.
(A) Model depicting three enzymes capable of mitochondrial AKG production from glutamate (GLU) and necessary cofactors. GPT2; glutamic-pyruvic transaminase 2, GDH; glutamate dehydrogenase, GOT2; …
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Figure 5—figure supplement 1—source data 1
Relative metabolite levels by LCMS in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig5-figsupp1-data1-v3.zip
Figure 6 with 1 supplement
GOT1 is required for increased aspartate synthesis in SDH impaired cells upon complex I co-inhibition.
(A) Western blot for GOT2 and Vinculin from WT 143B cells, GOT2 KO 143B cells, and the same GOT2 KO clone expressing GOT2 cDNA (AB). Vinculin is the loading control. (B) Proliferation rates of GOT2 …
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Figure 6—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 6.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig6-data1-v3.zip
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Figure 6—source data 2
Uncropped western blots for Figure 6A and E.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig6-data2-v3.zip
Figure 6—figure supplement 1
Contributions of components of the malate-aspartate shuttle to alternative aspartate synthesis in SDH-deficient cells.
(A) Proliferation rates of GOT2 KO and GOT2 AB 143B cells in pyruvate-free DMEM (n=3). (B) Relative malate levels by LCMS metabolomics of GOT1 KO 143B cells compared to GOT1 AB 143B cells, treated …
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Figure 6—figure supplement 1—source data 1
Proliferation rates and relative metabolite levels by LCMS in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig6-figsupp1-data1-v3.zip
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Figure 6—figure supplement 1—source data 2
Uncropped western blot for Figure 6—figure supplement 1D.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig6-figsupp1-data2-v3.zip
Figure 7 with 1 supplement
Adaptive CI activity loss supports proliferation in SDHB-null cells.
(A) Schematic showing how SDHB KO 143B clones were used to generate SDHB addback cells (AB), early passage SDHB KO cells (EP), or late passage (LP) SDHB KO cells. (B) Proliferation rates of …
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Figure 7—source data 1
Proliferation rates, relative metabolite levels by LCMS, volcano plot data, and complex I activity values in Figure 7.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig7-data1-v3.zip
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Figure 7—source data 2
Uncropped western blot for Figure 7I.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig7-data2-v3.zip
Figure 7—figure supplement 1
Characterization of adaptations in SDHB KO cells.
(A) Western blot for SDHB and tubulin from WT and SDHB KO HEK293T cells. Tubulin is used as a loading control. (B) Proliferation rates of SDHB KO HEK293T cells cultured in DMEM with 1 mM pyruvate …
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Figure 7—figure supplement 1—source data 1
Proliferation rates in Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig7-figsupp1-data1-v3.zip
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Figure 7—figure supplement 1—source data 2
Uncropped western blot for Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig7-figsupp1-data2-v3.zip
Figure 8 with 1 supplement
Complex I activity is deleterious in SDHB-mutant renal cell carcinoma cells.
(A) Western blot for NDUFA8 and tubulin from WT 143B, WT UOK269, and late-passage (LP) SDHB UOK269 cells. Tubulin is used as a loading control. (B) Mitochondrial oxygen consumption rate of WT UOK269 …
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Figure 8—source data 1
Proliferation rates, mitochondrial oxygen consumption values, complex I activity values, relative metabolite levels by LCMS, and tumor volumes in Figure 8.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig8-data1-v3.zip
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Figure 8—source data 2
Uncropped western blot for Figure 8A.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig8-data2-v3.zip
Figure 8—figure supplement 1
Characterization of WT and SDHB UOK269 cells.
(A) Complex I activity assay of extracts from WT 143Bs and WT UOK269 cells (n=3). (B) Fractional isotopologue distribution of aspartate measured by LCMS metabolomics of WT or SDHB UOK269 cells …
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Figure 8—figure supplement 1—source data 1
Proliferation rates, relative metabolite levels by LCMS, fractional isotopologue distribution values, and mitochondrial oxygen consumption values in Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/78654/elife-78654-fig8-figsupp1-data1-v3.zip
Author response image 1
Quantitative analysis of aspartate detection by LCMS.
(A) Detection of isotopically labeled aspartate by LCMS across a standard curve of aspartate levels. (B) Measurement of aspartate from 143B cells treated with vehicle, 50 nM rotenone, or 5 µM AA5, …
Author response image 2
Comparing relative changes in NAD+/NADH when adding pyruvate and/or rotenone, across studies.
NAD+/NADH measurements from the indicated figure panels in Gui et al. 2016 or this revised manuscript, with each made relative to vehicle treated cells cultured in pyruvate-free DMEM.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/78654/elife-78654-mdarchecklist1-v3.pdf
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Supplementary file 1
Ion counts from each metabolomics experiment conducted in this study.
Organized by tab for each figure; tables include cell line, treatment, and metabolite measured.
- https://cdn.elifesciences.org/articles/78654/elife-78654-supp1-v3.xlsx
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Supplementary file 2
Mitochondrial proteomics dataset used to generate Figure 7E-G, Figure 7—figure supplement 1F.
- https://cdn.elifesciences.org/articles/78654/elife-78654-supp2-v3.xlsx
