Succinate Dehydrogenase loss causes cascading metabolic effects that impair pyrimidine biosynthesis

Madeleine L. Hart, Kristian Davidsen, Serwah Danquah, Eric Zheng, David Sokolov, Lucas B. Sullivan

Posted on: 26 May 2025

Preprint posted on 19 February 2025

Not all aspartate limitation is created equal: An aspartate biosensor reveals that inhibiting succinate dehydrogenase results in defects in pyrimidine synthesis and replication stress, unique from other methods of aspartate limitation.

Selected by Hannah Pletcher

Categories: biochemistry, cancer biology, cell biology

A) General model of aspartate limitation established using aspartate biosensor; Fig. 1f of preprint. B) Model of specific aspartate limitation by SDH inhibition, which leads to pyrimidine synthesis impairment; Fig. 3a of preprint. Figures adapted from Hart et al., 2025 on bioRxiv; preprint is available under a CC-BY 4.0 International license.

Background:

While most of us think of amino acids as the building blocks for proteins, the amino acid aspartate also importantly fuels the synthesis of other amino acids and nucleotides. Recent work has shown that limiting aspartate by various pharmacological, genetic, and nutrient methods results in decreased cell proliferation (Birsoy et al., 2015; Hart et al., 2023; Sullivan et al., 2015), but the mechanism underlying this phenotype remained unclear. Hart and colleagues used a biosensor tool to obtain more detailed measurements of aspartate levels and cell proliferation over time compared to traditional mass spectrometry approaches. Using this novel method, the authors established a model for the effect of aspartate limitation on cell proliferation. In doing so, they uncovered that aspartate limitation by succinate dehydrogenase (SDH) inhibition causes defects in pyrimidine biosynthesis, leading to replication stress.

Key Findings:

Aspartate biosensor enables generation of aspartate limitation model: Using an aspartate biosensor (jAspSnFR3) and matched nuclear localized red fluorescent protein (NucRFP), the authors measured aspartate and proliferation with two methods of aspartate limitation: pharmacological limitation with a Complex I inhibitor (rotenone) and genetic limitation when GOT1/2 were knocked out and the cells were not supplemented with excess aspartate. These experiments showed that aspartate levels rapidly decreased within the first 24 hours of aspartate limitation. After 24 hours, a steady state was reached at a lower concentration of aspartate. Cell proliferation did not decrease until after 24 hours, suggesting a model in which consumption of aspartate (i.e., through proliferation) is only affected once a certain low threshold of aspartate is reached. Once the levels of aspartate get this low, a new steady state of aspartate acquisition and consumption is established.
SDH inhibition results in aspartate rebound: The authors tested a different method of aspartate limitation: pharmacological inhibition of SDH (Complex II) by Atpenin A5 (AA5). Here, a rebound in the aspartate level was seen after hitting the lowest level around 24 hours. The same rebound was not observed with cell proliferation, motivating the research team to further look into this.
SDH inhibition hinders pyrimidine synthesis: Since aspartate can build amino acids, proteins, and nucleotides, the authors tested the metabolic fates of aspartate with SDH inhibition to see if AA5 treatment decreased aspartate consumption into one of these fates, resulting in the aspartate rebound. Hart and colleagues found that defects in pyrimidine biosynthesis were apparent when cells were treated with AA5. When they added exogenous uridine (a pyrimidine) to these cells, the aspartate rebound was lost. This means that SDH inhibition impairs pyrimidine synthesis.
SDH inhibition accumulates succinate, inhibiting ATCase and therefore pyrimidine synthesis: How does SDH inhibition impair pyrimidine synthesis? Uridine nucleotides are a negative regulator of aspartate transcarbamoylase (ATCase) (Shin et al., 2023), suggesting that SDH inhibition impairs pyrimidine synthesis at this step. Bacterial ATCase is inhibited by succinate (Foote et al., 1985), and succinate accumulates when SDH is inhibited. When succinate accumulation was blocked by cotreatment of AA5 and rotenone, the aspartate decline was slowed and the aspartate rebound was reduced. Overall, aspartate rebounds when SDH is inhibited because succinate is inhibiting ATCase, combined with lower aspartate levels, resulting in limited pyrimidine synthesis.
Pyrimidine synthesis defects from SDH inhibition result in replication stress: Finally, the authors tested whether the pyrimidine synthesis impairment resulting from SDH inhibition led to replication stress. Indeed, they observed increases in cell volume, decreases in proliferation, and Chk1/2 phosphorylation, indicating ATR-dependent replication stress.

What I liked/why this new work is important:

This preprint is important both in terms of methodology and biological findings. The authors demonstrate that kinetic measurements of metabolism, such as with a fluorescent biosensor, can be advantageous compared to end point measurements like mass spectrometry. In addition, this preprint establishes a model for how aspartate limitation affects cell proliferation and highlights that distinct methods of aspartate limitation behave differently. This work is important not only for basic understanding of metabolism, but it also informs potential therapeutic strategies for cancers with mutated SDH. I appreciated the rigor and breadth of the study. Overall, the authors’ focus on method development and mechanistic biochemistry makes this preprint a very satisfying read.

Future Directions and Questions for the Authors:

When investigating how the metabolic fates of aspartate were affected by aspartate limitation, I found it compelling that GOT1/2 DKO resulted in defects in tRNA charging. Even though the changes in cell proliferation were minor, is this a phenotype you plan to investigate in the future?
I found the lack of study of mammalian ATCase striking. Do you think it would be more beneficial to study the enzymology of mammalian ATCase in vitro or the impact of other in vivo factors on ATCase activity, or both?
I really appreciated the rigor in testing if general TCA cycle dysfunction was sufficient to replicate the SDH inhibition phenotypes. Why did you choose FH KO as your model of TCA cycle dysfunction? Do you anticipate that other TCA cycle KO lines would behave similarly?

References:

Birsoy, K., Wang, T., Chen, W. W., Freinkman, E., Abu-Remaileh, M., & Sabatini, D. M. (2015). An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis. Cell, 162(3), 540–551. https://doi.org/10.1016/j.cell.2015.07.016

Foote, J., Lauritzen, A. M., & Lipscomb, W. N. (1985). Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate. The Journal of Biological Chemistry, 260(17), 9624–9629.

Hart, M. L., Davidsen, K., Danquah, S., Zheng, E., Sokolov, D., & Sullivan, L. B. (2025). Succinate Dehydrogenase loss causes cascading metabolic effects that impair pyrimidine biosynthesis. BioRxiv, 2025.02.18.638948. https://doi.org/10.1101/2025.02.18.638948

Hart, M. L., Quon, E., Vigil, A.-L. B., Engstrom, I. A., Newsom, O. J., Davidsen, K., Hoellerbauer, P., Carlisle, S. M., & Sullivan, L. B. (2023). Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells. ELife, 12, e78654. https://doi.org/10.7554/eLife.78654

Shin, J., Mir, H., Khurram, M. A., Fujihara, K. M., Dynlacht, B. D., Cardozo, T. J., & Possemato, R. (2023). Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle. Nature Metabolism, 5(2), 277–293. https://doi.org/10.1038/s42255-023-00735-9

Sullivan, L. B., Gui, D. Y., Hosios, A. M., Bush, L. N., Freinkman, E., & Vander Heiden, M. G. (2015). Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell, 162(3), 552–563. https://doi.org/10.1016/j.cell.2015.07.017

Tags: aspartate, biosensor, complex ii, metabolism, proliferation, pyrimidine synthesis, replication stress, succinate dehydrogenase

doi: https://doi.org/10.1242/prelights.40560

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Author's response

Lucas Sullivan shared

When investigating how the metabolic fates of aspartate were affected by aspartate limitation, I found it compelling that GOT1/2 DKO resulted in defects in tRNA charging. Even though the changes in cell proliferation were minor, is this a phenotype you plan to investigate in the future?

I agree that it is a thought-provoking result. To me there are two intriguing aspects: 1) That the effects are unevenly distributed across compartments and aspartate-related tRNA species, and 2) That cells can maintain proliferation while some proportion of tRNA species remain uncharged. For the latter point, my intuition would have been that any uncharged tRNA would have halted biosynthesis, but apparently that is not the case. There are some well-described effects of uncharged tRNAs (e.g. ribosomal collisions and integrated stress response signaling), so these data raise questions about whether these uncharged tRNA species impact cell signaling and cell function. While it is not in our immediate plans, I think it would be fascinating to understand how different tRNA variables – transcripts, base modifications, anticodon sequence, amino acid substrate, cellular compartment, and degree of aminoacylation – all influence the response to uncharged tRNAs at the signaling and functional level.

I found the lack of study of mammalian ATCase striking. Do you think it would be more beneficial to study the enzymology of mammalian ATCase in vitro or the impact of other in vivo factors on ATCase activity, or both?

We completely agree that it is a worthwhile goal to evaluate the purported mechanism of succinate inhibition of mammalian ATCase with enzymology studies. We did not tackle this specific question in this draft in the interest of communicating our findings as quickly as possible, but it is one of many next steps that we would love to pursue given enough time and resources. One important aspect of peer review is that expert colleagues can help us to focus our next experiments on those that they find most important for addressing study limitations and corroborating our most impactful findings. Whether that includes this specific experiment is up to the reviewers to decide!

I really appreciated the rigor in testing if general TCA cycle dysfunction was sufficient to replicate the SDH inhibition phenotypes. Why did you choose FH KO as your model of TCA cycle dysfunction? Do you anticipate that other TCA cycle KO lines would behave similarly?

Thank you! Our primary model is that the oxidative TCA cycle is a major route of aspartate synthesis and so disruption to any step in that pathway would presumably similarly cause aspartate limitation. That said, SDH inhibition is a bit unique since it has the added consequence of succinate accumulation, which we show competes with aspartate utilization at ATCase to further impact de novo pyrimidine synthesis. We chose FH KO as a secondary model of TCA cycle dysfunction because it also blocks for oxidative TCA cycle function but does not result in the same magnitude of succinate accumulation as SDH loss, allowing us to uncouple the effects of aspartate synthesis inhibition from succinate-mediated inhibition on ATCase activity. Whether or not other the disruption of other oxidative TCA cycle components (OGDH, SCS, MDH2, GOT2) would behave identically to FH loss is unclear. However, given my experience working in cell metabolism, my guess is they would not be identical and would instead tempt us into trying to understand all the fascinating heterogeneous responses to mitochondrial metabolic perturbations!

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