Abstract

The Tricarboxylic Acid Cycle (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis.

Data availability

All the transcriptomics. proteomics and uncropped blots data have been deposited in Dryad.

The following data sets were generated
    1. Ryan D
    (2021) Label-free proteomics - thenoyltrifluoroacetone (TTFA)
    Dryad Digital Repository, doi:10.5061/dryad.h44j0zpkt.
    1. Ryan D
    (2021) TruSeq stranded mRNA - Atpenin A5 (AA5)
    Dryad Digital Repository, doi:10.5061/dryad.08kprr536.
    1. Ryan D
    (2021) Western blot uncropped blots
    Dryad Digital Repository, doi:10.5061/dryad.08kprr537.

Article and author information

Author details

  1. Dylan Gerard Ryan

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Ming Yang

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Hiran A Prag

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Giovanny Rodriguez Blanco

    Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Efterpi Nikitopoulou

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Marc Segarra-Mondejar

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Christopher A Powell

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7501-0586
  8. Tim Young

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1831-3473
  9. Nils Burger

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Jan Lj Miljkovic

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Michal Minczuk

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Michael P Murphy

    MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1115-9618
  13. Alex von Kriegsheim

    Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Christian Frezza

    MRC Cancer Unit, Hutchison MRC Research Centre, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    cf366@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3293-7397

Funding

Medical Research Council (MRC_MC_UU_12022/6.)

  • Christian Frezza

H2020 European Research Council (ERC819920)

  • Dylan Gerard Ryan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Matthew G Vander Heiden, Massachusetts Institute of Technology, United States

Version history

  1. Preprint posted: July 27, 2021 (view preprint)
  2. Received: July 28, 2021
  3. Accepted: December 21, 2021
  4. Accepted Manuscript published: December 23, 2021 (version 1)
  5. Version of Record published: January 6, 2022 (version 2)
  6. Version of Record updated: February 2, 2022 (version 3)

Copyright

© 2021, Ryan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Dylan Gerard Ryan
  2. Ming Yang
  3. Hiran A Prag
  4. Giovanny Rodriguez Blanco
  5. Efterpi Nikitopoulou
  6. Marc Segarra-Mondejar
  7. Christopher A Powell
  8. Tim Young
  9. Nils Burger
  10. Jan Lj Miljkovic
  11. Michal Minczuk
  12. Michael P Murphy
  13. Alex von Kriegsheim
  14. Christian Frezza
(2021)
Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism
eLife 10:e72593.
https://doi.org/10.7554/eLife.72593

Share this article

https://doi.org/10.7554/eLife.72593

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    Chi-Ning Chuang, Hou-Cheng Liu ... Ting-Fang Wang
    Research Article

    Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

    1. Biochemistry and Chemical Biology
    2. Developmental Biology
    Sima Stroganov, Talia Harris ... Michal Neeman
    Research Article Updated

    Background:

    Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.

    Methods:

    Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry.

    Results:

    We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation.

    Conclusions:

    This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.

    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.