Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism
- University of Cambridge, United Kingdom
- University of Edinburgh, United Kingdom
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.
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Label-free proteomics - thenoyltrifluoroacetone (TTFA)Dryad Digital Repository, doi:10.5061/dryad.h44j0zpkt.
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Label-free proteomics - fumarate hydratase inhibitor (FHIN-1)Dryad Digital Repository, doi:10.5061/dryad.fttdz08t9.
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TruSeq stranded mRNA - Atpenin A5 (AA5)Dryad Digital Repository, doi:10.5061/dryad.08kprr536.
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TruSeq stranded mRNA_Fumarate hydratase inhibitor (FHIN-1)Dryad Digital Repository, doi:10.5061/dryad.bk3j9kdcq.
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Western blot uncropped blotsDryad Digital Repository, doi:10.5061/dryad.08kprr537.
Article and author information
Author details
Christopher A Powell
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
- Matthew G Vander Heiden, Massachusetts Institute of Technology, United States
Publication history
- Preprint posted: July 27, 2021 (view preprint)
- Received: July 28, 2021
- Accepted: December 21, 2021
- Accepted Manuscript published: December 23, 2021 (version 1)
- Version of Record published: January 6, 2022 (version 2)
- 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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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
Further reading
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- Structural Biology and Molecular Biophysics
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Mitochondrial ATP production in cardiac ventricular myocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local ventricular myocyte metabolic signaling. However, these two regulatory systems do not fully account for the physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product of the oxidation of nutrients that powers ATP production. It is a lipid soluble gas that rapidly permeates the inner mitochondrial membrane (IMM) and produces bicarbonate (HCO3-) in a reaction accelerated by carbonic anhydrase (CA). The bicarbonate level is tracked physiologically by a bicarbonate-activated adenylyl cyclase, soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular myocytes where it generates cAMP when activated by HCO3-. Our data strongly suggest that ATP production in these mitochondria is regulated by this cAMP signaling cascade operating within the inter-membrane space (IMS) by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein 1). Thus, mitochondrial ATP production is shown to be increased by bicarbonate-triggered sAC signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the cardiac mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. We propose that the bicarbonate and calcium signaling arms function in a resonant or complementary manner to match mitochondrial ATP production to the full range of energy consumption in cardiac ventricular myocytes in health and disease.
