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.
All the transcriptomics. proteomics and uncropped blots data have been deposited in Dryad.
Label-free proteomics - thenoyltrifluoroacetone (TTFA)Dryad Digital Repository, doi:10.5061/dryad.h44j0zpkt.
Label-free proteomics - fumarate hydratase inhibitor (FHIN-1)Dryad Digital Repository, doi:10.5061/dryad.fttdz08t9.
TruSeq stranded mRNA - Atpenin A5 (AA5)Dryad Digital Repository, doi:10.5061/dryad.08kprr536.
TruSeq stranded mRNA_Fumarate hydratase inhibitor (FHIN-1)Dryad Digital Repository, doi:10.5061/dryad.bk3j9kdcq.
Western blot uncropped blotsDryad Digital Repository, doi:10.5061/dryad.08kprr537.
- Christian Frezza
- Dylan Gerard Ryan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Matthew G Vander Heiden, Massachusetts Institute of Technology, United States
© 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.
The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.
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