GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway
- Max Planck Institute for Biology of Ageing, Germany
- JLP Health GmbH, Austria
- University of Cologne, Germany
- Austrian Academy of Sciences, Austria
- Altos Labs, United Kingdom
Abstract
The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP‑GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N‑acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP‑GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.
Data availability
Structural data reported in this study have been deposited in the Protein Data Bank with the accession codes 7NUT [https://doi.org/10.2210/pdb7NUT/pdb] and 7NUU [https://doi.org/10.2210/pdb7NUU/pdb].
Article and author information
Author details
Martin Sebastian Denzel
Funding
Bundesministerium für Bildung und Forschung (01GQ1423A EndoProtect)
- Sabine Ruegenberg
Bundesministerium für Bildung und Forschung (01GQ1423A EndoProtect)
- Stephan Miethe
Bundesministerium für Bildung und Forschung (01GQ1423A EndoProtect)
- Martin Sebastian Denzel
Deutsche Forschungsgemeinschaft (73111208-SFB 829)
- Ulrich Baumann
Deutsche Forschungsgemeinschaft (73111208-SFB 829)
- Martin Sebastian Denzel
H2020 European Research Council (ERC-2014-StG-640254-MetAGEn)
- Martin Sebastian Denzel
Max Planck Institute for Biology of Ageing (Open Access Funding)
- Virginia Kroef
Deutsche Forschungsgemeinschaft (SCHE1562/8-1)
- Bernhard Schermer
Deutsche Forschungsgemeinschaft (SFB1403, project number 414786233, A09)
- Bernhard Schermer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures have been performed in our specialized facility, followed all relevant animal welfare guidelines and regulations, and were approved by LANUV NRW 84-02.04.2015.A025.
Copyright
© 2022, Kroef 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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GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway
eLife 11:e69223.
https://doi.org/10.7554/eLife.69223
Further reading
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- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
