GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

  1. Virginia Kroef
  2. Sabine Ruegenberg
  3. Moritz Horn
  4. Kira Allmeroth
  5. Lena Ebert
  6. Seyma Bozkus
  7. Stephan Miethe
  8. Ulrich Elling
  9. Bernhard Schermer
  10. Ulrich Baumann
  11. Martin Sebastian Denzel  Is a corresponding author
  1. Max Planck Institute for Biology of Ageing, Germany
  2. JLP Health GmbH, Austria
  3. University of Cologne, Germany
  4. Austrian Academy of Sciences, Austria
  5. 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].

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Virginia Kroef

    Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3695-911X
  2. Sabine Ruegenberg

    Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5292-9610
  3. Moritz Horn

    JLP Health GmbH, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Kira Allmeroth

    Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2659-6776
  5. Lena Ebert

    University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Seyma Bozkus

    University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Stephan Miethe

    Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Ulrich Elling

    Vienna Biocenter, Austrian Academy of Sciences, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  9. Bernhard Schermer

    University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Ulrich Baumann

    University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Martin Sebastian Denzel

    Altos Labs, Cambridge, United Kingdom
    For correspondence
    mdenzel@altoslabs.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5691-3349

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.

Reviewing Editor

  1. Hening Lin, Cornell University, United States

Publication history

  1. Received: April 8, 2021
  2. Preprint posted: April 23, 2021 (view preprint)
  3. Accepted: February 28, 2022
  4. Accepted Manuscript published: March 1, 2022 (version 1)
  5. Accepted Manuscript updated: March 3, 2022 (version 2)
  6. Version of Record published: March 31, 2022 (version 3)

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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  1. Virginia Kroef
  2. Sabine Ruegenberg
  3. Moritz Horn
  4. Kira Allmeroth
  5. Lena Ebert
  6. Seyma Bozkus
  7. Stephan Miethe
  8. Ulrich Elling
  9. Bernhard Schermer
  10. Ulrich Baumann
  11. Martin Sebastian Denzel
(2022)
GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway
eLife 11:e69223.
https://doi.org/10.7554/eLife.69223
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