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.
Reviewing Editor
- Hening Lin, Cornell University, United States
Version history
- Received: April 8, 2021
- Preprint posted: April 23, 2021 (view preprint)
- Accepted: February 28, 2022
- Accepted Manuscript published: March 1, 2022 (version 1)
- Accepted Manuscript updated: March 3, 2022 (version 2)
- 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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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
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The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
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Background: High levels of circulating adiponectin are associated with increased insulin sensitivity, low prevalence of diabetes, and low body mass index (BMI); however, high levels of circulating adiponectin are also associated with increased mortality in the 60-70 age group. In this study, we aimed to clarify factors associated with circulating high-molecular-weight (cHMW) adiponectin levels and their association with mortality in the very old (85-89 years old) and centenarians.
Methods: The study included 812 (women: 84.4%) for centenarians and 1,498 (women: 51.7%) for the very old. The genomic DNA sequence data were obtained by whole genome sequencing or DNA microarray-imputation methods. LASSO and multivariate regression analyses were used to evaluate cHMW adiponectin characteristics and associated factors. All-cause mortality was analyzed in three quantile groups of cHMW adiponectin levels using Cox regression.
Results: The cHMW adiponectin levels were increased significantly beyond 100 years of age, were negatively associated with diabetes prevalence, and were associated with SNVs in CDH13 (p = 2.21 × 10-22) and ADIPOQ (p = 5.72 × 10-7). Multivariate regression analysis revealed that genetic variants, BMI, and high-density lipoprotein cholesterol (HDLC) were the main factors associated with cHMW adiponectin levels in the very old, whereas the BMI showed no association in centenarians. The hazard ratios for all-cause mortality in the intermediate and high cHMW adiponectin groups in very old men were significantly higher rather than those for all-cause mortality in the low level cHMW adiponectin group, even after adjustment with BMI. In contrast, the hazard ratios for all-cause mortality were significantly higher for high cHMW adiponectin groups in very old women, but were not significant after adjustment with BMI.
Conclusions: cHMW adiponectin levels increased with age until centenarians, and the contribution of known major factors associated with cHMW adiponectin levels, including BMI and HDLC, varies with age, suggesting that its physiological significance also varies with age in the oldest old.
Funding: This study was supported by grants from the Ministry of Health, Welfare, and Labour for the Scientific Research Projects for Longevity; a Grant-in-Aid for Scientific Research (No 21590775, 24590898, 15KT0009, 18H03055, 20K20409, 20K07792, 23H03337) from the Japan Society for the Promotion of Science; Keio University Global Research Institute (KGRI), Kanagawa Institute of Industrial Science and Technology (KISTEC), Japan Science and Technology Agency (JST) Research Complex Program 'Tonomachi Research Complex' Wellbeing Research Campus: Creating new values through technological and social innovation (JP15667051), the Program for an Integrated Database of Clinical and Genomic Information from the Japan Agency for Medical Research and Development (No. 16kk0205009h001, 17jm0210051h0001, 19dk0207045h0001); the medical-welfare-food-agriculture collaborative consortium project from the Japan Ministry of Agriculture, Forestry, and Fisheries; and the Biobank Japan Program from the Ministry of Education, Culture, Sports, and Technology.
