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Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

ABSTRACT: Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.

SUBMITTER: Ruegenberg S

PROVIDER: S-EPMC7000685 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

Ruegenberg Sabine S Horn Moritz M Pichlo Christian C Allmeroth Kira K Baumann Ulrich U Denzel Martin S MS

Nature communications 20200204 1

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in ma ...[more]

PMID: 32019926

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Project description:BackgroundThe antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.ResultsWe determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the ?-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells.ConclusionsThe present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.

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Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

Publications

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

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