The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

Longevity is often associated with stress resistance, but whether they are causally linked is incompletely understood. Here we investigate chemosensory defective Caenorhabditis elegans mutants that are long-lived and stress resistant. We find that mutants in the intraflagellar transport protein gene osm-3 were significantly protected from tunicamycin-induced ER stress. While osm-3 lifespan extension is dependent on the key longevity factor DAF-16/FOXO, tunicamycin resistance was not. osm-3 mutants are protected from bacterial pathogens, which is pmk-1 p38 MAP kinase dependent while TM resistance was pmk-1 independent. Expression of P-glycoprotein (PGP) xenobiotic detoxification genes was elevated in osm-3 mutants and their knockdown or inhibition with verapamil suppressed tunicamycin resistance. The nuclear hormone receptor nhr-8 was necessary to regulate a subset of PGPs. We thus identify a cell-nonautonomous regulation of xenobiotic detoxification and show that separate pathways are engaged to mediate longevity, pathogen resistance, and xenobiotic detoxification in osm-3 mutants.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and through phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the relative positioning of the two GFAT-1 domains to control its activity. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response, is implicated in cancer and diabetes, and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the first and rate-limiting HP enzyme. GFAT-1 activity is modulated through UDP-GlcNAc feedback inhibition and by kinase signaling, including Ser205 phosphorylation by protein kinase A (PKA). The consequence and molecular mechanism of GFAT-1 phosphorylation, however, remains poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans, leading to ER stress resistance. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and PKA-mediated Ser205 phosphorylation. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, GFAT-1 phosphorylation by PKA uncoupled the feedback loop of the HP and, depending on UDP-GlcNAc availability, phosphorylation by PKA results in lower or enhanced GFAT-1 activity in vivo. Mechanistically, our data indicate that the relative positioning of the two GFAT-1 domains might be affected by phosphorylation and we propose a model how Ser205 phosphorylation modulates the activity and feedback inhibition of GFAT-1.

27Longevity is often associated with stress resistance, but whether they are causally 28 linked is incompletely understood. Here we investigate chemosensory defective 29Caenorhabditis elegans mutants that are long-lived and stress resistant. We find that 30 mutants in the intraflagellar transport protein gene osm-3 are completely protected 31 from tunicamycin-induced ER stress. While osm-3 lifespan extension is fully dependent 32 on the key longevity factor DAF-16/FOXO, tunicamycin resistance is not. osm-3 33 mutants are protected from bacterial pathogens, which is fully pmk-1 p38 MAP kinase 34 dependent. Transcriptomic analysis revealed enhanced expression of P-glycoprotein 35 xenobiotic detoxification genes in osm-3 mutants and chemical inhibition of P-36 glycoproteins with verapamil suppressed tunicamycin resistance. Of note, the nuclear 37 hormone receptor nhr-8 is necessary and sufficient to regulate P-glycoproteins and 38 tunicamycin resistance. We thus identify a cell-nonautonomous regulation of 39 xenobiotic detoxification and show that separate pathways are engaged to mediate 40 longevity, pathogen resistance, and xenobiotic detoxification in osm-3 mutants. 41