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Hexosamine pathway and (ER) protein quality control
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ScienceDirect Hexosamine pathway and (ER) protein quality control Martin S Denzel1 and Adam Antebi1,2,3 Aminosugars produced in the hexosamine pathway (HP) are utilized in protein glycosylation reactions involved in protein maturation and cellular signaling. Recent evidence revealed a role of the HP in protein quality control and ageing. Elevation of the HP product UDP-N-acetylglucosamine in the nematode Caenorhabditis elegans results in resistance towards toxic aggregation-prone proteins, and extended lifespan. Glutamine-fructose 6 phosphate aminotransferase (GFAT-1), the HP’s key enzyme, is a target of the unfolded protein response (UPR). Thus, cardiac stress in mice results in GFAT-1 activation that triggers a cytoprotective response. Feeding of glucosamine to aged mice increases their life expectancy.Here we discuss HP activation and cellular protein quality control mechanisms that result in stress resistance and suppression of age-related proteotoxicity. Addresses 1 Max Planck Institute for Biology of Ageing, Joseph-Stelzmann-Str. 9b, 50931 Cologne, Germany 2 Department of Molecular and Cellular Biology, Huffington Center on Aging Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA 3 Cologne Excellence Cluster on Cellular Stress Responses in AgingAssociated Diseases (CECAD), University of Cologne, 50674 Cologne, Germany Corresponding author: Antebi, Adam ([email protected])
Current Opinion in Cell Biology 2015, 33:14–18 This review comes from a themed issue on Cell regulation Edited by Johan Auwerx and Jodi Nunnari
http://dx.doi.org/10.1016/j.ceb.2014.10.001 0955-0674/# 2015 Published by Elsevier Ltd.
The HP is a central integrator of energy status The HP is comprised of a series of anabolic reactions that generate the activated aminosugar UDP-N-acetylglucosamine (UDP-GlcNAc). The HP is controlled by its first and rate-limiting enzyme GFAT-1, which converts fructose 6-phosphate and glutamine into glucosamine 6-phosphate and glutamate. Subsequent enzymatic steps then lead to acetylation and activation using UTP to produce UDP-GlcNAc. Thus, the HP is positioned to sample all major components of central energy metabolism, including glucose, fatty acid, amino acid metabolism, aerobic respiration, and, through UTP, energy availability (Figure 1). Current Opinion in Cell Biology 2015, 33:14–18
Consistent with a role in integrating various aspects of energy metabolism, the HP’s key enzyme GFAT-1 is not only regulated by product inhibition through UDPGlcNAc [1,2], but also by the major energy sensing AMP-activated protein kinase (AMPK) through inhibitory phosphorylation [3]. Further, GFAT-1 kinases include protein kinase A (PKA) and Ca2+/calmodulindependent protein kinase (CaMKII) whose physiological effects on GFAT-1 activity remain unclear and appear to depend on cellular context and isoform expression [4].
Posttranslational modifications involving GlcNAc UDP-GlcNAc is an important substrate for at least four types of glycosylation reactions that fulfill diverse cellular functions. First, N-glycosylation is an ER-specific posttranslational modification process during which a highmannose glycan is preassembled on the lipid carrier dolichol phosphate and then transferred en bloc to consensus Asn-X-Ser/Thr of nascent ER proteins. UDPGlcNAc is the substrate for the first two critical biosynthetic steps in the preassembly of this glycan structure. Nglycosylation plays a major role in the structure and function of cell surface and secreted proteins. In addition, N-glycans are involved in protein maturation in the ER and signal a substrate proteins’ folding state [5]. Notably, inhibition of N-glycosylation by drugs such as tunicamycin triggers massive protein misfolding stress. Second, proteins undergo numerous described types of O-linked glycosylation, the most abundant form being mucin-type O-glycosylation [6]. This reaction occurs in the Golgi apparatus and, unlike N-glycosylation, involves the step-by-step addition of sugars. UDP-N-acetylgalactosamine (UDP-GalNAc), which is produced from UDPGlcNAc by the epimerase GALE-1 , is used in the first two steps of mucin type O-glycosylation. GalNAc based O-glycosylation has various functions, including protein processing and folding [7]. Third, O-GlcNAcylation involves the addition of a single b-D-N-acetylglucosamine moiety to serine or threonine residues of target proteins. This posttranslational modification has been suggested to have regulatory functions, and many substrate proteins have been identified. It has been shown that O-GlcNAcylation interacts with phosphorylation to regulate client protein activity [8]. Finally, GlcNAc contributes to major biomolecules across nature. Chitin, a long-chain polymer of GlcNAc molecules synthetized from UDP-GlcNAc, is the most abundant biopolymer in marine environments [9]. Additionally, GlcNAc is a major component of the peptidoglycan layer of the bacterial cell wall. www.sciencedirect.com
Hexosamine pathway and (ER) protein quality control Denzel and Antebi 15
Figure 1
ER-stress/UPR N-glycosylation Glucose
F6-P O-GlcNAcylation
ATP
GlcN 6-P
Amino Acids
GlcNAc 6-P
Acetyl CoA
GlcNAc 1-P
UTP
UDP-GlcNAc UDP-GalNAc
O-glycosylation Chitin biosynthesis Peptidoglycan synthesis
UDP-GlcNAc
Others? Energy input
Integration in HP
Output, posttranscriptional modifications Current Opinion in Cell Biology
The HP integrates metabolic status from a number of central metabolism intermediates. This includes glycolysis, fatty acid and amino acid metabolism, aerobic respiration, and, through UTP, energy availability. These precursors are used to build up UDP-GlcNAc and UDP-GalNAc, which subsequently are involved in a number of glycosylation reactions. These posttranslational modifications regulate protein folding, sorting, and cellular signaling. As a precursor for chitin and glycosaminoglycan, UDP-GlcNAc is a precursor for some of the most abundant biomolecules.
HP might be a prosurvival signal Given that HP activity requires a positive energy status and the availability of cellular biomolecules or molecular building blocks, its activity is likely to reflect beneficial environmental conditions. This raises the question how downstream N-glycosylation and O-glycosylation mediate biological responses to the environment. Indeed there is evidence that elevated O-GlcNAcylation has pro-survival function. This is true in the mouse heart, where ischemia/reperfusion stress induces O-GlcNAcylation and serves a protective role for the survival of cardiomyocytes [10]. Moreover, in cancer cells, OGlcNAcylation regulates metabolism via hypoxia-inducible factor 1 (HIF-1) and inhibition of O-GlcNAcylation leads to ER stress-induced apoptosis [11]. Consistently, elevated O-GlcNAc transferase (OGT-1) levels correlate with poor outcome in patients. Also in prostate cancer, OGT-1 is upregulated and was found to promote metastasis [12]. Finally, O-GlcNAcylation is antiapoptotic in pancreatic cancer cells [13]. Taken together, this points to a unified view that HP flux reflects the cell’s energy status and constitutes a pro-survival signal. Cancer cells, in which high glucose flux itself can be oncogenic [14], can benefit from this and optimize proliferation even under growth-induced stress. However, non-malignant cells are also protected. In the search for interventions that might slow age-related disease it will be important to www.sciencedirect.com
balance these effects to exclude the promotion of tumorigenesis.
HP activation in the context of known longevity pathways A number of lifespan extending conditions such as reduced insulin signaling, dietary restriction, and mitochondrial respiration imbalances are involved in sensing and signaling of low energy availability. These interventions extend lifespan by activating stress responses that increase organismal fitness. Does HP activation constitute yet another such stress response pathway to extend lifespan? Indeed, it was shown that ER stress activates GFAT-1 expression and UDP-GlcNAc production as part of the UPR [10], which then improves protein quality control to counter proteotoxicity and to reset ER homeostasis. This signaling is consistent with the expression of chaperones downstream of the UPR. Supporting the idea that the HP is a specific stress response triggered by ER proteotoxicity, we observed that HP-induced activation of ER-associated degradation (ERAD), proteasome activity, and autophagy all contributed to improved ER protein quality. This might occur directly via changes in glycosylation patterns within the ER. It was shown that N-glycan remodeling on certain target proteins, such as the glucagon receptor, can be the output of the HP [15], and elevation of insulin-like growth factor 1 receptor Current Opinion in Cell Biology 2015, 33:14–18
16 Cell regulation
(IGF-1R) glycosylation can affect its activity [16]. All of these observations point to a role of the HP as a protein stress response pathway (Figure 2). Conceivably, imbalanced anabolic processes might physiologically trigger stress pathways. For example, antibody producing B-cells ramp up ER stress mechanisms to cope with increased secretory demand [17]. Proper induction and dampening of such stress mechanisms would be critical to cellular homeostasis. Interestingly, the energy-stress induced AMPK is a negative regulator of GFAT-1 that suppresses HP flux when ATP becomes scarce, which is consistent with the anabolic nature of the HP. This reflects the specificity of cellular stress response pathways: energy stress will activate AMPK and suppress GFAT-1 while protein stress activates GFAT-1 and the HP. Alternative to the HP being a stress response pathway, HP activation itself might constitute cellular stress. HP activation might lead to energy imbalance by consuming metabolites, triggering a stress response. This view receives support from the observation that glucosamine (GlcN) supplementation causes a ROS response [18] that extends worm and mouse lifespan through a hormetic mechanism. To test the possibility that HP activation itself is a stress, it might be interesting to supplement with the final product of the HP, UDP-GlcNAc. In this case, none of the HP substrates would be depleted to induce a metabolic imbalance. So far, studies have only used HP intermediates such as GlcN and GlcNAc to mimic HP activation. Figure 2
toxic proteins
ERAD Proteasome
ER stress Autophagy O-glycosylation
AMPK
others?
F6P
GFAT-1
Gln
GlcN 6-P GlcNAc 6-P GlcNAc 1-P
Glu AcetylCoA
CoA
UTP
UDP-GlcNAc UDP
Current Opinion in Cell Biology
The HP is part of a stress response pathway that maintains protein homeostasis. ER protein misfolding triggers GFAT-1 transcription and activates the HP. Through incompletely understood mechanisms, subsequent UDP-GlcNAc elevation activates protein quality surveillance by inducing O-glycosylation, autophagy, ERAD, and proteasome activity. This counters ER protein misfolding stress and also alleviates toxicity caused by toxic aggregation prone proteins such as poly-glutamine peptides or Ab. Current Opinion in Cell Biology 2015, 33:14–18
How do UDP-GlcNAc levels influence protein quality control? A puzzling question that remains is how the levels of one sentinel molecule, UDP-GlcNAc, can orchestrate diverse responses to improve protein quality control. A number of mechanisms are imaginable: Changes in N-glycosylation: Given the role of Nglycosylation in protein quality control, it appears likely that enhancing this process might cause the observed effects on protein homeostasis. Enhanced Nglycosylation could globally relieve stress on the ER by mass action, and thereby unfetter other quality control systems. However, we have not found a global increase in levels of N-glycosylated proteins at steady state in Caenorhabditis elegans during HP activation [19]. Quite possibly, though, certain proteins might be over-glycosylated under these conditions to elicit the beneficial effects. Members of the ER associated degradation machinery, for example, are substrates for N-glycosylation [20]. These changes would also fulfill a signaling role as we found a coordinated improvement in protein homeostasis that included proteasome activity and autophagy. ERAD components would be optimally situated to fulfill all these functions. Consistent with this, preliminarily results indicate that an increased dose of the ERAD component SEL-1 is sufficient for lifespan extension in C. elegans [19]. Future experiments will address the role of glycosylation and ERAD activity. Elevation of O-GlcNAcylation: In accord with this hypothesis, Hill and colleagues detected increased O-GlcNAc modification levels in the ischemic heart, and inhibition of this process through OGT-1 knockdown led to decreased cellular protection[10]. Surprisingly, OGT-1 mutation in C. elegans has little effect on longevity, but it is currently unclear whether O-GlcNAcylation is truly abolished in these mutants. Cytosolic addition of GlcNAc moieties modulates the activity of a number of substrates, including signaling proteins in important pathways such as insulin signaling and the downstream transcription factor FOXO [21,22]. Thus it is possible that high energy sensing via the HP and subsequent O-GlcNAcylation might induce lifespan extension. Other prominent signaling proteins that are O-GlcNAcylated include, among others, beta catenin [23], FXR [24], NF-kB subunits [25,26], carbohydrate-responsive elementbinding protein (ChREBP) [27], as well as components of the circadian clock [28,29]. Elevation of mucin type O-glycosylation: Given the role of mucin type O-glycosylation in the secretory apparatus, it is possible that elevation of its precursor UDP-GalNAc/UDP-GlcNAc might lead to changes in the degree of glycosylation or in the abundance of substrate proteins. This might result in functional changes of substrate proteins, which www.sciencedirect.com
Hexosamine pathway and (ER) protein quality control Denzel and Antebi 17
include chemokines and regulators of cellular signaling cascades [30]. Function as an allosteric effector of other proteins: It is possible that the beneficial effects of HP metabolites arise not as a consequence of covalent post-translational modifications. Rather HP metabolites could non-covalently bind target proteins to allosterically regulate their activity. GFAT itself is modulated by such end-product inhibition [2]. Alternately, HP metabolites could serve as chemical chaperones, similar to phenylbutyric acid (PBA) or tauro-ursodeoxycholic acid (TUDCA) to stabilize protein folding. Chemical chaperones can either act broadly and interact with numerous substrates, or stabilize specific proteins [31]. Interestingly, chemical chaperones are implicated in countering ER stress [32,33]. In all of the above potential mechanisms, it will be interesting to see if single target proteins are responsible for the observed phenotypes, if there is a convergence of mechanisms, or if the phenotypes are a consequence of allover changes in glycosylation.
significance, too little is known at the mechanistic level about the molecular consequences of HP activation and elevation of UDP-GlcNAc levels. What are the major downstream events? What type of glycosylation is mostly affected and what are the major substrates that trigger downstream effects? Altogether, this makes GFAT-1 an attractive target for research into the ageing process with the goal of alleviating age-associated diseases.
Acknowledgements This work was supported by an EMBO fellowship and a Marie-Curie Career Integration Grants (M.S.D.), the Max Planck Society, BMBF/Sybacol, DFG/CECAD (A.A.). We thank Sarah Tremmel for helpful comments on the manuscript.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Small molecules in the regulation of ageing In addition to hexosamine metabolites, a number of biomolecules have been found to extend lifespan. These include spermidine, which stimulates autophagy [34], trehalose [35], a-ketoglutarate [36] and rapamycin [37]. It is conceivable that increasing concentrations of metabolites might lead to imbalances allover the metabolic network. Metabolic imbalance could trigger protective stress responses. This is most likely during manipulation with endogenous compounds such as UDP-GlcNAc and trehalose. It remains to be tested if supplementation with these compounds might elicit metabolic or signaling changes that result in longevity. These might include a hormetic process, during which exposure to a potentially toxic compound triggers a stress response that results in an organism’s increased lifespan. Importantly, the dose response of such molecules is critical to provide benefits. For example, GlcNAc extends C. elegans life span from 2 to 10 mM concentrations, but not at 25 mM. Conceivably higher levels may dampen protein quality control mechanisms or induce pathology. Indeed, in mammals, high levels of HP metabolites and high aminosugar flux are thought to trigger diabetic-like states [38,39].
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Assrir N, Richez C, Durand P, Guittet E, Badet B, Lescop E, BadetDenisot M-A: Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis. Biochimie 2014, 97:39-48. This paper identifies the UDP-GlcNAc binding pocket of GFAT-1 that is essential for product inhibition.
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Li Y, Roux C, Lazereg S, LeCaer J-P, Lapre´vote O, Badet B, BadetDenisot M-A: Identification of a novel serine phosphorylation site in human glutamine:fructose-6-phosphate amidotransferase isoform 1. Biochemistry 2007, 46:13163-13169.
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Tharanathan RN, Kittur FS: Chitin – the undisputed biomolecule of great potential. Crit Rev Food Sci Nutr 2003, 43:61-87.
Conclusion The HP demonstrates the intimate link between metabolic status, protein quality control, and thus systemic fitness that ultimately affects the ageing process. Cytoprotective downstream functions of HP activation are conserved: We observed improved protein surveillance in the nematode C. elegans [19], while in mice stressinduced activation of GFAT-1 results in cardioprotection through O-GlcNAcylation [10], and GlcN feeding extends mouse lifespan [18]. Despite this biological www.sciencedirect.com
Kato N, Dasgupta R, Smartt CT, Christensen BM: Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti. Insect Mol Biol 2002, 11:207-216.
10. Wang ZV, Deng Y, Gao N, Pedrozo Z, Li DL, Morales CR, Criollo A, Luo X, Tan W, Jiang N et al.: Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway. Cell 2014, 156:1179-1192. This study shows that GFAT-1 is activated by the UPR during cardiac stress in mice and that HP activation has cardioprotective function. 11. Ferrer CM, Lynch TP, Sodi VL, Falcone JN, Schwab LP, Peacock DL, Vocadlo DJ, Seagroves TN, Reginato MJ: OGlcNAcylation regulates cancer metabolism and survival Current Opinion in Cell Biology 2015, 33:14–18
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stress signaling via regulation of the HIF-1 pathway. Mol Cell 2014, 54:820-831. 12. Lynch TP, Ferrer CM, Jackson SR, Shahriari KS, Vosseller K, Reginato MJ: Critical role of O-linked N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis, and metastasis. J Biol Chem 2012, 287:11070-11081. 13. Ma Z, Vocadlo DJ, Vosseller K: Hyper-O-GlcNAcylation is antiapoptotic and maintains constitutive NF-B activity in pancreatic cancer cells. J Biol Chem 2013, 288:15121-15130. 14. Onodera Y, Nam J-M, Bissell MJ: Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways. J Clin Invest 2013, 124:367-384. This study shows that glucose uptake itself stimulates oncogenic pathways. These partially depend on GFAT-1, HP, and O-GlcNAcylation. 15. Johswich A, Longuet C, Pawling J, Rahman AA, Ryczko M, Drucker DJ, Dennis JW: N-Glycan remodeling on glucagon receptor is an effector of nutrient sensing by the hexosamine biosynthesis pathway. J Biol Chem 2014, 289:15927-15941. 16. Itkonen HM, Mills IG: N-Linked glycosylation supports crosstalk between receptor tyrosine kinases and androgen receptor. PLoS ONE 2013, 8:e65016. 17. Gass JN, Gifford NM, Brewer JW: Activation of an unfolded protein response during differentiation of antibody-secreting B cells. J Biol Chem 2002, 277:49047-49054. 18. Weimer S, Priebs J, Kuhlow D, Groth M, Priebe S, Mansfeld J, Merry TL, Dubuis SEB, Laube B, Pfeiffer AF et al.: Glucosamine supplementation extends life span of nematodes and of ageing mice. Nat Commun 2014, 5:1-12. Study showing that glucosamine supplementation can extend lifespan. In worms, this effect appeared to be independent of the HP. The authors suggest that it extends lifespan via inhibition of glycolysis and subsequent ROS signaling. 19. Denzel MS, Storm NJ, Gutschmidt A, Baddi R, Hinze Y, Jarosch E, Sommer T, Hoppe T, Antebi A: Hexosamine pathway metabolites enhance protein quality control and prolong life. Cell 2014, 156:1167-1178. Our study identified a number of GFAT-1 gain-of-function mutations that extend lifespan in C. elegans by improving protein quality control. 20. Saito Y, Yamanushi T, Oka T, Nakano A: Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy suppressors of ts mutants of Sar1 GTPase. J Biochem 1999, 125:130-137.
25. Xing D, Gong K, Feng W, Nozell SE, Chen Y-F, Chatham JC, Oparil S: O-GlcNAc modification of NFkB p65 inhibits TNF-ainduced inflammatory mediator expression in rat aortic smooth muscle cells. PLoS ONE 2011, 6:e24021. 26. Ramakrishnan P, Clark PM, Mason DE, Peters EC, HsiehWilson LC, Baltimore D: Activation of the transcriptional function of the NF-B protein c-Rel by O-GlcNAc glycosylation. Sci Signal 2013, 6:ra75. 27. Guinez C, Filhoulaud G, Rayah-Benhamed F, Marmier S, Dubuquoy C, Dentin R, Moldes M, Burnol AF, Yang X, Lefebvre T et al.: O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver. Diabetes 2011, 60:1399-1413. 28. Kaasik K, Kivima¨e S, Allen JJ, Chalkley RJ, Huang Y, Baer K, Kissel H, Burlingame AL, Shokat KM, Pta´cˇek LJ et al.: Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock. Cell Metabol 2013, 17:291-302. 29. Li M-D, Ruan H-B, Hughes ME, Lee J-S, Singh JP, Jones SP, Nitabach MN, Yang X: Short article. Cell Metabol 2013, 17:303-310. 30. Hang HC, Bertozzi CR: The chemistry and biology of mucintype O-linked glycosylation. Bioorgan Med Chem 2005, 13:5021-5034. 31. Perlmutter DH: Chemical chaperones: a pharmacological strategy for disorders of protein folding and trafficking. Pediatr Res 2002, 52:832-836. 32. Zode GS, Kuehn MH, Nishimura DY, Searby CC, Mohan K, Grozdanic SD, Bugge K, Anderson MG, Clark AF, Stone EM et al.: Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest 2011, 121:3542-3553. 33. Ozcan U: Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006, 313:1137-1140. 34. Eisenberg T, Knauer H, Schauer A, Bu¨ttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L et al.: Induction of autophagy by spermidine promotes longevity. Nature 2009, 11:1305-1314 [Internet]. 35. Honda Y, Tanaka M, Honda S: Trehalose extends longevity in the nematode Caenorhabditis elegans. Aging Cell 2010, 9:558-569.
21. Rahman MM, Stuchlick O, El-Karim EG, Stuart R, Kipreos ET, Wells L: Intracellular protein glycosylation modulates insulin mediated lifespan in C. elegans. Aging (Albany NY) 2010, 2:678-690.
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ScienceDirect Hexosamine pathway and (ER) protein quality control Martin S Denzel1 and Adam Antebi1,2,3 Aminosugars produced in the hexosamine pathway (HP) are utilized in protein glycosylation reactions involved in protein maturation and cellular signaling. Recent evidence revealed a role of the HP in protein quality control and ageing. Elevation of the HP product UDP-N-acetylglucosamine in the nematode Caenorhabditis elegans results in resistance towards toxic aggregation-prone proteins, and extended lifespan. Glutamine-fructose 6 phosphate aminotransferase (GFAT-1), the HP’s key enzyme, is a target of the unfolded protein response (UPR). Thus, cardiac stress in mice results in GFAT-1 activation that triggers a cytoprotective response. Feeding of glucosamine to aged mice increases their life expectancy.Here we discuss HP activation and cellular protein quality control mechanisms that result in stress resistance and suppression of age-related proteotoxicity. Addresses 1 Max Planck Institute for Biology of Ageing, Joseph-Stelzmann-Str. 9b, 50931 Cologne, Germany 2 Department of Molecular and Cellular Biology, Huffington Center on Aging Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA 3 Cologne Excellence Cluster on Cellular Stress Responses in AgingAssociated Diseases (CECAD), University of Cologne, 50674 Cologne, Germany Corresponding author: Antebi, Adam ([email protected])
Current Opinion in Cell Biology 2015, 33:14–18 This review comes from a themed issue on Cell regulation Edited by Johan Auwerx and Jodi Nunnari
http://dx.doi.org/10.1016/j.ceb.2014.10.001 0955-0674/# 2015 Published by Elsevier Ltd.
The HP is a central integrator of energy status The HP is comprised of a series of anabolic reactions that generate the activated aminosugar UDP-N-acetylglucosamine (UDP-GlcNAc). The HP is controlled by its first and rate-limiting enzyme GFAT-1, which converts fructose 6-phosphate and glutamine into glucosamine 6-phosphate and glutamate. Subsequent enzymatic steps then lead to acetylation and activation using UTP to produce UDP-GlcNAc. Thus, the HP is positioned to sample all major components of central energy metabolism, including glucose, fatty acid, amino acid metabolism, aerobic respiration, and, through UTP, energy availability (Figure 1). Current Opinion in Cell Biology 2015, 33:14–18
Consistent with a role in integrating various aspects of energy metabolism, the HP’s key enzyme GFAT-1 is not only regulated by product inhibition through UDPGlcNAc [1,2], but also by the major energy sensing AMP-activated protein kinase (AMPK) through inhibitory phosphorylation [3]. Further, GFAT-1 kinases include protein kinase A (PKA) and Ca2+/calmodulindependent protein kinase (CaMKII) whose physiological effects on GFAT-1 activity remain unclear and appear to depend on cellular context and isoform expression [4].
Posttranslational modifications involving GlcNAc UDP-GlcNAc is an important substrate for at least four types of glycosylation reactions that fulfill diverse cellular functions. First, N-glycosylation is an ER-specific posttranslational modification process during which a highmannose glycan is preassembled on the lipid carrier dolichol phosphate and then transferred en bloc to consensus Asn-X-Ser/Thr of nascent ER proteins. UDPGlcNAc is the substrate for the first two critical biosynthetic steps in the preassembly of this glycan structure. Nglycosylation plays a major role in the structure and function of cell surface and secreted proteins. In addition, N-glycans are involved in protein maturation in the ER and signal a substrate proteins’ folding state [5]. Notably, inhibition of N-glycosylation by drugs such as tunicamycin triggers massive protein misfolding stress. Second, proteins undergo numerous described types of O-linked glycosylation, the most abundant form being mucin-type O-glycosylation [6]. This reaction occurs in the Golgi apparatus and, unlike N-glycosylation, involves the step-by-step addition of sugars. UDP-N-acetylgalactosamine (UDP-GalNAc), which is produced from UDPGlcNAc by the epimerase GALE-1 , is used in the first two steps of mucin type O-glycosylation. GalNAc based O-glycosylation has various functions, including protein processing and folding [7]. Third, O-GlcNAcylation involves the addition of a single b-D-N-acetylglucosamine moiety to serine or threonine residues of target proteins. This posttranslational modification has been suggested to have regulatory functions, and many substrate proteins have been identified. It has been shown that O-GlcNAcylation interacts with phosphorylation to regulate client protein activity [8]. Finally, GlcNAc contributes to major biomolecules across nature. Chitin, a long-chain polymer of GlcNAc molecules synthetized from UDP-GlcNAc, is the most abundant biopolymer in marine environments [9]. Additionally, GlcNAc is a major component of the peptidoglycan layer of the bacterial cell wall. www.sciencedirect.com
Hexosamine pathway and (ER) protein quality control Denzel and Antebi 15
Figure 1
ER-stress/UPR N-glycosylation Glucose
F6-P O-GlcNAcylation
ATP
GlcN 6-P
Amino Acids
GlcNAc 6-P
Acetyl CoA
GlcNAc 1-P
UTP
UDP-GlcNAc UDP-GalNAc
O-glycosylation Chitin biosynthesis Peptidoglycan synthesis
UDP-GlcNAc
Others? Energy input
Integration in HP
Output, posttranscriptional modifications Current Opinion in Cell Biology
The HP integrates metabolic status from a number of central metabolism intermediates. This includes glycolysis, fatty acid and amino acid metabolism, aerobic respiration, and, through UTP, energy availability. These precursors are used to build up UDP-GlcNAc and UDP-GalNAc, which subsequently are involved in a number of glycosylation reactions. These posttranslational modifications regulate protein folding, sorting, and cellular signaling. As a precursor for chitin and glycosaminoglycan, UDP-GlcNAc is a precursor for some of the most abundant biomolecules.
HP might be a prosurvival signal Given that HP activity requires a positive energy status and the availability of cellular biomolecules or molecular building blocks, its activity is likely to reflect beneficial environmental conditions. This raises the question how downstream N-glycosylation and O-glycosylation mediate biological responses to the environment. Indeed there is evidence that elevated O-GlcNAcylation has pro-survival function. This is true in the mouse heart, where ischemia/reperfusion stress induces O-GlcNAcylation and serves a protective role for the survival of cardiomyocytes [10]. Moreover, in cancer cells, OGlcNAcylation regulates metabolism via hypoxia-inducible factor 1 (HIF-1) and inhibition of O-GlcNAcylation leads to ER stress-induced apoptosis [11]. Consistently, elevated O-GlcNAc transferase (OGT-1) levels correlate with poor outcome in patients. Also in prostate cancer, OGT-1 is upregulated and was found to promote metastasis [12]. Finally, O-GlcNAcylation is antiapoptotic in pancreatic cancer cells [13]. Taken together, this points to a unified view that HP flux reflects the cell’s energy status and constitutes a pro-survival signal. Cancer cells, in which high glucose flux itself can be oncogenic [14], can benefit from this and optimize proliferation even under growth-induced stress. However, non-malignant cells are also protected. In the search for interventions that might slow age-related disease it will be important to www.sciencedirect.com
balance these effects to exclude the promotion of tumorigenesis.
HP activation in the context of known longevity pathways A number of lifespan extending conditions such as reduced insulin signaling, dietary restriction, and mitochondrial respiration imbalances are involved in sensing and signaling of low energy availability. These interventions extend lifespan by activating stress responses that increase organismal fitness. Does HP activation constitute yet another such stress response pathway to extend lifespan? Indeed, it was shown that ER stress activates GFAT-1 expression and UDP-GlcNAc production as part of the UPR [10], which then improves protein quality control to counter proteotoxicity and to reset ER homeostasis. This signaling is consistent with the expression of chaperones downstream of the UPR. Supporting the idea that the HP is a specific stress response triggered by ER proteotoxicity, we observed that HP-induced activation of ER-associated degradation (ERAD), proteasome activity, and autophagy all contributed to improved ER protein quality. This might occur directly via changes in glycosylation patterns within the ER. It was shown that N-glycan remodeling on certain target proteins, such as the glucagon receptor, can be the output of the HP [15], and elevation of insulin-like growth factor 1 receptor Current Opinion in Cell Biology 2015, 33:14–18
16 Cell regulation
(IGF-1R) glycosylation can affect its activity [16]. All of these observations point to a role of the HP as a protein stress response pathway (Figure 2). Conceivably, imbalanced anabolic processes might physiologically trigger stress pathways. For example, antibody producing B-cells ramp up ER stress mechanisms to cope with increased secretory demand [17]. Proper induction and dampening of such stress mechanisms would be critical to cellular homeostasis. Interestingly, the energy-stress induced AMPK is a negative regulator of GFAT-1 that suppresses HP flux when ATP becomes scarce, which is consistent with the anabolic nature of the HP. This reflects the specificity of cellular stress response pathways: energy stress will activate AMPK and suppress GFAT-1 while protein stress activates GFAT-1 and the HP. Alternative to the HP being a stress response pathway, HP activation itself might constitute cellular stress. HP activation might lead to energy imbalance by consuming metabolites, triggering a stress response. This view receives support from the observation that glucosamine (GlcN) supplementation causes a ROS response [18] that extends worm and mouse lifespan through a hormetic mechanism. To test the possibility that HP activation itself is a stress, it might be interesting to supplement with the final product of the HP, UDP-GlcNAc. In this case, none of the HP substrates would be depleted to induce a metabolic imbalance. So far, studies have only used HP intermediates such as GlcN and GlcNAc to mimic HP activation. Figure 2
toxic proteins
ERAD Proteasome
ER stress Autophagy O-glycosylation
AMPK
others?
F6P
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Gln
GlcN 6-P GlcNAc 6-P GlcNAc 1-P
Glu AcetylCoA
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UDP-GlcNAc UDP
Current Opinion in Cell Biology
The HP is part of a stress response pathway that maintains protein homeostasis. ER protein misfolding triggers GFAT-1 transcription and activates the HP. Through incompletely understood mechanisms, subsequent UDP-GlcNAc elevation activates protein quality surveillance by inducing O-glycosylation, autophagy, ERAD, and proteasome activity. This counters ER protein misfolding stress and also alleviates toxicity caused by toxic aggregation prone proteins such as poly-glutamine peptides or Ab. Current Opinion in Cell Biology 2015, 33:14–18
How do UDP-GlcNAc levels influence protein quality control? A puzzling question that remains is how the levels of one sentinel molecule, UDP-GlcNAc, can orchestrate diverse responses to improve protein quality control. A number of mechanisms are imaginable: Changes in N-glycosylation: Given the role of Nglycosylation in protein quality control, it appears likely that enhancing this process might cause the observed effects on protein homeostasis. Enhanced Nglycosylation could globally relieve stress on the ER by mass action, and thereby unfetter other quality control systems. However, we have not found a global increase in levels of N-glycosylated proteins at steady state in Caenorhabditis elegans during HP activation [19]. Quite possibly, though, certain proteins might be over-glycosylated under these conditions to elicit the beneficial effects. Members of the ER associated degradation machinery, for example, are substrates for N-glycosylation [20]. These changes would also fulfill a signaling role as we found a coordinated improvement in protein homeostasis that included proteasome activity and autophagy. ERAD components would be optimally situated to fulfill all these functions. Consistent with this, preliminarily results indicate that an increased dose of the ERAD component SEL-1 is sufficient for lifespan extension in C. elegans [19]. Future experiments will address the role of glycosylation and ERAD activity. Elevation of O-GlcNAcylation: In accord with this hypothesis, Hill and colleagues detected increased O-GlcNAc modification levels in the ischemic heart, and inhibition of this process through OGT-1 knockdown led to decreased cellular protection[10]. Surprisingly, OGT-1 mutation in C. elegans has little effect on longevity, but it is currently unclear whether O-GlcNAcylation is truly abolished in these mutants. Cytosolic addition of GlcNAc moieties modulates the activity of a number of substrates, including signaling proteins in important pathways such as insulin signaling and the downstream transcription factor FOXO [21,22]. Thus it is possible that high energy sensing via the HP and subsequent O-GlcNAcylation might induce lifespan extension. Other prominent signaling proteins that are O-GlcNAcylated include, among others, beta catenin [23], FXR [24], NF-kB subunits [25,26], carbohydrate-responsive elementbinding protein (ChREBP) [27], as well as components of the circadian clock [28,29]. Elevation of mucin type O-glycosylation: Given the role of mucin type O-glycosylation in the secretory apparatus, it is possible that elevation of its precursor UDP-GalNAc/UDP-GlcNAc might lead to changes in the degree of glycosylation or in the abundance of substrate proteins. This might result in functional changes of substrate proteins, which www.sciencedirect.com
Hexosamine pathway and (ER) protein quality control Denzel and Antebi 17
include chemokines and regulators of cellular signaling cascades [30]. Function as an allosteric effector of other proteins: It is possible that the beneficial effects of HP metabolites arise not as a consequence of covalent post-translational modifications. Rather HP metabolites could non-covalently bind target proteins to allosterically regulate their activity. GFAT itself is modulated by such end-product inhibition [2]. Alternately, HP metabolites could serve as chemical chaperones, similar to phenylbutyric acid (PBA) or tauro-ursodeoxycholic acid (TUDCA) to stabilize protein folding. Chemical chaperones can either act broadly and interact with numerous substrates, or stabilize specific proteins [31]. Interestingly, chemical chaperones are implicated in countering ER stress [32,33]. In all of the above potential mechanisms, it will be interesting to see if single target proteins are responsible for the observed phenotypes, if there is a convergence of mechanisms, or if the phenotypes are a consequence of allover changes in glycosylation.
significance, too little is known at the mechanistic level about the molecular consequences of HP activation and elevation of UDP-GlcNAc levels. What are the major downstream events? What type of glycosylation is mostly affected and what are the major substrates that trigger downstream effects? Altogether, this makes GFAT-1 an attractive target for research into the ageing process with the goal of alleviating age-associated diseases.
Acknowledgements This work was supported by an EMBO fellowship and a Marie-Curie Career Integration Grants (M.S.D.), the Max Planck Society, BMBF/Sybacol, DFG/CECAD (A.A.). We thank Sarah Tremmel for helpful comments on the manuscript.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Small molecules in the regulation of ageing In addition to hexosamine metabolites, a number of biomolecules have been found to extend lifespan. These include spermidine, which stimulates autophagy [34], trehalose [35], a-ketoglutarate [36] and rapamycin [37]. It is conceivable that increasing concentrations of metabolites might lead to imbalances allover the metabolic network. Metabolic imbalance could trigger protective stress responses. This is most likely during manipulation with endogenous compounds such as UDP-GlcNAc and trehalose. It remains to be tested if supplementation with these compounds might elicit metabolic or signaling changes that result in longevity. These might include a hormetic process, during which exposure to a potentially toxic compound triggers a stress response that results in an organism’s increased lifespan. Importantly, the dose response of such molecules is critical to provide benefits. For example, GlcNAc extends C. elegans life span from 2 to 10 mM concentrations, but not at 25 mM. Conceivably higher levels may dampen protein quality control mechanisms or induce pathology. Indeed, in mammals, high levels of HP metabolites and high aminosugar flux are thought to trigger diabetic-like states [38,39].
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Conclusion The HP demonstrates the intimate link between metabolic status, protein quality control, and thus systemic fitness that ultimately affects the ageing process. Cytoprotective downstream functions of HP activation are conserved: We observed improved protein surveillance in the nematode C. elegans [19], while in mice stressinduced activation of GFAT-1 results in cardioprotection through O-GlcNAcylation [10], and GlcN feeding extends mouse lifespan [18]. Despite this biological www.sciencedirect.com
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