Alzheimer disease (AD) is a growing problem for aging populations worldwide. Despite significant efforts, no therapeutics are available that stop or slow progression of AD, which has driven interest ...in the basic causes of AD and the search for new therapeutic strategies. Longitudinal studies have clarified that defects in glucose metabolism occur in patients exhibiting Mild Cognitive Impairment (MCI) and glucose hypometabolism is an early pathological change within AD brain. Further, type 2 diabetes mellitus (T2DM) is a strong risk factor for the development of AD. These findings have stimulated interest in the possibility that disrupted glucose regulated signaling within the brain could contribute to the progression of AD. One such process of interest is the addition of O-linked N-acetylglucosamine (O-GlcNAc) residues onto nuclear and cytoplasmic proteins within mammals. O-GlcNAc is notably abundant within brain and is present on hundreds of proteins including several, such as tau and the amyloid precursor protein, which are involved in the pathophysiology AD. The cellular levels of O-GlcNAc are coupled to nutrient availability through the action of just two enzymes. O-GlcNAc transferase (OGT) is the glycosyltransferase that acts to install O-GlcNAc onto proteins and O-GlcNAcase (OGA) is the glycoside hydrolase that acts to remove O-GlcNAc from proteins. Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is the donor sugar substrate for OGT and its levels vary with cellular glucose availability because it is generated from glucose through the hexosamine biosynthetic pathway (HBSP). Within the brains of AD patients O-GlcNAc levels have been found to be decreased and aggregates of tau appear to lack O-GlcNAc entirely. Accordingly, glucose hypometabolism within the brain may result in disruption of the normal functions of O-GlcNAc within the brain and thereby contribute to downstream neurodegeneration. While this hypothesis remains largely speculative, recent studies using different mouse models of AD have demonstrated the protective benefit of pharmacologically increased brain O-GlcNAc levels. In this review we summarize the state of knowledge in the area of O-GlcNAc as it pertains to AD while also addressing some of the basic biochemical roles of O-GlcNAc and how these might contribute to protecting against AD and other neurodegenerative diseases.
Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of ...glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKβ were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC.
Background: Cancer cells rely on energy metabolism that requires increased glucose uptake and constitutive NF-κB activity for survival.
Results: Pancreatic cancer cells display elevated O-GlcNAcylation, reduction of which inhibits cell survival and oncogenic NF-κB signaling.
Conclusion: Hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB activation in pancreatic cancer.
Significance: Targeting hyper-O-GlcNAcylation may serve as a novel therapeutic intervention in pancreatic cancer.
The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked β-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation ...by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via hypoxia-inducible factor 1 (HIF-1α) and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation, and interaction with von Hippel-Lindau protein (pVHL), resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of endoplasmic reticulum (ER) stress and cancer cell apoptosis mediated through C/EBP homologous protein (CHOP). HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects. Human breast cancers with high levels of HIF-1α contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.
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•Reducing OGT and O-GlcNAcylation in cancer cells decreases cancer glycolysis•OGT regulates stability of HIF-1α via regulation of α-ketoglutarate levels•Reducing OGT levels or activity induces ER stress and apoptosis in cancer cells•Stable HIF-1α mutant or GLUT1 overexpression rescues OGT-mediated phenotypes
Multiple cancers contain elevated levels of O-GlcNAc transferase (OGT). Ferrer et al. show that OGT and O-GlcNAcylation play a critical role in cancer-driven glycolysis and stress survival signaling by regulating the degradation of the hypoxia-inducible factor 1 (HIF-1α).
Dynamic modification of proteins with O-GlcNAc modification at serine and threonine residues of proteins is carried out by OGT and removal of O-GlcNAc is carried out by OGA. O-GlcNAc levels respond ...to nutrient availability including glucose, which is assimilated via the hexosamine biosynthetic pathway (HBSP) to form uridine diphospho-N-acetylglucosamine (UDP-O-GlcNAc). The mechanisms and structures of these enzymes are being uncovered as is the basis for the substrate specificity of these enzymes. Display omitted
► O-GlcNAc levels in cells respond to availability of nutrients including glucose. ► OGT and OGA are the two enzymes processing hundreds of O-GlcNAc modified proteins. ► Catalysis by OGA and OGT is being studied using kinetic and structural approaches. ► OGA processes metabolic derivatives of O-GlcNAc from proteins. ► The substrate specificity and regulation of OGA and OGT merit further attention.
The addition of N-acetylglucosamine (GlcNAc) O-linked to serine and threonine residues of proteins is known as O-GlcNAc. This post-translational modification is found within multicellular eukaryotes on hundreds of nuclear and cytoplasmic proteins. O-GlcNAc transferase (OGT) installs O-GlcNAc onto target proteins and O-GlcNAcase (OGA) removes O-GlcNAc. Their combined action makes O-GlcNAc reversible and serves to regulate cellular O-GlcNAc levels. Here I review select recent literature on the catalytic mechanism of these enzymes and studies on the molecular basis by which these enzymes identify and process their substrates. Molecular level understanding of how these enzymes work, and the basis for their specificity, should aid understanding how O-GlcNAc contributes to diverse cellular processes ranging from cellular signaling through to transcriptional regulation.
The pathological hallmark of Parkinson's disease (PD) is Lewy bodies that form within the brain from aggregated forms of α‐synuclein (α‐syn). These toxic α‐syn aggregates are transferred from cell to ...cell by release of fibrils from dying neurons into the extracellular environment, followed by their subsequent uptake by neighboring cells. This process leads to spreading of the pathology throughout the brain in a prion‐like manner. Identifying new pathways that hinder the internalization of such α‐syn fibrils is of high interest for their downstream potential exploitation as a way to create disease‐modifying therapeutics for PD. Here, we show that Thiamet‐G, a highly selective pharmacological agent that inhibits the glycoside hydrolase O‐GlcNAcase (OGA), blunts the cellular uptake of α‐syn fibrils. This effect correlates with increased nucleocytoplasmic levels of O‐linked N‐acetylglucosamine (O‐GlcNAc)‐modified proteins, and genetic knockdown of OGA expression closely phenocopies both these effects. These reductions in the uptake of α‐syn fibrils caused by inhibition of OGA are both concentration‐ and time‐dependent and are observed in multiple cell lines including mouse primary cortical neurons. Moreover, treatment of cells with the OGT inhibitor, 5SGlcNHex, increases the level of uptake of α‐syn PFFs, further supporting O‐GlcNAcylation of proteins driving these effects. Notably, this effect is mediated through an unknown mechanism that does not involve well‐characterized endocytotic pathways. These data suggest one mechanism by which OGA inhibitors might exert their protective effects in prion‐like neuropathologies and support exploration of OGA inhibitors as a potential disease‐modifying approach to treat PD.
Increases in cellular levels of O‐linked N‐acetylglucosamine (O‐GlcNAc)‐modified proteins, induced by inhibition of the glycoside hydrolase O‐GlcNAcase (OGA) using a highly selective inhibitor, blunt the uptake of α‐synuclein fibrils. This effect is phenocopied by siRNA‐mediated knockdown of OGA, and inhibition of OGT increases the uptake of α‐synuclein fibrils. The effects are mediated through a pathway that is independent of general endocytosis.
Glycoconjugates are ubiquitous biomolecules found in all kingdoms of life. These diverse structures are metabolically responsive and occur in a cell line- and protein-specific manner, conferring ...tissue type-specific properties. Glycans have essential roles in diverse processes, including, for example, intercellular signaling, inflammation, protein quality control, glucohomeostasis and cellular adhesion as well as cell differentiation and proliferation. Many mysteries remain in the field, however, and uncovering the physiological roles of various glycans remains a key pursuit. Realizing this aim necessitates the ability to subtly and selectively manipulate the series of different glycoconjugates both in cells and in vivo. Selective small-molecule inhibitors of glycan processing enzymes hold great potential for such manipulation as well as for determining the function of 'orphan' carbohydrate-processing enzymes. In this review, we discuss recent advances and existing inhibitors, the prospects for small-molecule inhibitors and the challenges associated with generating high-quality chemical probes for these families of enzymes. The coordinated efforts of chemists, biochemists and biologists will be crucial for creating and characterizing inhibitors that are useful tools both for advancing a basic understanding of glycobiology in mammals as well as for validating new potential therapeutic targets within this burgeoning field.
Regional glucose hypometabolism is a defining feature of Alzheimer disease (AD). One emerging link between glucose hypometabolism and progression of AD is the nutrient-responsive post-translational ...O-GlcNAcylation of nucleocytoplasmic proteins. O-GlcNAc is abundant in neurons and occurs on both tau and amyloid precursor protein. Increased brain O-GlcNAcylation protects against tau and amyloid-β peptide toxicity. Decreased O-GlcNAcylation occurs in AD, suggesting that glucose hypometabolism may impair the protective roles of O-GlcNAc within neurons and enable neurodegeneration. Here, we review how O-GlcNAc may link cerebral glucose hypometabolism to progression of AD and summarize data regarding the protective role of O-GlcNAc in AD models.
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•Good cell active competitive inhibitors of O-GlcNAc processing enzymes are available.•In vitro assays for O-GlcNAc processing enzymes are established.•OGA inhibitors are advancing ...into the clinic.•Improved inhibitors and cell-based assays are needed to advance the field.
O-linked N-acetylglucosamine (O-GlcNAc) is protein modification that is emerging as a regulator of diverse aspects of cellular physiology. Aberrant O-GlcNAcylation has been linked to several diseases, spurring the creation of methods to detect and perturb the activity of the two enzymes that govern this modification — O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Here we summarize assays used for these two enzymes. We also detail the latest structure-guided development of inhibitors of these two enzymes and touch on selected reports that underscore the utility of inhibitors as tools for uncovering the diverse roles of O-GlcNAc in cell function. Finally, we summarize recent reports on the potential therapeutic benefits of antagonizing these enzymes and comment on outstanding challenges within the field.
Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, ...psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.
Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the ...nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.
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