Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for ...disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate-GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.
To maintain homeostasis under variable nutrient conditions, cells rapidly and robustly respond to fluctuations through adaptable signaling networks. Evidence suggests that the O-linked ...N-acetylglucosamine (O-GlcNAc) posttranslational modification of serine and threonine residues functions as a critical regulator of intracellular signaling cascades in response to nutrient changes. O-GlcNAc is a highly regulated, reversible modification poised to integrate metabolic signals and acts to influence many cellular processes, including cellular signaling, protein stability, and transcription. This review describes the role O-GlcNAc plays in governing both integrated cellular processes and the activity of individual proteins in response to nutrient levels. Moreover, we discuss the ways in which cellular changes in O-GlcNAc status may be linked to chronic diseases such as type 2 diabetes, neurodegeneration, and cancers, providing a unique window through which to identify and treat disease conditions.
Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the ...hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key ‘hallmarks of cancer’. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb’s cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a ‘vicious cycle’. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.
Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger ...often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid beta-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD entry and recovery and uncover genetic interactions that provide insight into the role of OGT and OGA.
Proteostasis is essential in the mammalian brain where post‐mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites ...for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient‐sensitive nucleocytoplasmic post‐translational modification O‐linked N‐acetylglucosamine (O‐GlcNAc) regulates protein homeostasis. The O‐GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O‐GlcNAc transferase‐ and O‐GlcNAcase‐knockout alleles have helped define the role O‐GlcNAc plays in development as well as age‐associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O‐GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O‐GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O‐GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a ‘mannose timer’ to monitor protein folding, we propose that cytoplasmic proteostasis relies on an ‘O‐GlcNAc timer’ to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O‐GlcNAc‐dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain‐selective knockout mouse models will be an important tool for understanding the role of O‐GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury.
Proteostasis is essential in the mammalian brain where post‐mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient‐sensitive nucleocytoplasmic post‐translational modification O‐linked N‐acetylglucosamine (O‐GlcNAc) regulates protein homeostasis. This cyclic modification is coordinately regulated with other PTMs such as phosphorylation to regulate the required intricacies of cellular processes. Deregulation of PTMs including O‐GlcNAc leads to several pathologies that are associated with neurodegeneration.
O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and ...essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.
The NRF2/sMAF protein complex regulates the oxidative stress response by occupying cis-acting enhancers containing an antioxidant response element (ARE). Integrating genome-wide maps of NRF2/sMAF ...occupancy with disease-susceptibility loci, we discovered eight polymorphic AREs linked to 14 highly ranked disease-risk SNPs in individuals of European ancestry. Among these SNPs was rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple experiments and its strong-binding allele associated with higher mRNA levels in cell lines and human brain tissue. Induction of MAPT transcription by NRF2 was confirmed using a human neuroblastoma cell line and a Nrf2-deficient mouse model. Most importantly, rs242561 displayed complete linkage disequilibrium with a highly protective allele identified in multiple GWASs of progressive supranuclear palsy, Parkinson’s disease, and corticobasal degeneration. These observations suggest a potential role for NRF2/sMAF in tauopathies and a possible role for NRF2 pathway activators in disease prevention.
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•Genome-wide NRF2/sMAF occupancy defines highly ranked disease-associated SNPs•SNPs in NRF2 binding sites are rare in the human genome due to negative selection•A SNP in MAPT shows allele-specific binding to NRF2, enhanced MAPT transcription•Strong NRF2 binding site is linked with reduced risk of parkinsonian diseases
Wang et al. find that a polymorphic NRF2/sMAF binding site in MAPT is strongly associated with differential risk for parkinsonian disorders and provide evidence that it is occupied by NRF2/sMAF proteins in human and mouse cells. The stronger-binding allele enhances MAPT expression in the human brain and associates with a protective effect in GWAS analyses.
The dynamic carbohydrate post-translational modification (PTM)
-linked β-
-acetyl glucosamine (
-GlcNAc) is found on thousands of proteins throughout the nucleus and cytoplasm, and rivals ...phosphorylation in terms of the number of substrates and pathways influenced.
-GlcNAc is highly conserved and essential in most organisms, with disruption of
-GlcNAc cycling linked to diseases ranging from cancer to neurodegeneration. Nuclear pore proteins were the first identified
-GlcNAc-modified substrates, generating intense and ongoing interest in understanding the role of
-GlcNAc cycling in nuclear pore complex structure and function. Recent advances in detecting and altering O-GlcNAcylation levels have provided insights into many mechanisms by which O-GlcNAcylation influences the nucleocytoplasmic localization and stability of protein targets. The emerging view is that the multifunctional enzymes of
-GlcNAc cycling are critical nutrient-sensing components of a complex network of signaling cascades involving multiple PTMs. Furthermore,
-GlcNAc plays a role in maintaining the structural integrity of the nuclear pore and regulating its function as the gatekeeper of nucleocytoplasmic trafficking.
The National Institute of General Medical Sciences (NIGMS) at the U.S. National Institutes of Health (NIH) is committed to supporting the safety of the nation's biomedical research and training ...environments. Institutional training grants affect many trainees and can have a broad influence across their parent institutions, making them good starting points for our initial efforts to promote the development and maintenance of robust cultures of safety at U.S. academic institutions. In this Perspective, we focus on laboratory safety, although many of the strategies we describe for improving laboratory safety are also applicable to other forms of safety including the prevention of harassment, intimidation, and discrimination. We frame the problem of laboratory safety using a number of recent examples of tragic accidents, highlight some of the lessons that have been learned from these and other events, discuss what NIGMS is doing to address problems related to laboratory safety, and outline steps that institutions can take to improve their safety cultures.
The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a ...polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.
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•A common SNP can influence p53’s regulation of a key target gene, the KITLG gene•The SNP in the p53-binding site can significantly influence human cancer risk•The SNP in the p53-binding site has undergone positive natural selection in humans•Similar SNPs in p53-binding sites are rare in the genome due to negative selection
The polymorphism in the KITLG regulatory region associates with differential testicular cancer risk (higher with the G allele, which is more prevalent in Caucasians than in African American males) but also shows signs of positive natural selection, perhaps due to the role of KIT in UV protection in light skinned Caucasians.
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