Bisecting N-Acetylglucosamine Correlates with Phospho-Tau181 in Subjective Cognitive Decline but not in Control Cases.

Background: The N-glycan structure bisecting N-acetylglucosamine (bisecting GlcNAc) is present on several N-glycans that are elevated in Alzheimer's disease (AD), and previous studies have shown that bisecting GlcNAc levels correlate with total tau and phospho-tau181 in cerebrospinal fluid at early stages of AD. A recent population-based study showed that bisecting GlcNAc correlates with total tau also in blood and that this correlation could predict conversion to dementia. Objective: In this study, we have further investigated how bisecting GlcNAc relates to total tau and phospho-tau 181 in cerebrospinal fluid samples from controls and cases with early cognitive deficits, stratified by amyloid/tau status and gender. Methods: Relative levels of bisecting GlcNAc in cerebrospinal fluid were measured by an enzyme-linked lectin assay in individuals with subjective cognitive decline, mild cognitive impairment and controls from the Norwegian Dementia Disease Initiation cohort. Results: As in our previous study, the correlation between bisecting GlcNAc and total tau or phospho-tau181 was particularly strong in the subjective cognitive decline group. The correlation was observed in amyloid negative and tau negative as well as amyloid positive and tau positive individuals, both in females and in males. Interestingly, among the amyloid negative and tau negative individuals, the correlation was observed in individuals with subjective cognitive decline but not in the controls. Conclusions: Thus, bisecting GlcNAc could be a biomarker for early cognitive decline.

A protein O-GlcNAc glycosyltransferase regulates the antioxidative response in Yersinia pestis.

Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins is commonly associated with a variety of stress responses and cellular processes in eukaryotes, but its potential roles in bacteria are unclear. Here, we show that protein HmwC acts as an O-GlcNAc transferase (OGT) responsible for O-GlcNAcylation of multiple proteins in Yersinia pestis, a flea-borne pathogen responsible for plague. We identify 64 O-GlcNAcylated proteins (comprising 65 sites) with differential abundance under conditions mimicking the mammalian host (Mh) and flea vector (Fv) environments. Deletion of hmwC, encoding a putative OGT, structurally distinct from any existing member of the GT41 family, results in reduced O-GlcNAcylation, reduced growth, and alterations in virulence properties and survival under stress. Purified HmwC can modify target proteins in vitro using UDP-GlcNAc as sugar donor. One of the target proteins, OsdY, promotes Y. pestis survival under oxidative stress conditions. Thus, our results support that regulation of antioxidative responses through O-GlcNAcylation may be a conserved process shared by prokaryotes and eukaryotes.

Integrating HexNAcQuest with Glycoproteomics Data Analysis Software to Distinguish HexNAc Isomers on Peptides.

Recently, HexNAcQuest was developed to help distinguish peptides modified by HexNAc isomers, more specifically O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc, Tn antigen). To facilitate its usage (particularly for datasets from glycoproteomics studies), herein we present a detailed protocol. It describes example cases and procedures for which users might need to use HexNAcQuest to distinguish these two modifications.

Chronic rapid eye movement sleep deprivation aggravates the pathogenesis of Alzheimer's disease by decreasing brain O-GlcNAc cycling in mice.

This study investigated the role of O-GlcNAc cycling in Alzheimer's disease-related changes in brain pathophysiology induced by chronic REM sleep deprivation (CSD) in mice. CSD increased amyloid beta (Aß) and p-Tau accumulation and impaired learning and memory (L/M) function. CSD decreased dendritic length and spine density. CSD also increased the intensity of postsynaptic density protein-95 (PSD-95) staining. All of these Alzheimer's disease (AD) pathogenic changes were effectively reversed through glucosamine (GlcN) treatment by enhancing O-GlcNAcylation. Interestingly, the lelvel of O-GlcNAcylated-Tau (O-Tau) exhibited an opposite trend compared to p-Tau, as it was elevated by CSD and suppressed by GlcN treatment. CSD increased neuroinflammation, as indicated by elevated levels of glial fibrillary acidic protein and IBA-1-positive glial cells in the brain, which were suppressed by GlcN treatment. CSD promoted the phosphorylation of GSK3ß and led to an upregulation in the expression of endoplasmic reticulum (ER) stress regulatory proteins and genes. These alterations were effectively suppressed by GlcN treatment. Minocycline not only suppressed neuroinflammation induced by CSD, but it also rescued the decrease in O-GlcNAc levels caused by CSD. Minocycline also reduced AD neuropathy without affecting CSD-induced ER stress. Notably, overexpressing O-GlcNAc transferase in the dentate gyrus region of the mouse brain rescued CSD-induced cognitive dysfunction, neuropathy, neuroinflammation, and ER stress responses. Collectively, our findings reveal that dysregulation of O-GlcNAc cycling underlies CSD-induced AD pathology and demonstrate that restoration of OGlcNAcylation protects against CSD-induced neurodegeneration.

O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among them, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here, we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72), and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive, while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

O-GlcNAc signaling: Implications for stress-induced adaptive response pathway in the tumor microenvironment.

The tumor microenvironment (TME) consists of tumor cells, non-tumor cells, extracellular matrix, and signaling molecules, which can contribute to tumor initiation, progression, and therapy resistance. In response to starvation, hypoxia, and drug treatments, tumor cells undergo a variety of deleterious endogenous stresses, such as hypoxia, DNA damage, and oxidative stress. In this context, to survive the difficult situation, tumor cells evolve multiple conserved adaptive responses, including metabolic reprogramming, DNA damage checkpoints, homologous recombination, up-regulated antioxidant pathways, and activated unfolded protein responses. In the last decades, the protein O-GlcNAcylation has emerged as a crucial causative link between glucose metabolism and tumor progression. Here, we discuss the relevant pathways that regulate the above responses. These pathways are adaptive adjustments induced by endogenous stresses in cells. In addition, we systematically discuss the role of O-GlcNAcylation-regulated stress-induced adaptive response pathways (SARPs) in TME remodeling, tumor progression, and treatment resistance. We also emphasize targeting O-GlcNAcylation through compounds that modulate OGT or OGA activity to inhibit tumor progression. It seems that targeting O-GlcNAcylated proteins to intervene in TME may be a novel approach to improve tumor prognosis.

ROK family regulator NagC promotes prodigiosin biosynthesis independent of N-acetylglucosamine in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 is an important model strain for the study of prodigiosin production, whose prodigiosin biosynthesis genes (pigA-O) are arranged in an operon. Several transcription factors have been shown to control the transcription of the pig operon. However, since the regulation of prodigiosin biosynthesis is complex, the regulatory mechanism for this process has not been well established. In most γ-proteobacteria, the ROK family regulator NagC acts as a global transcription factor in response to N-acetylglucosamine (GlcNAc). In Serratia sp. ATCC 39006, NagC represses the transcription of two divergent operons, nagE and nagBAC, which encode proteins involved in the transport and metabolism of GlcNAc. Moreover, NagC directly binds to a 21-nt region that partially overlaps the -10 and -35 regions of the pig promoter and promotes the transcription of prodigiosin biosynthesis genes, thereby increasing prodigiosin production. Although NagC still acts as both repressor and activator in Serratia sp. ATCC 39006, its transcriptional regulatory activity is independent of GlcNAc. NagC was first found to regulate antibiotic biosynthesis in Gram-negative bacteria, and NagC-mediated regulation is not responsive to GlcNAc, which contributes to future studies on the regulation of secondary metabolism by NagC in other bacteria. IMPORTANCE: The ROK family transcription factor NagC is an important global regulator in the γ-proteobacteria. A large number of genes involved in the transport and metabolism of sugars, as well as those associated with biofilm formation and pathogenicity, are regulated by NagC. In all of these regulations, the transcriptional regulatory activity of NagC responds to the supply of GlcNAc in the environment. Here, we found for the first time that NagC can regulate antibiotic biosynthesis, whose transcriptional regulatory activity is independent of GlcNAc. This suggests that NagC may respond to more signals and regulate more physiological processes in Gram-negative bacteria.

Role of O-linked N-acetylglucosamine protein modification in oxidative stress-induced autophagy: a novel target for bone remodeling.

O-linked N-acetylglucosamine protein modification (O-GlcNAcylation) is a dynamic post-translational modification (PTM) involving the covalent binding of serine and/or threonine residues, which regulates bone cell homeostasis. Reactive oxygen species (ROS) are increased due to oxidative stress in various pathological contexts related to bone remodeling, such as osteoporosis, arthritis, and bone fracture. Autophagy serves as a scavenger for ROS within bone marrow-derived mesenchymal stem cells, osteoclasts, and osteoblasts. However, oxidative stress-induced autophagy is affected by the metabolic status, leading to unfavorable clinical outcomes. O-GlcNAcylation can regulate the autophagy process both directly and indirectly through oxidative stress-related signaling pathways, ultimately improving bone remodeling. The present interventions for the bone remodeling process often focus on promoting osteogenesis or inhibiting osteoclast absorption, ignoring the effect of PTM on the overall process of bone remodeling. This review explores how O-GlcNAcylation synergizes with autophagy to exert multiple regulatory effects on bone remodeling under oxidative stress stimulation, indicating the application of O-GlcNAcylation as a new molecular target in the field of bone remodeling.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation.

N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.

Effects of the pleiotropic regulator DasR on lincomycin production in Streptomyces lincolnensis.

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.

Enzymatic Synthesis of Novel Terpenoid Glycoside Derivatives Decorated with N-Acetylglucosamine Catalyzed by UGT74AC1.

The addition of the O-linked N-acetylglucosamine (O-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1His18A/Asp111A in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.

N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

O-GlcNAcylation controls pro-fibrotic transcriptional regulatory signaling in myofibroblasts.

Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked ß-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.

The non-catalytic domains of O-GlcNAc cycling enzymes present new opportunities for function-specific control.

O-GlcNAcylation is an essential protein glycosylation governed by two O-GlcNAc cycling enzymes: O-GlcNAc transferase (OGT) installs a single sugar moiety N-acetylglucosamine (GlcNAc) on protein serine and threonine residues, and O-GlcNAcase (OGA) removes them. Aberrant O-GlcNAcylation has been implicated in various diseases. However, the large repertoire of more than 1000 O-GlcNAcylated proteins and the elusive mechanisms of OGT/OGA in substrate recognition present significant challenges in targeting the dysregulated O-GlcNAcylation for therapeutic development. Recently, emerging evidence suggested that the non-catalytic domains play critical roles in regulating the functional specificity of OGT/OGA via modulating their protein interactions and substrate recognition. Here, we discuss recent studies on the structures, mechanisms, and related tools of the OGT/OGA non-catalytic domains, highlighting new opportunities for function-specific control.

Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy.

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

O-GlcNAcase regulates pluripotency states of human embryonic stem cells.

Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine. Despite the recent progress, molecular events regulating hESC pluripotency, especially the transition between naive and primed states, still remain unclear. Here we show that naive hESCs display lower levels of O-linked N-acetylglucosamine (O-GlcNAcylation) than primed hESCs. O-GlcNAcase (OGA), the key enzyme catalyzing the removal of O-GlcNAc from proteins, is highly expressed in naive hESCs and is important for naive pluripotency. Depletion of OGA accelerates naive-to-primed pluripotency transition. OGA is transcriptionally regulated by EP300 and acts as a transcription regulator of genes important for maintaining naive pluripotency. Moreover, we profile protein O-GlcNAcylation of the two pluripotency states by quantitative proteomics. Together, this study identifies OGA as an important factor of naive pluripotency in hESCs and suggests that O-GlcNAcylation has a broad effect on hESCs homeostasis.