Downregulation of O-GlcNAc transferase activity impairs basal autophagy and late endosome positioning under nutrient-rich conditions in human colon cells.

Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

O-GlcNAc Dynamics: The Sweet Side of Protein Trafficking Regulation in Mammalian Cells.

The transport of proteins between the different cellular compartments and the cell surface is governed by the secretory pathway. Alternatively, unconventional secretion pathways have been described in mammalian cells, especially through multivesicular bodies and exosomes. These highly sophisticated biological processes rely on a wide variety of signaling and regulatory proteins that act sequentially and in a well-orchestrated manner to ensure the proper delivery of cargoes to their final destination. By modifying numerous proteins involved in the regulation of vesicular trafficking, post-translational modifications (PTMs) participate in the tight regulation of cargo transport in response to extracellular stimuli such as nutrient availability and stress. Among the PTMs, O-GlcNAcylation is the reversible addition of a single N-acetylglucosamine monosaccharide (GlcNAc) on serine or threonine residues of cytosolic, nuclear, and mitochondrial proteins. O-GlcNAc cycling is mediated by a single couple of enzymes: the O-GlcNAc transferase (OGT) which catalyzes the addition of O-GlcNAc onto proteins, and the O-GlcNAcase (OGA) which hydrolyses it. Here, we review the current knowledge on the emerging role of O-GlcNAc modification in the regulation of protein trafficking in mammalian cells, in classical and unconventional secretory pathways.

Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control.

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin ß1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin ß1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

Involvement of the pro-oncogenic enzyme fatty acid synthase in the hallmarks of cancer: a promising target in anti-cancer therapies.

An accelerated de novo lipogenesis (DNL) flux is a common characteristic of cancer cells required to sustain a high proliferation rate. The DNL enzyme fatty acid synthase (FASN) is overexpressed in many cancers and is pivotal for the increased production of fatty acids. There is increasing evidences of the involvement of FASN in several hallmarks of cancer linked to its ability to promote cell proliferation via membranes biosynthesis. In this review we discuss about the implication of FASN in the resistance to cell death and in the deregulation of cellular energetics by increasing nucleic acids, protein and lipid synthesis. FASN also promotes cell proliferation, cell invasion, metastasis and angiogenesis by enabling the building of lipid rafts and consequently to the localization of oncogenic receptors such as HER2 and c-Met in membrane microdomains. Finally, FASN is involved in immune escape by repressing the activation of pro-inflammatory cells and promoting the recruitment of M2 macrophages and T regulatory cells in the tumor microenvironment. Here, we provide an overview of the involvement of the pro-oncogenic enzyme in the hallmarks of cancer making FASN a promising target in anti-cancer therapy to circumvent resistance to chemotherapies.

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylatedglycoconjugates: a comparative study.

Sialylation of cell surface glycans plays an essential role in cell-cell interaction and communication of cells with their microenvironment. Among the tools that have been developed for the study of sialylation in living cells, metabolic oligosaccharide engineering (MOE) exploits the biosynthetic pathway of sialic acid (Sia) to incorporate unnatural monosaccharides into nascent sialylatedglycoconjugates, followed by their detection by a bioorthogonal ligation of a molecular probe. Among bioorthogonal reactions, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the only ligation where both reactive tags can be switched on the chemical reporter or on the probe, making this reaction very flexible and adaptable to various labeling strategies. Azide- and alkyne-modified ManNAc and Sia reporters have been widely used, but per-O-acetylated ManNAz (Ac4ManNAz) remains the most popular choice so far for tracking intracellular processing of sialoglycans and cell surface sialylation in various cells. Taking advantage of CuAAC, we compared the metabolic incorporation of ManNAl, ManNAz, SiaNAl, SiaNAz and Ac4ManNAz in the human colon cell lines CCD841CoN, HT29 and HCT116, and in the two gold standard cell lines, HEK293 and HeLa. Using complementary approaches, we showed marked differences in the efficiency of labeling of sialoglycoproteins between the different chemical reporters in a given cell line, and that switching the azide and alkyne bioorthogonal tags on the analogs highly impacted their metabolic incorporation in the human colon cell lines. Our results also indicated that ManNAz was the most promiscuous metabolized reporter to study sialylation in these cells.

[Fatty acid synthase, a "multi-FASet" enzyme]. / L'acide gras synthase, une enzyme « multi-FASette ¼.

In mammals, fatty acids are supplied by diet or synthesized de novo by fatty acid synthase (FASN). Beyond its key role in energy storage, FASN is involved in many biological processes. It actively participates in the synthesis of membrane components necessary for cell division, protein modification, cell signaling and cell proliferation. In this review, we discuss the various physiological functions of FASN as well as its involvement in cancer, the expression of the lipogenic enzyme being particularly high in this disease.

Title: L'acide gras synthase, une enzyme « multi-FASette ¼. Abstract: Chez les mammifères, les acides gras sont fournis par l'alimentation ou sont synthétisés de novo par l'acide gras synthase (FASN pour fatty acid synthase). Au-delà de son rôle clé dans le stockage de l'énergie, FASN est impliquée dans de nombreux processus biologiques. Elle participe activement à la synthèse des composants membranaires nécessaires à la division cellulaire, à la modification des protéines, à la signalisation et à la prolifération cellulaires. Dans cette revue, nous discutons des diverses fonctions physiologiques de FASN ainsi que de son implication dans les cancers, l'expression de cette enzyme lipogénique étant particulièrement élevée dans cette maladie.

Evaluation of the expression of fatty acid synthase and O-GlcNAc transferase in patients with liver cancer by exploration of transcriptome databases and experimental approaches.

Tumor occurrence and development are closely related to metabolism abnormalities. One of the metabolic networks that is dysregulated during carcinogenesis is the fatty acid synthesis pathway, which is mainly controlled by fatty acid synthase (FASN). We previously demonstrated in proliferating HepG2 liver cancer cells that FASN expression depends on the catalytic activity of O-GlcNAc transferase (OGT) and the activation of the mechanistic/mammalian target of rapamycin (mTOR) pathway. The aim of the present study was to go further in these investigations by analyzing datasets and tissues of patients with liver cancer. To that purpose, transcriptome databases were explored, and reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used. Database analyses revealed that FASN and OGT gene expression was higher in certain cancer tissues, including liver hepatocellular carcinoma, compared with that in non-cancerous tissues. At the protein level, FASN expression was higher in the liver cancer-derived cell lines HepG2 and Hep3B compared with the immortalized human hepatocytes IHH cell line. However, neither the expression of OGT nor of its product O-GlcNAcylation showed any significant difference among the three hepatic cell lines. Subsequently, the expression of FASN and OGT at the protein and mRNA levels was evaluated in human liver cancer and non-tumoral tissues from the same patients with different liver lesions. The results from western blotting demonstrated a significant increase in OGT ands O-GlcNAcylation expression in liver cancer tissues independently of the type of lesion characterizing the non-tumoral counterpart. As previously reported for HepG2 proliferating cells, the protein level of FASN was positively correlated with the activation of mTOR and, although a rather upward trend, a high variability in its expression was monitored between patients. However, the results from immunohistochemistry showed no particular modification for OGT and O-GlcNAcylation expression and a significant increase in FASN expression in cancer tissues compared with that in adjacent non-tumoral tissues. Non-significant changes were observed for FASN and OGT mRNA levels between tumoral and non-tumoral samples, with a high variability between patients. Taken together, these results demonstrated that FASN expression was higher in hepatic cancer tissues in comparison with non-tumoral tissues. Furthermore, OGT expression and activity were shown to vary greatly between cell or cancer type, making any generalization difficult.

Thymidylate synthase O-GlcNAcylation: a molecular mechanism of 5-FU sensitization in colorectal cancer.

Alteration of O-GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.

Mitochondrial O-GlcNAc Transferase Interacts with and Modifies Many Proteins and Its Up-Regulation Affects Mitochondrial Function and Cellular Energy Homeostasis.

O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT's role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.

O-GlcNAcylation Prediction: An Unattained Objective.

BACKGROUND: O-GlcNAcylation is an essential post-translational modification (PTM) in mammalian cells. It consists in the addition of a N-acetylglucosamine (GlcNAc) residue onto serines or threonines by an O-GlcNAc transferase (OGT). Inhibition of OGT is lethal, and misregulation of this PTM can lead to diverse pathologies including diabetes, Alzheimer's disease and cancers. Knowing the location of O-GlcNAcylation sites and the ability to accurately predict them is therefore of prime importance to a better understanding of this process and its related pathologies. PURPOSE: Here, we present an evaluation of the current predictors of O-GlcNAcylation sites based on a newly built dataset and an investigation to improve predictions. METHODS: Several datasets of experimentally proven O-GlcNAcylated sites were combined, and the resulting meta-dataset was used to evaluate three prediction tools. We further defined a set of new features following the analysis of the primary to tertiary structures of experimentally proven O-GlcNAcylated sites in order to improve predictions by the use of different types of machine learning techniques. RESULTS: Our results show the failure of currently available algorithms to predict O-GlcNAcylated sites with a precision exceeding 9%. Our efforts to improve the precision with new features using machine learning techniques do succeed for equal proportions of O-GlcNAcylated and non-O-GlcNAcylated sites but fail like the other tools for real-life proportions where ~1.4% of S/T are O-GlcNAcylated. CONCLUSION: Present-day algorithms for O-GlcNAcylation prediction narrowly outperform random prediction. The inclusion of additional features, in combination with machine learning algorithms, does not enhance these predictions, emphasizing a pressing need for further development. We hypothesize that the improvement of prediction algorithms requires characterization of OGT's partners.

Dual regulation of fatty acid synthase (FASN) expression by O-GlcNAc transferase (OGT) and mTOR pathway in proliferating liver cancer cells.

Fatty acid synthase (FASN) participates in many fundamental biological processes, including energy storage and signal transduction, and is overexpressed in many cancer cells. We previously showed in a context of lipogenesis that FASN is protected from degradation by its interaction with O-GlcNAc transferase (OGT) in a nutrient-dependent manner. We and others also reported that OGT and O-GlcNAcylation up-regulate the PI3K/AKT/mTOR pathway that senses mitogenic signals and nutrient availability to drive cell cycle. Using biochemical and microscopy approaches, we show here that FASN co-localizes with OGT in the cytoplasm and, to a lesser extent, in the membrane fraction. This interaction occurs in a cell cycle-dependent manner, following the pattern of FASN expression. Moreover, we show that FASN expression depends on OGT upon serum stimulation. The level of FASN also correlates with the activation of the PI3K/AKT/mTOR pathway in hepatic cell lines, and in livers of obese mice and in a chronically activated insulin and mTOR signaling mouse model (PTEN-null mice). These results indicate that FASN is under a dual control of O-GlcNAcylation and mTOR pathways. In turn, blocking FASN with the small-molecule inhibitor C75 reduces both OGT and O-GlcNAcylation levels, and mTOR activation, highlighting a novel reciprocal regulation between these actors. In addition to the role of O-GlcNAcylation in tumorigenesis, our findings shed new light on how aberrant activity of FASN and mTOR signaling may promote the emergence of hepatic tumors.

Exploring the Potential of ß-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator ß-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of ß-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of ß-catenin in HeLa cells. The changes in O-GlcNAcylation of ß-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.

Identification of lipid raft glycoproteins obtained from boar spermatozoa.

The surface of the spermatozoa is coated with glycoproteins the redistribution of which during in vitro capacitation plays a key role in the subsequent fertilization process. Lipid rafts are membrane microdomains involved in signal transduction through receptors and include or recruit specific types of proteins and glycoproteins. Few studies have focused on identifying glycoproteins resident in the lipid rafts of spermatozoa. Proteins associated with lipid rafts modify their localization during capacitation. The objective of the study was to identify the glycoproteins associated with lipid rafts of capacitated boar spermatozoa through a lectin-binding assay coupled to mass spectrometry approach. From the proteomic profiles generated by the raft proteins extractions, we observed that after capacitation the intensity of some bands increased while that of others decreased. To determine whether the proteins obtained from lipid rafts are glycosylated, lectin blot assays were performed. Protein bands with a good resolution and showing significant glycosylation modifications after capacitation were analyzed by mass spectrometry. The bands of interest had an apparent molecular weight of 64, 45, 36, 34, 24, 18 and 15 kDa. We sequenced the 7 bands and 20 known or potential glycoproteins were identified. According to us, for ten of them this is the first time that their association with sperm lipid rafts is described (ADAM5, SPMI, SPACA1, Seminal plasma protein pB1, PSP-I, MFGE8, tACE, PGK2, SUCLA2, MDH1). Moreover, LYDP4, SPAM-1, HSP60, ZPBP1, AK1 were previously reported in lipid rafts of mouse and human spermatozoa but not in boar spermatozoa. We also found and confirmed the presence of ACR, ACRBP, AWN, AQN3 and PRDX5 in lipid rafts of boar spermatozoa. This paper provides an overview of the glycosylation pattern in lipid rafts of boar spermatozoa before and after capacitation. Further glycomic analysis is needed to determine the type and the variation of glycan chains of the lipid rafts glycoproteins on the surface of spermatozoa during capacitation and acrosome reaction.

OGT Controls the Expression and the Glycosylation of E-cadherin, and Affects Glycosphingolipid Structures in Human Colon Cell Lines.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation, and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affects overall N- and O-glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells.

O-GlcNAcylation Is Involved in the Regulation of Stem Cell Markers Expression in Colon Cancer Cells.

The dynamic O-linked-N-acetylglucosamine posttranslational modification of nucleocytoplasmic proteins has emerged as a key regulator of diverse cellular processes including several hallmarks of cancer. However, the role played by this modification in the establishment of CSC phenotype has been poorly studied so far and remains unclear. In this study we confirmed the previous reports showing that colon cancer cells exhibit higher O-GlcNAc basal levels than non-malignant cells, and investigated the role played by O-GlcNAcylation in the regulation of CSC phenotype. We found that the modification of O-GlcNAcylation levels by pharmacological inhibition of the O-GlcNAc-transferase enzyme that adds O-GlcNAc (OGT), but not of the enzyme that removes it (OGA), increased the expression of all stem cell markers tested in our colon malignant cell lines, and induced the appearance of a double positive (CD44+/CD133+) small stem cell-like subpopulation (which corresponded to 1-10%) that displayed very aggressive malignant phenotype such as increased clonogenicity and spheroid formation abilities in 3D culture. We reasoned that OGT inhibition would mimic in the tumor the presence of severe nutritional stress, and indeed, we demonstrated that nutritional stress reproduced in colon cancer cells the effects obtained with OGT inhibition. Thus, our data strongly suggests that stemness is regulated by HBP/O-GlcNAcylation nutrient sensing pathway, and that O-GlcNAc nutrient sensor represents an important survival mechanism in cancer cells under nutritional stressful conditions.

Identification of O-Glcnacylated Proteins in Trypanosoma cruzi.

Originally an anthropozoonosis in the Americas, Chagas disease has spread from its previous borders through migration. It is caused by the protozoan Trypanosoma cruzi. Differences in disease severity have been attributed to a natural pleomorphism in T. cruzi. Several post-translational modifications (PTMs) have been studied in T. cruzi, but to date no work has focused on O-GlcNAcylation, a highly conserved monosaccharide-PTM of serine and threonine residues mainly found in nucleus, cytoplasm, and mitochondrion proteins. O-GlcNAcylation is thought to regulate protein function analogously to protein phosphorylation; indeed, crosstalk between both PTMs allows the cell to regulate its functions in response to nutrient levels and stress. Herein, we demonstrate O-GlcNAcylation in T. cruzi epimastigotes by three methods: by using specific antibodies against the modification in lysates and whole parasites, by click chemistry labeling, and by proteomics. In total, 1,271 putative O-GlcNAcylated proteins and six modification sequences were identified by mass spectrometry (data available via ProteomeXchange, ID PXD010285). Most of these proteins have structural and metabolic functions that are essential for parasite survival and evolution. Furthermore, O-GlcNAcylation pattern variations were observed by antibody detection under glucose deprivation and heat stress conditions, supporting their possible role in the adaptive response. Given the numerous biological processes in which O-GlcNAcylated proteins participate, its identification in T. cruzi proteins opens a new research field in the biology of Trypanosomatids, improve our understanding of infection processes and may allow us to identify new therapeutic targets.

Cyclin D1 Stability Is Partly Controlled by O-GlcNAcylation.

Cyclin D1 is the regulatory partner of the cyclin-dependent kinases (CDKs) CDK4 or CDK6. Once associated and activated, the cyclin D1/CDK complexes drive the cell cycle entry and G1 phase progression in response to extracellular signals. To ensure their timely and accurate activation during cell cycle progression, cyclin D1 turnover is finely controlled by phosphorylation and ubiquitination. Here we show that the dynamic and reversible O-linked ß-N-Acetyl-glucosaminylation (O-GlcNAcylation) regulates also cyclin D1 half-life. High O-GlcNAc levels increase the stability of cyclin D1, while reduction of O-GlcNAcylation strongly decreases it. Moreover, elevation of O-GlcNAc levels through O-GlcNAcase (OGA) inhibition significantly slows down the ubiquitination of cyclin D1. Finally, biochemical and cell imaging experiments in human cancer cells reveal that the O-GlcNAc transferase (OGT) binds to and glycosylates cyclin D1. We conclude that O-GlcNAcylation promotes the stability of cyclin D1 through modulating its ubiquitination.

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex.

The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα-interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.