Sharma NS, Gupta VK, Dauer P, Kesh K, Hadad R, Giri B, Chandra A, Dudeja V, Slawson C, Banerjee S, Vickers SM, Saluja A, Banerjee S.
O-GlcNAc modification of Sox2 regulates self-renewal in pancreatic cancer by promoting its stability.
Abstract: Pancreatic adenocarcinoma (PDAC) claims more than 90% of the patients diagnosed with the disease owing to its aggressive biology that is manifested by high rate of tumor recurrence. Aberrant upregulation in the transcriptional activity of proteins involved in self-renewal like Sox2, Oct4 and Nanog is instrumental in these recurrence phenomena. In cancer, Sox2 is aberrantly "turned-on" leading to activation of downstream genes those results in relapse of the tumor. Molecular mechanisms that regulate the activity of Sox2 in PDAC are not known. In the current study, we have studied the how glycosylation of Sox2 by O-GlcNAc transferase (OGT) can affect its transcriptional activity and thus regulate self-renewal in cancer. Methods: RNA-Seq analysis of CRISPR-OGTi PDAC cells indicated a deregulation of differentiation and self-renewal pathways in PDAC. Pancreatic tumor burden following inhibition of OGT in vivo was done by using small molecule inhibitor, OSMI, on subcutaneous implantation of PDAC cells. Sox2 activity assay was performed by Dual Luciferase Reporter Assay kit. Results: Our study shows for the first time that in PDAC, glycosylation of Sox2 by OGT stabilizes it in the nucleus. Site directed mutagenesis of this site (S246A) prevents this modification. We further show that inhibition of OGT delayed initiation of pancreatic tumors by inhibition of Sox2. We also show that targeting OGT in vivo with a small molecule-inhibitor OSMI, results in decreased tumor burden in PDAC. Conclusion: Understanding this mechanism of SOX2 regulation by its glycosylation is expected to pave the way for development of novel therapy that has the potential to eradicate the cells responsible for tumor-recurrence.
Machacek M, Saunders H, Zhang Z, Tan EP, Li J, Li T, Villar MT, Artigues A, Lydic T, Cork G, Slawson C, Fields PE.
Elevated O-GlcNAcylation enhances pro-inflammatory Th17 function by altering the intracellular lipid microenvironment.
The Journal of biological chemistry2019
Abstract: Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.
Trinca GM, Goodman ML, Papachristou EK, D'Santos CS, Chalise P, Madan R, Slawson C, Hagan CR.
O-GlcNAc-Dependent Regulation of Progesterone Receptor Function in Breast Cancer.
Hormones & cancer2018
Abstract: Emerging clinical trial data implicate progestins in the development of breast cancer. While the role for the progesterone receptor (PR) in this process remains controversial, it is clear that PR, a steroid-activated nuclear receptor, alters the transcriptional landscape of breast cancer. PR interacts with many different types of proteins, including transcriptional co-activators and co-repressors, transcription factors, nuclear receptors, and proteins that post-translationally modify PR (i.e., kinases and phosphatases). Herein, we identify a novel interaction between PR and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of a single N-acetylglucosamine sugar, referred to as O-GlcNAc, to acceptor serines and threonines in target proteins. This interaction between PR and OGT leads to the post-translational modification of PR by O-GlcNAc. Moreover, we show that O-GlcNAcylated PR is more transcriptionally active on PR-target genes, despite the observation that PR messenger RNA and protein levels are decreased when O-GlcNAc levels are high. O-GlcNAcylation in breast cancer is clinically relevant, as we show that O-GlcNAc levels are higher in breast cancer as compared to matched normal tissues, and PR-positive breast cancers have higher levels of OGT. These data predict that under conditions where O-GlcNAc levels are high (breast cancer), PR, through an interaction with the modifying enzyme OGT, will exhibit increased O-GlcNAcylation and potentiated transcriptional activity. Therapeutic strategies aimed at altering cellular O-GlcNAc levels may have profound effects on PR transcriptional activity in breast cancer.
Tan EP, McGreal SR, Graw S, Tessman R, Koppel SJ, Dhakal P, Zhang Z, Machacek M, Zachara NE, Koestler DC, Peterson KR, Thyfault JP, Swerdlow RH, Krishnamurthy P, DiTacchio L, Apte U, Slawson C.
Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.
The Journal of biological chemistry2017
Abstract: Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.
de Queiroz RM, Madan R, Chien J, Dias WB, Slawson C.
Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells.
The Journal of biological chemistry2016
Abstract: O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.
Zhang Z, Costa FC, Tan EP, Bushue N, DiTacchio L, Costello CE, McComb ME, Whelan SA, Peterson KR, Slawson C.
O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.
The Journal of biological chemistry2016
Abstract: One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter.
Ramirez-Correa GA, Ma J, Slawson C, Zeidan Q, Lugo-Fagundo NS, Xu M, Shen X, Gao WD, Caceres V, Chakir K, DeVine L, Cole RN, Marchionni L, Paolocci N, Hart GW, Murphy AM.
Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle.
Abstract: Contractile dysfunction and increased deposition of O-linked β-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined β-elimination/Michael addition with tandem mass tags (TMT)-labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca(2+) in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.
Gao X, Wang X, Pham TH, Feuerbacher LA, Lubos ML, Huang M, Olsen R, Mushegian A, Slawson C, Hardwidge PR.
NleB, a bacterial effector with glycosyltransferase activity, targets GAPDH function to inhibit NF-κB activation.
Cell host & microbe2013
Abstract: Modulation of NF-κB-dependent responses is critical to the success of attaching/effacing (A/E) human pathogenic E. coli (EPEC and EHEC) and the natural mouse pathogen Citrobacter rodentium. NleB, a highly conserved type III secretion system effector of A/E pathogens, suppresses NF-κB activation, but the underlying mechanisms are unknown. We identified the mammalian glycolysis enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an NleB-interacting protein. Further, we discovered that GAPDH interacts with the TNF receptor-associated factor 2 (TRAF2), a protein required for TNF-α-mediated NF-κB activation, and regulates TRAF2 polyubiquitination. During infection, NleB functions as a translocated N-acetyl-D-glucosamine (O-GlcNAc) transferase that modifies GAPDH. NleB-mediated GAPDH O-GlcNAcylation disrupts the TRAF2-GAPDH interaction to suppress TRAF2 polyubiquitination and NF-κB activation. Eliminating NleB O-GlcNAcylation activity attenuates C. rodentium colonization of mice. These data identify GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by A/E pathogens to inhibit NF-κB-dependent host innate immune responses.
Wells L, Slawson C, Hart GW.
The E2F-1 associated retinoblastoma-susceptibility gene product is modified by O-GlcNAc.
Abstract: The retinoblastoma-susceptibility gene product (pRB) is a classical tumor suppressor. pRB regulates a number of cellular processes including proliferation, differentiation, and apoptosis. One of the essential mechanisms by which pRB, and the related p107 and p130 family members, act is through its interactions with the E2F class of transcription factors. E2F-1 transcription is necessary for entry into S-phase during the cell-cycle. pRB binds E2F-1 and represses transcription via recruitment of a histone deacetylase complex and by preventing co-activator complexes from binding E2F-1. Current dogma suggests that phosphorylation of pRB during mid- to late-G1 leads to release of E2F-1 and E2F-1 dependent transcriptional activation of essential S-phase genes. Here we show that pRB, and the related p107 protein, are modified by O-linked β-N-acetylglucosamine (O-GlcNAc) in an in vitro transcription/translation system. Furthermore, we show in vivo that pRB is more heavily glycosylated in G1 of the cell-cycle when pRB is known to be in an active, hypophosphorylated state. Finally, we demonstrate that E2F-1 associated pRB is modified by O-GlcNAc. These studies suggest that regulation of pRB function(s) may be controlled by dynamic O-GlcNAc modification, as well as phosphorylation.
Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW.
Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis.
Abstract: Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
Li X, Molina H, Huang H, Zhang YY, Liu M, Qian SW, Slawson C, Dias WB, Pandey A, Hart GW, Lane MD, Tang QQ.
O-linked N-acetylglucosamine modification on CCAAT enhancer-binding protein beta: role during adipocyte differentiation.
The Journal of biological chemistry2009
Abstract: CCAAT enhancer-binding protein (C/EBP)beta is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPbeta undergoes sequential phosphorylation during 3T3-L1 adipocyte differentiation. Phosphorylation on Thr(188) by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser(184)/Thr(179) by GSK3beta, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPbeta. Here we show that C/EBPbeta is modified by O-GlcNAc, a dynamic single sugar modification found on nucleocytoplasmic proteins. The GlcNAcylation sites are Ser(180) and Ser(181), which are in the regulation domain and are very close to the phosphorylation sites (Thr(188), Ser(184), and Thr(179)) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser(180) and Ser(181) prevents phosphorylation on Thr(188), Ser(184), and Thr(179), as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPbeta delayed the adipocyte differentiation program. Mutation of both Ser(180) and Ser(181) to Ala significantly increase the transcriptional activity of C/EBPbeta. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPbeta.
Slawson C, Lakshmanan T, Knapp S, Hart GW.
A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin.
Molecular biology of the cell2008
Abstract: O-linked beta-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.
Slawson C, Zachara NE, Vosseller K, Cheung WD, Lane MD, Hart GW.
Perturbations in O-linked beta-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis.
The Journal of biological chemistry2005
Abstract: The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification that is rapidly responsive to morphogens, hormones, nutrients, and cellular stress. Here we show that O-GlcNAc is an important regulator of the cell cycle. Increased O-GlcNAc (pharmacologically or genetically) results in growth defects linked to delays in G2/M progression, altered mitotic phosphorylation, and cyclin expression. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc, induces a mitotic exit phenotype accompanied by a delay in mitotic phosphorylation, altered cyclin expression, and pronounced disruption in nuclear organization. Overexpression of the O-GlcNAc transferase, the enzyme that adds O-GlcNAc, results in a polyploid phenotype with faulty cytokinesis. Notably, O-GlcNAc transferase is concentrated at the mitotic spindle and midbody at M phase. These data suggest that dynamic O-GlcNAc processing is a pivotal regulatory component of the cell cycle, controlling cell cycle progression by regulating mitotic phosphorylation, cyclin expression, and cell division.