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Papanicolaou KN, Jung J, Ashok D, Zhang W, Modaressanavi A, Avila E, Foster DB, Zachara NE, O'Rourke B. Inhibiting O-GlcNAcylation impacts p38 and Erk1/2 signaling and perturbs cardiomyocyte hypertrophy. The Journal of biological chemistry 2023 299(3) 36642184
Abstract:
The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.
O-GlcNAc proteins:
M3K7, CREB1, MK03, MP2K2, HSPB1, MK01, MK14, MP2K1, CDC37
Species: Rattus norvegicus
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Sager RA, Woodford MR, Backe SJ, Makedon AM, Baker-Williams AJ, DiGregorio BT, Loiselle DR, Haystead TA, Zachara NE, Prodromou C, Bourboulia D, Schmidt LS, Linehan WM, Bratslavsky G, Mollapour M. Post-translational Regulation of FNIP1 Creates a Rheostat for the Molecular Chaperone Hsp90. Cell reports 2019 26(5) 30699359
Abstract:
The molecular chaperone Hsp90 stabilizes and activates client proteins. Co-chaperones and post-translational modifications tightly regulate Hsp90 function and consequently lead to activation of clients. However, it is unclear whether this process occurs abruptly or gradually in the cellular context. We show that casein kinase-2 phosphorylation of the co-chaperone folliculin-interacting protein 1 (FNIP1) on priming serine-938 and subsequent relay phosphorylation on serine-939, 941, 946, and 948 promotes its gradual interaction with Hsp90. This leads to incremental inhibition of Hsp90 ATPase activity and gradual activation of both kinase and non-kinase clients. We further demonstrate that serine/threonine protein phosphatase 5 (PP5) dephosphorylates FNIP1, allowing the addition of O-GlcNAc (O-linked N-acetylglucosamine) to the priming serine-938. This process antagonizes phosphorylation of FNIP1, preventing its interaction with Hsp90, and consequently promotes FNIP1 lysine-1119 ubiquitination and proteasomal degradation. These findings provide a mechanism for gradual activation of the client proteins through intricate crosstalk of post-translational modifications of the co-chaperone FNIP1.
O-GlcNAc proteins:
FNIP1
Species: Homo sapiens
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Drake WR, Hou CW, Zachara NE, Grimes CL. New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT. Journal of bioenergetics and biomembranes 2018 50(3) 29671171
Abstract:
O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.
O-GlcNAc proteins:
NOD2, NOD1
Species: Homo sapiens
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Kim DI, Cutler JA, Na CH, Reckel S, Renuse S, Madugundu AK, Tahir R, Goldschmidt HL, Reddy KL, Huganir RL, Wu X, Zachara NE, Hantschel O, Pandey A. BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation. Journal of proteome research 2018 17(2) 29249144
Abstract:
Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.
O-GlcNAc proteins:
CALU, RLA2, SAP, SON, NU214, NU153, HCFC1, NUP98, F193A, NOTC2, BPTF, UBP2L, AMOT, UBAP2, RPRD2, LIN54, ZC3HE, POGZ, EMSY, YTHD3, WNK1, CDK12, MINP1, HYOU1, S23IP
Species: Homo sapiens
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Taparra K, Wang H, Malek R, Lafargue A, Barbhuiya MA, Wang X, Simons BW, Ballew M, Nugent K, Groves J, Williams RD, Shiraishi T, Verdone J, Yildirir G, Henry R, Zhang B, Wong J, Wang KK, Nelkin BD, Pienta KJ, Felsher D, Zachara NE, Tran PT. O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis. The Journal of clinical investigation 2018 128(11) 30130254
Abstract:
Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.
O-GlcNAc proteins:
MYC
Species: Homo sapiens
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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 chemistry 2017 292(36) 28739801
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.
O-GlcNAc proteins:
NF2L2
Species: Homo sapiens
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Lee A, Miller D, Henry R, Paruchuri VD, O'Meally RN, Boronina T, Cole RN, Zachara NE. Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress. Journal of proteome research 2016 15(12) 27669760
Abstract:
O-Linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel "G5-lectibody" immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic reversed phase chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins that were identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggests that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cell's response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.
O-GlcNAc proteins:
E9Q3G8, ATX2, PTN23, NU214, UBP2L, EMSY, CNOT2, ANR17, CILK1
Species: Mus musculus
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Hou CW, Mohanan V, Zachara NE, Grimes CL. Identification and biological consequences of the O-GlcNAc modification of the human innate immune receptor, Nod2. Glycobiology 2016 26(1) 26369908
Abstract:
Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular receptor that can sense the bacterial peptidoglycan component, muramyl dipeptide. Upon activation, Nod2 induces the production of various inflammatory molecules such as cytokines and chemokines. Genetic linkage analysis identified and revealed three major mutations in Nod2 that are associated with the development of Crohn's disease. The objective of this study is to further characterize this protein by determining whether Nod2 is posttranslationally modified by O-N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one type of posttranslational modification in which the O-GlcNAc transferase transfers GlcNAc from UDP-GlcNAc to selected serine and threonine residues of intracellular proteins. We found that wild-type Nod2 and a Nod2 Crohn's-associated variant are O-GlcNAcylated and this modification affects Nod2's ability to signal via the nuclear factor kappa B pathway.
O-GlcNAc proteins:
NOD2
Species: Homo sapiens
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Zachara NE, Molina H, Wong KY, Pandey A, Hart GW. The dynamic stress-induced "O-GlcNAc-ome" highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino acids 2011 40(3) 20676906
Abstract:
The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.
O-GlcNAc proteins:
NU214, HCFC1, TPIS, ZFR
Species: Homo sapiens
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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 chemistry 2005 280(38) 16027160
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.
O-GlcNAc proteins:
SP1, TYY1
Species: Homo sapiens
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O'Donnell N, Zachara NE, Hart GW, Marth JD. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Molecular and cellular biology 2004 24(4) 14749383
Abstract:
The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling pathways.
O-GlcNAc proteins:
SP1, GSK3B
Species: Mus musculus
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