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Regulation of Protein Degradation by O-GlcNAcylation: Crosstalk with Ubiquitination*

  • Hai-Bin Ruan
    Affiliations
    Program in Integrative Cell Signaling and Neurobiology of Metabolism and Section of Comparative Medicine, Department of Cellular & Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520;
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  • Yongzhan Nie
    Affiliations
    State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 127 West Changle Road, Xi'an, Shaanxi 710032, China;
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  • Xiaoyong Yang
    Correspondence
    To whom correspondence should be addressed: Section of Comparative Medicine, Yale University School of Medicine, P.O. Box 208016, New Haven, CT 06520, Tel.:1-203-737-1446, Fax:1-203-785-7499,
    Affiliations
    Program in Integrative Cell Signaling and Neurobiology of Metabolism and Section of Comparative Medicine, Department of Cellular & Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520;

    Department of Cellular & Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520
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  • Author Footnotes
    * This work was supported by NIH R01 DK089098, American Diabetes Association, and Ellison Medical Foundation to X.Y.; a Brown-Coxe Postdoctoral Fellowship to H.B.R.; and NSFC 81120108005/81225003 to Y.N.
Open AccessPublished:July 03, 2013DOI:https://doi.org/10.1074/mcp.R113.029751
      The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Misfolded, damaged, and unwanted proteins are tagged with a chain of ubiquitin moieties for degradation by the proteasome, which is critical for cellular homeostasis. In this review, we summarize the current knowledge of the interplay between O-GlcNAcylation and ubiquitination in the control of protein degradation. Understanding the mechanisms of action of O-GlcNAcylation in the ubiquitin-proteosome system shall facilitate the development of therapeutics for human diseases such as cancer, metabolic syndrome, and neurodegenerative diseases.

      THE UBIQUITIN-PROTEASOME SYSTEM

      Ubiquitination is a post-translational process by which ubiquitin is covalently attached to the lysine residues of target proteins. Ubiquitin is an 8.5-kDa protein that exists in all eukaryotic cells (
      • Goldstein G.
      • Scheid M.
      • Hammerling U.
      • Schlesinger D.H.
      • Niall H.D.
      • Boyse E.A.
      Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.
      ). It is encoded in mammals by four genes. The UBB and UBC genes encode polyubiquitin precursors, whereas UBA52 and RPS27A genes encode fusion proteins composed of a ubiquitin and the ribosomal proteins L40 and S27a, respectively (
      • Kimura Y.
      • Tanaka K.
      Regulatory mechanisms involved in the control of ubiquitin homeostasis.
      ). The ubiquitination process consists a cascade of reactions: (i) activation of ubiquitin by the ubiquitin-activating enzyme (E1), (ii) transfer of ubiquitin from E1 to a ubiquitin-conjugating enzyme (E2), and (iii) recognizing target proteins and mediating transfer of ubiquitin from E2 to the target by a ubiquitin ligase (E3) (
      • Hershko A.
      • Ciechanover A.
      The ubiquitin system.
      ).
      Polyubiquitination (at least four subunits) through lysine 48 (K48) of ubiquitin normally marks target proteins for proteasomal degradation. The ubiquitin-proteasome system (UPS)
      The abbreviations used are:
      ChREBP
      carbohydrate-response element-binding protein
      CK2
      casein kinase 2
      CRTC2
      cAMP-response element-binding-protein-regulated transcription coactivator
      DUB
      deubiquitinase
      ERβ
      estrogen receptor β
      HCF-1
      host cell factor C1
      O-GlcNAc
      O-linked β-N-acetylglucosamine
      OGT
      O-GlcNAc transferase
      PGC-1α
      peroxisome proliferator-activated receptor gamma co-activator 1-α
      PTM
      post-translational modification
      UPS
      ubiquitin-proteasome system.
      is the key machinery by which cells dispose of misfolded and damaged proteins in order to maintain cellular homeostasis. In addition, monoubiquitination through K48 or polyubiquitination through other lysine residues of ubiquitin regulates distinct cellular processes, including subcellular localization, endocytosis, and enzymatic activity (
      • Ikeda F.
      • Dikic I.
      Atypical ubiquitin chains: new molecular signals.
      ,
      • Hicke L.
      Protein regulation by monoubiquitin.
      ).
      Crosstalk between different types of post-translational modifications (PTMs) encodes a wealth of biological information. It is known that ubiquitination and other forms of PTMs are mutually regulated. A large body of evidence shows that phosphorylation and ubiquitination are connected either positively or negatively (
      • Hunter T.
      The age of crosstalk: phosphorylation, ubiquitination, and beyond.
      ,
      • Gao M.
      • Karin M.
      Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli.
      ). Regulatory crosstalk between lysine acetylation and ubiquitination has been shown to control protein stability (
      • Caron C.
      • Boyault C.
      • Khochbin S.
      Regulatory cross-talk between lysine acetylation and ubiquitination: role in the control of protein stability.
      ). Crosstalk between histone methylation and ubiquitination is involved in gene expression and protein stability (
      • Shukla A.
      • Chaurasia P.
      • Bhaumik S.R.
      Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability.
      ).

      O-GlcNAc Modification and Its Interplay with Other PTMs

      Thousands of cytoplasmic and nuclear proteins are modified by a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety at serine (S) or threonine (T) residues, termed O-GlcNAcylation (
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ,
      • Hanover J.A.
      • Krause M.W.
      • Love D.C.
      Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation.
      ). O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), whereas the reverse reaction is mediated by O-GlcNAcase (OGA, NCOAT, or MGEA5). UDP-GlcNAc, the donor substrate for O-GlcNAcylation, is derived from extracellular glucose through the hexosamine biosynthetic pathway. Because UDP-GlcNAc and protein O-GlcNAc levels in the cell fluctuate with the availability of glucose, free fatty acids, uridine, and the amino acid glutamine, O-GlcNAc is proposed as a nutrient sensor and metabolic regulator (
      • Wells L.
      • Vosseller K.
      • Hart G.W.
      A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.
      ,
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ). This dynamic and reversible modification is emerging as a key regulator of diverse cellular processes, such as signal transduction, transcription, translation, and cytoskeletal functions (
      • Yang X.
      • Su K.
      • Roos M.D.
      • Chang Q.
      • Paterson A.J.
      • Kudlow J.E.
      O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability.
      ,
      • Yang X.
      • Zhang F.
      • Kudlow J.E.
      Recruitment of O-GlcNAc transferase to promoters by corepressor mSin3A: coupling protein O-GlcNAcylation to transcriptional repression.
      ,
      • Yang X.
      • Ongusaha P.P.
      • Miles P.D.
      • Havstad J.C.
      • Zhang F.
      • So W.V.
      • Kudlow J.E.
      • Michell R.H.
      • Olefsky J.M.
      • Field S.J.
      • Evans R.M.
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
      ). Aberrant O-GlcNAcylation has been implicated in a spectrum of human diseases, including diabetes, cancer, cardiovascular disease, and Alzheimer disease.
      Since its discovery in 1984, O-GlcNAcylation has been extensively studied in relationship with phosphorylation (
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ,
      • Torres C.R.
      • Hart G.W.
      Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc.
      ). Interplay between O-GlcNAcylation and other PTMs is emerging as an important area of investigation. It has been shown that OGT overexpression alters the acetylation and methylation of histones and the activity of an arginine methyltransferase, CARM1 (
      • Sakabe K.
      • Hart G.W.
      O-GlcNAc transferase regulates mitotic chromatin dynamics.
      ). Allison et al. show that O-GlcNAcylation of RelA at T305 is required in order for p300-mediated acetylation at K310 to fully activate NF-κB transcription (
      • Allison D.F.
      • Wamsley J.J.
      • Kumar M.
      • Li D.
      • Gray L.G.
      • Hart G.W.
      • Jones D.R.
      • Mayo M.W.
      Modification of RelA by O-linked N-acetylglucosamine links glucose metabolism to NF-kappaB acetylation and transcription.
      ). O-GlcNAcylation of a histone lysine methyltranferase, MLL5, promotes methylation of H3K4 to facilitate retinoic-acid-induced granulopoiesis (
      • Fujiki R.
      • Chikanishi T.
      • Hashiba W.
      • Ito H.
      • Takada I.
      • Roeder R.G.
      • Kitagawa H.
      • Kato S.
      GlcNAcylation of a histone methyltransferase in retinoic-acid-induced granulopoiesis.
      ). Recent studies reveal that the ten-eleven translocation proteins TET2 and TET3 form a complex with OGT that sustains H3K4 methylation through O-GlcNAcylating host cell factor C1 (HCF-1), a component of the H3K4 methyltransferase SET1/COMPASS complex (
      • Deplus R.
      • Delatte B.
      • Schwinn M.K.
      • Defrance M.
      • Mendez J.
      • Murphy N.
      • Dawson M.A.
      • Volkmar M.
      • Putmans P.
      • Calonne E.
      • Shih A.H.
      • Levine R.L.
      • Bernard O.
      • Mercher T.
      • Solary E.
      • Urh M.
      • Daniels D.L.
      • Fuks F.
      TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS.
      ). Meanwhile, a growing body of evidence demonstrates that O-GlcNAcylation regulates mono- and polyubiquitination, protein stability, and proteasome function, which is the focus of this review.

      O-GlcNAcylation Regulates Protein Ubiquitination via Phosphorylation

      Because O-GlcNAcylation can affect phosphorylation (
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ) and phosphorylation can regulate ubiquitination (
      • Hunter T.
      The age of crosstalk: phosphorylation, ubiquitination, and beyond.
      ), it is conceivable that O-GlcNAcylation controls protein ubiquitination and stability through interplay with phosphorylation (Table I, Fig. 1A), as exemplified below.
      Table IList of proteins for which stability is regulated by O-GlcNAc signaling
      ProteinO-GlcNAc siteExpression and stabilityUbiquitinationMechanismFunctionReference
      p53S149Increased by O-GlcNAcDecreased by O-GlcNAcReduce phosphorylation at T155Cancer(
      • Yang W.H.
      • Kim J.E.
      • Nam H.W.
      • Ju J.W.
      • Kim H.S.
      • Kim Y.S.
      • Cho J.W.
      Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability.
      )
      Δ-lactoferrinS10Increased by O-GlcNAcDecreased by O-GlcNAcCompete with phosphorylation at S10Cell cycle arrest and apoptosis(
      • Hardiville S.
      • Hoedt E.
      • Mariller C.
      • Benaissa M.
      • Pierce A.
      O-GlcNAcylation/phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin.
      )
      Snail1S112Increased by O-GlcNAcDecreased by O-GlcNAcBlock phosphorylationEpithelial-mesenchymal transition(
      • Park S.Y.
      • Kim H.S.
      • Kim N.H.
      • Ji S.
      • Cha S.Y.
      • Kang J.G.
      • Ota I.
      • Shimada K.
      • Konishi N.
      • Nam H.W.
      • Hong S.W.
      • Yang W.H.
      • Roth J.
      • Yook J.I.
      • Cho J.W.
      Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.
      )
      ERβS16Increased by O-GlcNAcN/DCompete with phosphorylation at S16Transcription(
      • Cheng X.
      • Hart G.W.
      Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta: post-translational regulation of turnover and transactivation activity.
      )
      CK2αS347Reduced by O-GlcNAcN/DReduce phosphorylation at T344Cell proliferation(
      • Tarrant M.K.
      • Rho H.S.
      • Xie Z.
      • Jiang Y.L.
      • Gross C.
      • Culhane J.C.
      • Yan G.
      • Qian J.
      • Ichikawa Y.
      • Matsuoka T.
      • Zachara N.
      • Etzkorn F.A.
      • Hart G.W.
      • Jeong J.S.
      • Blackshaw S.
      • Zhu H.
      • Cole P.A.
      Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
      )
      CRTC2S70, S171N/DN/DCompete with phosphorylation at S70, S171Gluconeogenesis(
      • Dentin R.
      • Liu Y.
      • Koo S.H.
      • Hedrick S.
      • Vargas T.
      • Heredia J.
      • Yates 3rd, J.
      • Montminy M.
      Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2.
      ,
      • Dentin R.
      • Hedrick S.
      • Xie J.
      • Yates 3rd, J.
      • Montminy M.
      Hepatic glucose sensing via the CREB coactivator CRTC2.
      )
      PGC-1αS333Increased by O-GlcNAcDecreased by O-GlcNAcRecruit BAP1Gluconeogenesis(
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      )
      ClockSite N/DIncreased by O-GlcNAcDecreased by O-GlcNAcRecruit BAP1Circadian rhythm(
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      )
      Bmal1S418Increased by O-GlcNAcDecreased by O-GlcNAcRecruit BAP1Circadian rhythm(
      • Ma Y.T.
      • Luo H.
      • Guan W.J.
      • Zhang H.
      • Chen C.
      • Wang Z.
      • Li J.D.
      O-GlcNAcylation of BMAL1 regulates circadian rhythms in NIH3T3 fibroblasts.
      ,
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      )
      ChREBPSite N/DIncreased by O-GlcNAcDecreased by O-GlcNAcLipogenesis(
      • Guinez C.
      • Filhoulaud G.
      • Rayah-Benhamed F.
      • Marmier S.
      • Dubuquoy C.
      • Dentin R.
      • Moldes M.
      • Burnol A.F.
      • Yang X.
      • Lefebvre T.
      • Girard J.
      • Postic C.
      O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver.
      ,
      • Ido-Kitamura Y.
      • Sasaki T.
      • Kobayashi M.
      • Kim H.J.
      • Lee Y.S.
      • Kikuchi O.
      • Yokota-Hashimoto H.
      • Iizuka K.
      • Accili D.
      • Kitamura T.
      Hepatic FoxO1 integrates glucose utilization and lipid synthesis through regulation of Chrebp O-glycosylation.
      )
      Keratins 8/18 (K8/18)K8 (N/D)Reduced by O-GlcNAcIncreased by O-GlcNAcFilament architecture(
      • Srikanth B.
      • Vaidya M.M.
      • Kalraiya R.D.
      O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.
      )
      K18 (S29/S30/S48)
      A20Site N/DReduced by high glucoseIncreased by high glucoseAtherosclerosis(
      • Shrikhande G.V.
      • Scali S.T.
      • da Silva C.G.
      • Damrauer S.M.
      • Csizmadia E.
      • Putheti P.
      • Matthey M.
      • Arjoon R.
      • Patel R.
      • Siracuse J.J.
      • Maccariello E.R.
      • Andersen N.D.
      • Monahan T.
      • Peterson C.
      • Essayagh S.
      • Studer P.
      • Guedes R.P.
      • Kocher O.
      • Usheva A.
      • Veves A.
      • Kaczmarek E.
      • Ferran C.
      O-glycosylation regulates ubiquitination and degradation of the anti-inflammatory protein A20 to accelerate atherosclerosis in diabetic ApoE-null mice.
      )
      β-cateninSite N/DIncreased by HBP fluxN/DCell proliferation(
      • Olivier-Van Stichelen S.
      • Guinez C.
      • Mir A.M.
      • Perez-Cervera Y.
      • Liu C.
      • Michalski J.C.
      • Lefebvre T.
      The hexosamine biosynthetic pathway and O-GlcNAcylation drive the expression of beta-catenin and cell proliferation.
      )
      p67eIF2S60/T62/S63Increased by O-GlcNAcN/DProtein synthesis(
      • Datta R.
      • Choudhury P.
      • Ghosh A.
      • Datta B.
      A glycosylation site, 60SGTS63, of p67 is required for its ability to regulate the phosphorylation and activity of eukaryotic initiation factor 2alpha.
      )
      FoxM1Not O-GlcNAcylatedIncreased by OGTN/DBreast cancer(
      • Caldwell S.A.
      • Jackson S.R.
      • Shahriari K.S.
      • Lynch T.P.
      • Sethi G.
      • Walker S.
      • Vosseller K.
      • Reginato M.J.
      Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1.
      )
      PlakoglobinSite N/DIncreased by OGTN/DCell–cell adhesion(
      • Hu P.
      • Berkowitz P.
      • Madden V.J.
      • Rubenstein D.S.
      Stabilization of plakoglobin and enhanced keratinocyte cell-cell adhesion by intracellular O-glycosylation.
      )
      Sp1Site N/DReduced upon starvationN/DTranscription(
      • Han I.
      • Kudlow J.E.
      Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility.
      )
      Nkx2.5Site N/DReduced by OGA inhibitorsN/DDiabetic cardiomyopathy(
      • Kim H.S.
      • Woo J.S.
      • Joo H.J.
      • Moon W.K.
      Cardiac transcription factor Nkx2.5 is downregulated under excessive O-GlcNAcylation condition.
      )
      SirT1Not known whether O-GlcNAcylatedCytosolic level decreased by HBPN/Dβ cell apoptosis(
      • Lafontaine-Lacasse M.
      • Dore G.
      • Picard F.
      Hexosamines stimulate apoptosis by altering SIRT1 action and levels in rodent pancreatic beta-cells.
      )
      N/D, not determined.

      p53

      The expression of the tumor suppressor p53 is tightly controlled by proteasomal degradation so as to maintain low levels under normal conditions and rapidly accumulate upon DNA damage (
      • Vousden K.H.
      p53: death star.
      ). The fate of p53 is dictated by a variety of PTMs, including phosphorylation, acetylation, methylation, ubiquitination, and O-GlcNAcylation (
      • Kruse J.P.
      • Gu W.
      SnapShot: p53 posttranslational modifications.
      ). Cho and colleagues demonstrate that the treatment of MCF-7 cells with an OGA inhibitor increases the level of O-GlcNAcylated p53 and decreases cell viability (
      • Yang W.H.
      • Kim J.E.
      • Nam H.W.
      • Ju J.W.
      • Kim H.S.
      • Kim Y.S.
      • Cho J.W.
      Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability.
      ). O-GlcNAcylation of p53 at S149 inhibits phosphorylation at T155 by the COP9 signalosome, thereby reducing p53 ubiquitination and degradation.

      Δ-lactoferrin

      Δ-lactoferrin is a transcription factor that induces cell cycle arrest by up-regulating the expression of genes including Skp1, DcpS, and Bax (
      • Mariller C.
      • Hardiville S.
      • Hoedt E.
      • Huvent I.
      • Pina-Canseco S.
      • Pierce A.
      Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor.
      ). Δ-lactoferrin expression is down-regulated in cancer cells, whereas its high-level expression is correlated with a good prognosis in human breast cancer (
      • Mariller C.
      • Hardiville S.
      • Hoedt E.
      • Huvent I.
      • Pina-Canseco S.
      • Pierce A.
      Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor.
      ). It has been shown that Δ-lactoferrin is reciprocally O-GlcNAcylated and phosphorylated at S10 (
      • Mariller C.
      • Hardiville S.
      • Hoedt E.
      • Benaissa M.
      • Mazurier J.
      • Pierce A.
      Proteomic approach to the identification of novel delta-lactoferrin target genes: characterization of DcpS, an mRNA scavenger decapping enzyme.
      ,
      • Hardiville S.
      • Hoedt E.
      • Mariller C.
      • Benaissa M.
      • Pierce A.
      O-GlcNAcylation/phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin.
      ). O-GlcNAcylation stabilizes Δ-lactoferrin and retains a basal level of transcriptional activity. Upon activation, Δ-lactoferrin is phosphorylated at S10, which promotes transcription and subsequent degradation through K379 polyubiquitination (
      • Hardiville S.
      • Hoedt E.
      • Mariller C.
      • Benaissa M.
      • Pierce A.
      O-GlcNAcylation/phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin.
      ). These studies point to the idea that protein functions can be precisely controlled by dynamic and coordinated changes in O-GlcNAcylation, phosphorylation, and ubiquitination.

      Snail1

      The zinc-finger protein Snail1 regulates epithelial-mesenchymal transition and tumor progression by repressing the transcription of E-cadherin, a major component of cell adhesion junctions (
      • Lee J.M.
      • Dedhar S.
      • Kalluri R.
      • Thompson E.W.
      The epithelial-mesenchymal transition: new insights in signaling, development, and disease.
      ). It has been shown that phosphorylation of Snail1 by casein kinase 1 and glycogen synthase kinase-3β promotes the ubiquitination and proteasomal degradation of Snail1 (
      • Xu Y.
      • Lee S.H.
      • Kim H.S.
      • Kim N.H.
      • Piao S.
      • Park S.H.
      • Jung Y.S.
      • Yook J.I.
      • Park B.J.
      • Ha N.C.
      Role of CK1 in GSK3beta-mediated phosphorylation and degradation of snail.
      ). Park et al. show that O-GlcNAcylation of Snail1 at S112 decreases glycogen synthase kinase-3β-mediated phosphorylation and increases the stability of the protein (
      • Park S.Y.
      • Kim H.S.
      • Kim N.H.
      • Ji S.
      • Cha S.Y.
      • Kang J.G.
      • Ota I.
      • Shimada K.
      • Konishi N.
      • Nam H.W.
      • Hong S.W.
      • Yang W.H.
      • Roth J.
      • Yook J.I.
      • Cho J.W.
      Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.
      ). Consistently, the pharmacological inhibition of OGA by PUGNAc increases the half-life of Snail1 by inhibiting ubiquitination. In response to hyperglycemia, O-GlcNAc modification of Snail1 down-regulates E-cadherin transcription and therefore promotes cell migration and invasive programs (
      • Park S.Y.
      • Kim H.S.
      • Kim N.H.
      • Ji S.
      • Cha S.Y.
      • Kang J.G.
      • Ota I.
      • Shimada K.
      • Konishi N.
      • Nam H.W.
      • Hong S.W.
      • Yang W.H.
      • Roth J.
      • Yook J.I.
      • Cho J.W.
      Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.
      ).

      Estrogen Receptor β

      The nuclear receptor estrogen receptor β (ERβ) mediates many aspects of estrogen action, including reproduction, inflammation, behavior, and energy metabolism (
      • Harris H.A.
      Estrogen receptor-beta: recent lessons from in vivo studies.
      ). Cheng and Hart demonstrate that ERβ is reciprocally modified by O-GlcNAcylation and phosphorylation at S16. The S16A mutant devoid of both modifications reduces ERβ turnover, whereas the S16E mutant mimicking constitutive phosphorylation has an increased turnover rate (
      • Cheng X.
      • Hart G.W.
      Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta: post-translational regulation of turnover and transactivation activity.
      ). A simple interpretation of these results is that O-GlcNAcylation protects the S16 site from phosphorylation and Pro-Glu-Ser-Thr region–mediated proteasomal degradation. However, whether this process involves the ubiquitination of ERβ remains unclear.

      Casein Kinase 2-α

      Casein kinase 2 (CK2) is a serine/threonine protein kinase that has been implicated in cell proliferation, DNA repair, circadian rhythm, and other cellular processes (
      • St-Denis N.A.
      • Litchfield D.W.
      Protein kinase CK2 in health and disease: from birth to death: the role of protein kinase CK2 in the regulation of cell proliferation and survival.
      ). Tarrant et al. demonstrate that phosphorylation of the catalytic subunit of CK2α at T344 increases protein stability by promoting the interaction with Pin1 (
      • Tarrant M.K.
      • Rho H.S.
      • Xie Z.
      • Jiang Y.L.
      • Gross C.
      • Culhane J.C.
      • Yan G.
      • Qian J.
      • Ichikawa Y.
      • Matsuoka T.
      • Zachara N.
      • Etzkorn F.A.
      • Hart G.W.
      • Jeong J.S.
      • Blackshaw S.
      • Zhu H.
      • Cole P.A.
      Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
      ). Moreover, the proximal S347 is modified by O-GlcNAc, which antagonizes T344 phosphorylation and leads to proteasomal degradation of CK2α (
      • Tarrant M.K.
      • Rho H.S.
      • Xie Z.
      • Jiang Y.L.
      • Gross C.
      • Culhane J.C.
      • Yan G.
      • Qian J.
      • Ichikawa Y.
      • Matsuoka T.
      • Zachara N.
      • Etzkorn F.A.
      • Hart G.W.
      • Jeong J.S.
      • Blackshaw S.
      • Zhu H.
      • Cole P.A.
      Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
      ). It will be interesting to explore the role of ubiquitination of CK2α in this context.

      cAMP-response Element-binding-protein-regulated Transcription Coactivator 2

      cAMP-response element-binding-protein-regulated transcription coactivator 2 (CRTC2) is a transcriptional coactivator for cAMP-response element-binding protein and an important regulator of gluconeogenesis in the liver (
      • Koo S.H.
      • Flechner L.
      • Qi L.
      • Zhang X.
      • Screaton R.A.
      • Jeffries S.
      • Hedrick S.
      • Xu W.
      • Boussouar F.
      • Brindle P.
      • Takemori H.
      • Montminy M.
      The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.
      ). Under fasting conditions, glucagon induces dephosphorylation of CRTC2 at S171, resulting in CRTC2 translocation into the nucleus to activate the transcription of gluconeogenic genes (
      • Koo S.H.
      • Flechner L.
      • Qi L.
      • Zhang X.
      • Screaton R.A.
      • Jeffries S.
      • Hedrick S.
      • Xu W.
      • Boussouar F.
      • Brindle P.
      • Takemori H.
      • Montminy M.
      The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.
      ). During feeding, insulin activates the Ser/Thr kinase SIK2 to phosphorylate CRTC2 at S171. Subsequently, phosphorylated CRTC2 translocates to the cytoplasm and undergoes ubiquitination-dependent degradation (
      • Dentin R.
      • Liu Y.
      • Koo S.H.
      • Hedrick S.
      • Vargas T.
      • Heredia J.
      • Yates 3rd, J.
      • Montminy M.
      Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2.
      ). Dentin et al. reveal that O-GlcNAcylation of CRTC2 at S70 and S171 competes with phosphorylation to suppress cytoplasmic sequestration, thereby contributing to hyperglycemia-induced hepatic gluconeogenesis (
      • Dentin R.
      • Hedrick S.
      • Xie J.
      • Yates 3rd, J.
      • Montminy M.
      Hepatic glucose sensing via the CREB coactivator CRTC2.
      ). Whether O-GlcNAcylation has a direct effect on CRTC2 ubiquitination and stability warrants further investigation.

      O-GlcNAcylation Stabilizes Proteins by Recruiting Deubiquitinase

      Using a proteomic approach, Ruan et al. recently identified a large number of putative OGT-binding proteins. Many proteins in the ubiquitination pathway, including ubiquitin precursors, E3 ubiquitin ligases, and deubiquitinases (DUBs), are enriched (Table II). Although the functions of these interactions have not been determined, these findings raise the possibility that O-GlcNAc signaling directly modulates the ubiquitin system (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ).
      Table IIPutative OGT-binding proteins involved in ubiquitination
      FamilySymbolName
      UbiquitinRPS27AUbiquitin and ribosomal protein S27a precursor
      UbiquitinUBA52Ubiquitin and ribosomal protein L40 precursor
      UbiquitinUBBUbiquitin B precursor
      UbiquitinUBCUBC ubiquitin C
      E3
      Putative E3s are annotated by Li et al. (85).
      DDB1DNA damage-binding protein 1
      E3HUWE1HECT, UBA, and WWE domain containing 1, E3 ubiquitin-protein ligase
      E3MYCBP2MYC binding protein 2, E3 ubiquitin-protein ligase
      E3PRPF19Pre-mRNA-processing factor 19
      DUB
      The inventory of DUBs is described by Nijman et al. (86).
      BAP1BRCA1 associated protein 1
      DUBOTUD4OTU domain-containing protein 4
      DUBPRPF8Pre-mRNA-processing-splicing factor 8
      DUBUSP9XUbiquitin specific protease 9
      Data from Ruan et al. (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ).
      a Putative E3s are annotated by Li et al. (
      • Li W.
      • Bengtson M.H.
      • Ulbrich A.
      • Matsuda A.
      • Reddy V.A.
      • Orth A.
      • Chanda S.K.
      • Batalov S.
      • Joazeiro C.A.
      Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.
      ).
      b The inventory of DUBs is described by Nijman et al. (
      • Nijman S.M.
      • Luna-Vargas M.P.
      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
      • Sixma T.K.
      • Bernards R.
      A genomic and functional inventory of deubiquitinating enzymes.
      ).

      Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-α

      Peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) is a key transcriptional cofactor that promotes mitochondrial biogenesis and hepatic gluconeogenesis. It integrates multiple metabolic signals and is extensively regulated by PTMs, including phosphorylation, methylation, acetylation, ubiquitination, and O-GlcNAcylation (
      • Fernandez-Marcos P.J.
      • Auwerx J.
      Regulation of PGC-1alpha, a nodal regulator of mitochondrial biogenesis.
      ). Ruan et al. demonstrate that OGT forms a glucose-sensitive complex with HCF-1 (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ). As a scaffold protein, HCF-1 recruits OGT to O-GlcNAcylate PGC-1α at S333. BAP1 is a DUB known to interact with HCF-1 through an HCF-1 binding motif (
      • Misaghi S.
      • Ottosen S.
      • Izrael-Tomasevic A.
      • Arnott D.
      • Lamkanfi M.
      • Lee J.
      • Liu J.
      • O'Rourke K.
      • Dixit V.M.
      • Wilson A.C.
      Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1.
      ,
      • Machida Y.J.
      • Machida Y.
      • Vashisht A.A.
      • Wohlschlegel J.A.
      • Dutta A.
      The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1.
      ). Ruan et al. further show that O-GlcNAcylation of PGC-1α facilitates the recruitment of BAP1 that deubiquitinates and stabilizes PGC-1α. This is the first demonstration that O-GlcNAcylation regulates protein ubiquitination and stability through a DUB. Diabetic animals have increased levels of HCF-1 and BAP1 in the liver, which is associated with increased PGC-1α levels and gluconeogenesis. Knockdown of OGT and HCF-1 improves glucose metabolism in diabetic db/db mice (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ). This study elucidates the role of the antagonism between O-GlcNAcylation and ubiquitination as a regulatory mechanism governing metabolic homeostasis.

      BMAL1/CLOCK

      The circadian clock functions to align physiological and behavioral processes with daily environmental cycles (
      • Bass J.
      Circadian topology of metabolism.
      ). The molecular clock involves a transcriptional feedback loop in which BMAL1 and CLOCK activate the Period (Per1, -2, and -3) and Cryptochrome (Cry1 and -2) genes. PERs and CRYs accumulate rhythmically and form an inhibitory complex against BMAL1/CLOCK to repress their own transcription (
      • Ko C.H.
      • Takahashi J.S.
      Molecular components of the mammalian circadian clock.
      ). The pace of the clock is controlled by various regulatory mechanisms, including PTMs of core clock proteins (
      • Bass J.
      • Takahashi J.S.
      Circadian integration of metabolism and energetics.
      ).
      The core clock proteins, including BMAL1, CLOCK, PER, and CRY, have been shown to be modified by O-GlcNAc in Drosophila and mammals (
      • Kim E.Y.
      • Jeong E.H.
      • Park S.
      • Jeong H.J.
      • Edery I.
      • Cho J.W.
      A role for O-GlcNAcylation in setting circadian clock speed.
      ,
      • Ma Y.T.
      • Luo H.
      • Guan W.J.
      • Zhang H.
      • Chen C.
      • Wang Z.
      • Li J.D.
      O-GlcNAcylation of BMAL1 regulates circadian rhythms in NIH3T3 fibroblasts.
      ,
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      ,
      • Kaasik K.
      • Kivimae S.
      • Allen J.J.
      • Chalkley R.J.
      • Huang Y.
      • Baer K.
      • Kissel H.
      • Burlingame A.L.
      • Shokat K.M.
      • Ptacek L.J.
      • Fu Y.H.
      Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock.
      ). BMAL1 and CLOCK are rhythmically modified and stabilized by O-GlcNAcylation (
      • Ma Y.T.
      • Luo H.
      • Guan W.J.
      • Zhang H.
      • Chen C.
      • Wang Z.
      • Li J.D.
      O-GlcNAcylation of BMAL1 regulates circadian rhythms in NIH3T3 fibroblasts.
      ,
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      ). Li et al. further demonstrate that the OGT–BAP1 complex O-GlcNAcylates and deubiquitinates BMAL1 and CLOCK to control the amplitude of circadian oscillation in response to nutrient availability. Disruption of O-GlcNAc signaling in mouse liver perturbs the diurnal rhythm of glucose metabolism (
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      ).
      Taken together, the studies by Ruan et al. and Li et al. define the OGT–HCF-1–BAP1 complex as a key modulator of ubiquitination, suggesting a novel mechanism by which O-GlcNAcylation controls protein stability (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ,
      • Li M.D.
      • Ruan H.B.
      • Hughes M.E.
      • Lee J.S.
      • Singh J.P.
      • Jones S.P.
      • Nitabach M.N.
      • Yang X.
      O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.
      ) (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1Modes of interaction between O-GlcNAcylation and ubiquitination. A, O-GlcNAcylation of the substrate antagonizes phosphorylation at the same or adjacent site to control ubiquitination. B, O-GlcNAcylation provides a docking site for DUBs that deubiquitinate the substrate. C, O-GlcNAcylation of H2B recruits an E3 complex to monoubiquitinate H2B. D, targeting the ubiquitin-proteasome system. OGT physically interacts with ubiquitin precursors, E3s, and DUBs. O-GlcNAcylation has been found on E1s, E3s, DUBs, and 19S and 20S proteasomes.

      O-GlcNAcylation Regulates Protein Ubiquitination via Unknown Mechanisms

      Carbohydrate-response Element-binding Protein

      Carbohydrate-response element-binding protein (ChREBP) is a basic helix-loop-helix leucine zipper transcription factor that regulates glucose and lipid metabolism in a glucose-dependent manner (
      • Postic C.
      • Dentin R.
      • Denechaud P.D.
      • Girard J.
      ChREBP, a transcriptional regulator of glucose and lipid metabolism.
      ). Under low glucose conditions, phosphorylated, inactive ChREBP primarily resides in the cytoplasm. High glucose levels trigger ChREBP dephosphorylation at S196 by PP2A and translocation into the nucleus, followed by dephosphorylation at T666 to induce the transcriptional activity of ChREBP. However, dephosphorylation at these sites is not sufficient for the constitutive activation of ChREBP, suggesting additional layers of regulation (
      • Postic C.
      • Dentin R.
      • Denechaud P.D.
      • Girard J.
      ChREBP, a transcriptional regulator of glucose and lipid metabolism.
      ). Guinez et al. show that ChREBP is modified by O-GlcNAc, which stabilizes ChREBP to increase the transcription of lipogenic genes (
      • Guinez C.
      • Filhoulaud G.
      • Rayah-Benhamed F.
      • Marmier S.
      • Dubuquoy C.
      • Dentin R.
      • Moldes M.
      • Burnol A.F.
      • Yang X.
      • Lefebvre T.
      • Girard J.
      • Postic C.
      O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver.
      ). OGT overexpression induces lipogenesis by increasing ChREBP levels, whereas OGA overexpression prevents hepatic steatosis in db/db mice (
      • Guinez C.
      • Filhoulaud G.
      • Rayah-Benhamed F.
      • Marmier S.
      • Dubuquoy C.
      • Dentin R.
      • Moldes M.
      • Burnol A.F.
      • Yang X.
      • Lefebvre T.
      • Girard J.
      • Postic C.
      O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver.
      ). Recently, another study showed that FoxO1 reduces ChREBP stability by inhibiting O-GlcNAcylation and promoting ubiquitination of the protein (
      • Ido-Kitamura Y.
      • Sasaki T.
      • Kobayashi M.
      • Kim H.J.
      • Lee Y.S.
      • Kikuchi O.
      • Yokota-Hashimoto H.
      • Iizuka K.
      • Accili D.
      • Kitamura T.
      Hepatic FoxO1 integrates glucose utilization and lipid synthesis through regulation of Chrebp O-glycosylation.
      ). How O-GlcNAcylation of ChREBP affects its ubiquitination is not known.

      Keratins 8 and 18

      Type I and II keratin proteins are expressed in specific pairs in various tissues during development and differentiation (
      • Kim S.
      • Coulombe P.A.
      Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm.
      ). Keratin pair 8/18 is widely studied in terms of the regulation of protein interaction, ubiquitination, and filament organization by phosphorylation (
      • Omary M.B.
      • Ku N.O.
      • Tao G.Z.
      • Toivola D.M.
      • Liao J.
      “Heads and tails” of intermediate filament phosphorylation: multiple sites and functional insights.
      ). Keratins 8 and 18 are also highly O-GlcNAcylated (
      • Srikanth B.
      • Vaidya M.M.
      • Kalraiya R.D.
      O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.
      ). O-GlcNAcylation increases keratin 8/18 solubility, ubiquitination, and proteasomal degradation. O-GlcNAc-deficient keratin 18 is more stable and causes changes in the filament architecture. There does not seem to be a reciprocal relationship between O-GlcNAcylation and phosphorylation of keratin 8/18 in vivo. The mechanism by which O-GlcNAcylation increases ubiquitination is still a mystery (
      • Srikanth B.
      • Vaidya M.M.
      • Kalraiya R.D.
      O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.
      ).

      A20

      The zinc-finger protein A20 is a negative regulator of NF-κB signaling that has been shown to suppress apoptosis and inflammation. Shrikhande et al. reported that hyperglycemia promotes the O-GlcNAcylation, ubiquitination, and degradation of A20, which accelerate atherosclerosis in diabetic mice (
      • Shrikhande G.V.
      • Scali S.T.
      • da Silva C.G.
      • Damrauer S.M.
      • Csizmadia E.
      • Putheti P.
      • Matthey M.
      • Arjoon R.
      • Patel R.
      • Siracuse J.J.
      • Maccariello E.R.
      • Andersen N.D.
      • Monahan T.
      • Peterson C.
      • Essayagh S.
      • Studer P.
      • Guedes R.P.
      • Kocher O.
      • Usheva A.
      • Veves A.
      • Kaczmarek E.
      • Ferran C.
      O-glycosylation regulates ubiquitination and degradation of the anti-inflammatory protein A20 to accelerate atherosclerosis in diabetic ApoE-null mice.
      ). Interestingly, A20 has both ubiquitin ligase and deubiquitinase activities, suggesting that regulation of the A20 protein level by O-GlcNAcylation is a control point for the ubiquitination of A20 target proteins.
      As listed in Table I, the hexosamine/O-GlcNAc pathway can modulate the stability of many other proteins either positively or negatively. For example, O-GlcNAc signaling increases the stability of β-catenin (
      • Olivier-Van Stichelen S.
      • Guinez C.
      • Mir A.M.
      • Perez-Cervera Y.
      • Liu C.
      • Michalski J.C.
      • Lefebvre T.
      The hexosamine biosynthetic pathway and O-GlcNAcylation drive the expression of beta-catenin and cell proliferation.
      ), p67eIF2 (
      • Datta R.
      • Choudhury P.
      • Ghosh A.
      • Datta B.
      A glycosylation site, 60SGTS63, of p67 is required for its ability to regulate the phosphorylation and activity of eukaryotic initiation factor 2alpha.
      ), FoxM1 (
      • Caldwell S.A.
      • Jackson S.R.
      • Shahriari K.S.
      • Lynch T.P.
      • Sethi G.
      • Walker S.
      • Vosseller K.
      • Reginato M.J.
      Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1.
      ), plakoglobin (
      • Hu P.
      • Berkowitz P.
      • Madden V.J.
      • Rubenstein D.S.
      Stabilization of plakoglobin and enhanced keratinocyte cell-cell adhesion by intracellular O-glycosylation.
      ), and Sp1 (
      • Han I.
      • Kudlow J.E.
      Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility.
      ) but decreases the stability of Nkx2.5 (
      • Kim H.S.
      • Woo J.S.
      • Joo H.J.
      • Moon W.K.
      Cardiac transcription factor Nkx2.5 is downregulated under excessive O-GlcNAcylation condition.
      ) and cytosolic SirT1 (
      • Lafontaine-Lacasse M.
      • Dore G.
      • Picard F.
      Hexosamines stimulate apoptosis by altering SIRT1 action and levels in rodent pancreatic beta-cells.
      ). However, whether the UPS is involved in the regulation of these proteins has not been determined.

      O-GlcNAcylation Facilitates Monoubiquitination

      Monoubiquitination is a dynamic and reversible PTM involved in nonproteolytic functions (
      • Weake V.M.
      • Workman J.L.
      Histone ubiquitination: triggering gene activity.
      ). Histones are well-known targets of monoubiquitination. H2B monoubiquitination at K120 has been shown to regulate transcription initiation and elongation (
      • Weake V.M.
      • Workman J.L.
      Histone ubiquitination: triggering gene activity.
      ). Several recent studies suggest that O-GlcNAc is also part of the histone code (
      • Sakabe K.
      • Wang Z.
      • Hart G.W.
      Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code.
      ,
      • Fujiki R.
      • Hashiba W.
      • Sekine H.
      • Yokoyama A.
      • Chikanishi T.
      • Ito S.
      • Imai Y.
      • Kim J.
      • He H.H.
      • Igarashi K.
      • Kanno J.
      • Ohtake F.
      • Kitagawa H.
      • Roeder R.G.
      • Brown M.
      • Kato S.
      GlcNAcylation of histone H2B facilitates its monoubiquitination.
      ,
      • Zhang S.
      • Roche K.
      • Nasheuer H.P.
      • Lowndes N.F.
      Modification of histones by sugar beta-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.
      ,
      • Fong J.J.
      • Nguyen B.L.
      • Bridger R.
      • Medrano E.E.
      • Wells L.
      • Pan S.
      • Sifers R.N.
      beta-N-acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3.
      ,
      • Chen Q.
      • Chen Y.
      • Bian C.
      • Fujiki R.
      • Yu X.
      TET2 promotes histone O-GlcNAcylation during gene transcription.
      ). Therefore, it is appealing to determine the interactions between O-GlcNAcylation and other histone markers. Fujiki et al. have demonstrated that O-GlcNAcylation of H2B at S112, which is sensitive to glucose availability, promotes K120 monoubiquitination by anchoring the BRE1A/1B complex, an E3 ligase (Fig. 1C) (
      • Fujiki R.
      • Hashiba W.
      • Sekine H.
      • Yokoyama A.
      • Chikanishi T.
      • Ito S.
      • Imai Y.
      • Kim J.
      • He H.H.
      • Igarashi K.
      • Kanno J.
      • Ohtake F.
      • Kitagawa H.
      • Roeder R.G.
      • Brown M.
      • Kato S.
      GlcNAcylation of histone H2B facilitates its monoubiquitination.
      ). Genome-wide analysis reveals that H2B S112 O-GlcNAcylation is associated with transcribed gene loci, many of which overlap with K120 monoubiquitination (
      • Fujiki R.
      • Hashiba W.
      • Sekine H.
      • Yokoyama A.
      • Chikanishi T.
      • Ito S.
      • Imai Y.
      • Kim J.
      • He H.H.
      • Igarashi K.
      • Kanno J.
      • Ohtake F.
      • Kitagawa H.
      • Roeder R.G.
      • Brown M.
      • Kato S.
      GlcNAcylation of histone H2B facilitates its monoubiquitination.
      ). It will be interesting to determine whether O-GlcNAcylation regulates the monoubiquitination of non-histone proteins.

      O-GlcNAcylation Regulates the Ubiquitination Process

      The preceding sections outline individual proteins that are covalently modified and regulated by O-GlcNAcylation and ubiquitination. There is also evidence that O-GlcNAcylation modulates global ubiquitination (
      • Guinez C.
      • Mir A.M.
      • Dehennaut V.
      • Cacan R.
      • Harduin-Lepers A.
      • Michalski J.C.
      • Lefebvre T.
      Protein ubiquitination is modulated by O-GlcNAc glycosylation.
      ). Thermal stress induces a rapid increase in both O-GlcNAcylation and ubiquitination. Increasing O-GlcNAc levels via glucosamine or PUGNAc promotes ubiquitination, whereas decreasing O-GlcNAc levels via forskolin, glucose deprivation, or OGT knockdown reduces ubiquitination (
      • Guinez C.
      • Mir A.M.
      • Dehennaut V.
      • Cacan R.
      • Harduin-Lepers A.
      • Michalski J.C.
      • Lefebvre T.
      Protein ubiquitination is modulated by O-GlcNAc glycosylation.
      ). However, increasing ubiquitination levels via the proteasome inhibitor has no obvious effect on global O-GlcNAcylation. These results suggest that O-GlcNAcylation affects ubiquitination, but not vice versa. The authors also found that the E1 enzyme Uba1 is O-GlcNAcylated, suggesting the possible regulation of protein ubiquitination by E1 O-GlcNAcylation (
      • Guinez C.
      • Mir A.M.
      • Dehennaut V.
      • Cacan R.
      • Harduin-Lepers A.
      • Michalski J.C.
      • Lefebvre T.
      Protein ubiquitination is modulated by O-GlcNAc glycosylation.
      ).
      A large number of O-GlcNAcylated proteins/peptides and O-GlcNAcylation sites have been discovered using proteomic approaches. We searched published O-GlcNAcylated protein datasets for those involved in the ubiquitination process including ubiquitin precursors, E1s, E2s, E3s, and DUBs, and we found that many of theses proteins are modified by O-GlcNAc (Table III). Although the functions of O-GlcNAcylation of these proteins have not been fully characterized, it is conceivable that O-GlcNAcylation regulates protein ubiquitination through multiple nodes of the UPS (Fig. 1D).
      Table IIIO-GlcNAcylated proteins involved in ubiquitination
      FamilySymbolDescriptionSpeciesSiteReference
      E1Uba1Ubiquitin activating enzyme 1Human, DrosophilaN/D(
      • Guinez C.
      • Mir A.M.
      • Dehennaut V.
      • Cacan R.
      • Harduin-Lepers A.
      • Michalski J.C.
      • Lefebvre T.
      Protein ubiquitination is modulated by O-GlcNAc glycosylation.
      ,
      • Drougat L.
      • Olivier-Van Stichelen S.
      • Mortuaire M.
      • Foulquier F.
      • Lacoste A.S.
      • Michalski J.C.
      • Lefebvre T.
      • Vercoutter-Edouart A.S.
      Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition.
      ,
      • Sprung R.
      • Nandi A.
      • Chen Y.
      • Kim S.C.
      • Barma D.
      • Falck J.R.
      • Zhao Y.
      Tagging-via-substrate strategy for probing O-GlcNAc modified proteins.
      )
      E3
      Putative E3s are annotated by Li et al. (85).
      CblCasitas B-lineage lymphoma proto-oncogeneHumanS601(
      • Hahne H.
      • Sobotzki N.
      • Nyberg T.
      • Helm D.
      • Borodkin V.S.
      • van Aalten D.M.
      • Agnew B.
      • Kuster B.
      Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.
      )
      Cnot4CCR4-NOT transcription complex subunit 4MouseS316, T331, T573(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Fbxo2F-box protein 2RatN/D(
      • Clark P.M.
      • Dweck J.F.
      • Mason D.E.
      • Hart C.R.
      • Buck S.B.
      • Peters E.C.
      • Agnew B.J.
      • Hsieh-Wilson L.C.
      Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.
      )
      Fbxo41F-box protein 41MouseS386, T387(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Hecd1HECT domain-containing protein 1MouseN/D(
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Hectd1HECT domain containing 1MouseS1350(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Herc1HECT domain and RCC1-like domain-containing protein 1MouseS3011(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Hic1Hypermethylated in cancer 1HumanN/D(
      • Lefebvre T.
      • Pinte S.
      • Guerardel C.
      • Deltour S.
      • Martin-Soudant N.
      • Slomianny M.C.
      • Michalski J.C.
      • Leprince D.
      The tumor suppressor HIC1 (hypermethylated in cancer 1) is O-GlcNAc glycosylated.
      )
      Huwe1HECT, UBA, and WWE domain containing 1RatN/D(
      • Clark P.M.
      • Dweck J.F.
      • Mason D.E.
      • Hart C.R.
      • Buck S.B.
      • Peters E.C.
      • Agnew B.J.
      • Hsieh-Wilson L.C.
      Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.
      )
      Kcmf1Differentially expressed in branching tubulogenesis 91MouseS262(
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Nedd4Neural precursor cell expressed, developmentally down-regulated 4MouseS371, T375(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      ,
      • Zaro B.W.
      • Yang Y.Y.
      • Hang H.C.
      • Pratt M.R.
      Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4–1.
      )
      Prpf19Pre-mRNA-processing factor 19HumanT169(
      • Hahne H.
      • Moghaddas Gholami A.
      • Kuster B.
      Discovery of O-GlcNAc-modified proteins in published large-scale proteome data.
      )
      Rc3h2Ring finger and CCCH-type zinc finger domains 2MouseT471, T472, S592, T841 or S844, S901(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Ring1RING finger protein 1HumanN/D(
      • Teo C.F.
      • Ingale S.
      • Wolfert M.A.
      • Elsayed G.A.
      • Not L.G.
      • Chatham J.C.
      • Wells L.
      • Boons G.J.
      Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.
      )
      Rnf2RING finger protein 2HumanN/D(
      • Teo C.F.
      • Ingale S.
      • Wolfert M.A.
      • Elsayed G.A.
      • Not L.G.
      • Chatham J.C.
      • Wells L.
      • Boons G.J.
      Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.
      )
      Rnf123Ring finger protein 123MouseS1078(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Sf3b3Splicing factor 3b, subunit 3HumanN/D(
      • Nandi A.
      • Sprung R.
      • Barma D.K.
      • Zhao Y.
      • Kim S.C.
      • Falck J.R.
      • Zhao Y.
      Global identification of O-GlcNAc-modified proteins.
      )
      Sh3rf1SH3 domain-containing RING finger protein 1MouseT512, T91, T92, S526, T527(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Trim33Tripartite motif-containing 33MouseS650(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Tulp4Tubby like protein 4MouseT943(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Ubr4Ubiquitin protein ligase E3 component n-recognin 4MouseS2577(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      UnklRING finger protein unkempt-likeMouseS451, T459(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      Zbtb20Zinc finger and BTB domain containing 20MouseS268, T465, T480(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Zfp598Zinc finger protein 598MouseOne site among S560, T563, and T564(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      DUB
      The inventory of DUBs is described by Nijman et al. (86).
      Bap1BRCA1 associated protein 1HumanN/D(
      • Teo C.F.
      • Ingale S.
      • Wolfert M.A.
      • Elsayed G.A.
      • Not L.G.
      • Chatham J.C.
      • Wells L.
      • Boons G.J.
      Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.
      )
      Usp11Ubiquitin specific peptidase 11MouseTwo sites between S617 and S619(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Usp24Ubiquitin specific peptidase 24MouseS3(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Usp31Ubiquitin specific peptidase 31MouseOne site between T1163 and T1165(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Usp5Ubiquitin specific peptidase 5RatN/D(
      • Clark P.M.
      • Dweck J.F.
      • Mason D.E.
      • Hart C.R.
      • Buck S.B.
      • Peters E.C.
      • Agnew B.J.
      • Hsieh-Wilson L.C.
      Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.
      )
      Usp8Ubiquitin specific peptidase 8MouseS218 and one site among S227/T231/S233(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      )
      Vcpip1Valosin containing protein (p97)/p47 complex interacting protein 1MouseT1072, S1075(
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation andphosphorylation at the murine synapse.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      )
      a Putative E3s are annotated by Li et al. (
      • Li W.
      • Bengtson M.H.
      • Ulbrich A.
      • Matsuda A.
      • Reddy V.A.
      • Orth A.
      • Chanda S.K.
      • Batalov S.
      • Joazeiro C.A.
      Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.
      ).
      b The inventory of DUBs is described by Nijman et al. (
      • Nijman S.M.
      • Luna-Vargas M.P.
      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
      • Sixma T.K.
      • Bernards R.
      A genomic and functional inventory of deubiquitinating enzymes.
      ).

      O-GlcNAc Signaling Modulates Proteasome Activity

      The 26S proteasome is responsible for the destruction of polyubiquitinated proteins. In addition to its roles in regulating protein ubiquitination, O-GlcNAc has long been proposed to directly modulate proteasome activity. Both the 20S catalytic particle and the 19S regulatory particle of the proteasome are known to be O-GlcNAcylated (
      • Sumegi M.
      • Hunyadi-Gulyas E.
      • Medzihradszky K.F.
      • Udvardy A.
      26S proteasome subunits are O-linked N-acetylglucosamine-modified in Drosophila melanogaster.
      ). Zhang et al. demonstrated that O-GlcNAcylation of Rpt2 ATPase in the 19S proteasome inhibits proteasome function, which may serve as a mechanism controlling cellular levels of amino acids in response to metabolic changes such as starvation and nutrient overload (
      • Zhang F.
      • Su K.
      • Yang X.
      • Bowe D.B.
      • Paterson A.J.
      • Kudlow J.E.
      O-GlcNAc modification is an endogenous inhibitor of the proteasome.
      ) (Fig. 1D). It should be noted that many of the studies listed in Table I were performed using indiscriminate approaches (i.e. OGT overexpression or OGA inhibition) that affect global O-GlcNAc levels. Thus, the stabilization of certain proteins by O-GlcNAc signaling could be, at least in part, attributed to proteasomal inhibition (Table I).
      The short form of O-GlcNAcase (OGA-S), which lacks the histone acetyltransferase domain, accumulates on the surface of lipid droplets (
      • Keembiyehetty C.N.
      • Krzeslak A.
      • Love D.C.
      • Hanover J.A.
      A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome.
      ). Selective knockdown of OGA-S results in global proteasome inhibition and increased levels of perilipin-2 and -3. These findings suggest that proteasomal modulation links O-GlcNAc signaling to lipid droplet maturation (
      • Keembiyehetty C.N.
      • Krzeslak A.
      • Love D.C.
      • Hanover J.A.
      A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome.
      ).

      O-GlcNAcylation and Ubiquitination in Physiology and Pathogenesis

      During starvation, increased proteasomal degradation in the muscle supplies amino acids to the liver for de novo glucose synthesis (gluconeogenesis). In the starved muscle, reduced O-GlcNAc signaling promotes the ability of the proteasome to degrade polyubiquitinated proteins (
      • Zhang F.
      • Paterson A.J.
      • Huang P.
      • Wang K.
      • Kudlow J.E.
      Metabolic control of proteasome function.
      ). Meanwhile, O-GlcNAc signaling in the liver promotes gluconeogenesis by deubiquitinating and stabilizing PGC-1α and CRTC2 (
      • Ruan H.B.
      • Han X.
      • Li M.D.
      • Singh J.P.
      • Qian K.
      • Azarhoush S.
      • Zhao L.
      • Bennett A.M.
      • Samuel V.T.
      • Wu J.
      • Yates 3rd, J.R.
      • Yang X.
      O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1alpha stability.
      ,
      • Dentin R.
      • Hedrick S.
      • Xie J.
      • Yates 3rd, J.
      • Montminy M.
      Hepatic glucose sensing via the CREB coactivator CRTC2.
      ,
      • Ruan H.B.
      • Singh J.P.
      • Li M.D.
      • Wu J.
      • Yang X.
      Cracking the O-GlcNAc code in metabolism.
      ). Thus, O-GlcNAcylation acts in concert with ubiquitination in multiple organs to control glucose metabolism.
      Obesity and diabetes are associated with increased O-GlcNAcylation in multiple tissues (
      • Ruan H.B.
      • Singh J.P.
      • Li M.D.
      • Wu J.
      • Yang X.
      Cracking the O-GlcNAc code in metabolism.
      ). Glucotoxicity, which is caused by diabetic hyperglycemia, leads to hepatic steatosis, β cell failure, cardiomyopathy, and atherosclerosis. As shown in Table I, O-GlcNAcylation regulates the ubiquitination and/or stability of ChREBP, SirT1, Nkx2.5, and A20. Therefore, glucotoxcity may contribute to metabolic syndrome through aberrant O-GlcNAcylation and ubiquitination of those proteins in various tissues.
      Many neurodegenerative diseases are associated with the formation of ubiquitin-conjugated protein aggregates in pathological inclusion bodies. For example, hyperphosphorylated Tau is the main component of neurofibrillary tangles, and amyloid-β is the major component of senile plaques in Alzheimer disease. α-synuclein aggregates form Lewy bodies in Parkinson disease, mutant Huntington proteins form inclusion bodies in Huntington disease, and skein-like inclusions exist in amyotrophic lateral sclerosis (
      • Deng H.X.
      • Chen W.
      • Hong S.T.
      • Boycott K.M.
      • Gorrie G.H.
      • Siddique N.
      • Yang Y.
      • Fecto F.
      • Shi Y.
      • Zhai H.
      • Jiang H.
      • Hirano M.
      • Rampersaud E.
      • Jansen G.H.
      • Donkervoort S.
      • Bigio E.H.
      • Brooks B.R.
      • Ajroud K.
      • Sufit R.L.
      • Haines J.L.
      • Mugnaini E.
      • Pericak-Vance M.A.
      • Siddique T.
      Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.
      ,
      • Huang Q.
      • Figueiredo-Pereira M.E.
      Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications.
      ,
      • Dennissen F.J.
      • Kholod N.
      • van Leeuwen F.W.
      The ubiquitin proteasome system in neurodegenerative diseases: culprit, accomplice or victim?.
      ,
      • Gong B.
      • Kielar C.
      • Morton A.J.
      Temporal separation of aggregation and ubiquitination during early inclusion formation in transgenic mice carrying the Huntington's disease mutation.
      ). The common cellular mechanism for these pathological conditions is the defective UPS. O-GlcNAc is also involved in many neurodegenerative diseases (
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ,
      • Gong C.X.
      • Liu F.
      • Iqbal K.
      O-GlcNAc cycling modulates neurodegeneration.
      ,
      • Shan X.
      • Vocadlo D.J.
      • Krieger C.
      Reduced protein O-glycosylation in the nervous system of the mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis.
      ). O-GlcNAc signaling modulates phosphorylation of Tau and the processing of the amyloid-β precursor protein, and treatment of the OGA inhibitor hinders the progression of Alzheimer disease (
      • Gong C.X.
      • Liu F.
      • Iqbal K.
      O-GlcNAc cycling modulates neurodegeneration.
      ,
      • Yuzwa S.A.
      • Shan X.
      • Macauley M.S.
      • Clark T.
      • Skorobogatko Y.
      • Vosseller K.
      • Vocadlo D.J.
      Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.
      ). A recent study shows that O-GlcNAc cycling influences proteasome and autophagy pathways in C. elegans models of neurodegenerative diseases (
      • Wang P.
      • Lazarus B.D.
      • Forsythe M.E.
      • Love D.C.
      • Krause M.W.
      • Hanover J.A.
      O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases.
      ). Further studies should define the casual role of O-GlcNAcylation and ubiquitination in neurodegeneration, ideally using murine models and human patient samples.
      The defective UPS causes the accumulation of misfolded or mutated proteins, which in turn contribute to tumor formation and progression (
      • Fulda S.
      • Rajalingam K.
      • Dikic I.
      Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications.
      ). E3 ligases such as Cbl and DUBs such as BAP1 and A20 have been implicated in cancer development (
      • Fulda S.
      • Rajalingam K.
      • Dikic I.
      Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications.
      ,
      • Dey A.
      • Seshasayee D.
      • Noubade R.
      • French D.M.
      • Liu J.
      • Chaurushiya M.S.
      • Kirkpatrick D.S.
      • Pham V.C.
      • Lill J.R.
      • Bakalarski C.E.
      • Wu J.
      • Phu L.
      • Katavolos P.
      • LaFave L.M.
      • Abdel-Wahab O.
      • Modrusan Z.
      • Seshagiri S.
      • Dong K.
      • Lin Z.
      • Balazs M.
      • Suriben R.
      • Newton K.
      • Hymowitz S.
      • Garcia-Manero G.
      • Martin F.
      • Levine R.L.
      • Dixit V.M.
      Loss of the tumor suppressor BAP1 causes myeloid transformation.
      ). Many drugs that target the UPS machinery have shown promise in clinical trials of cancer therapy (
      • Fulda S.
      • Rajalingam K.
      • Dikic I.
      Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications.
      ). Notably, the oncogene Cbl and the tumor suppressor BAP1 are O-GlcNAcylated (Table III). O-GlcNAc has also been implicated in cancer biology by targeting transcription factors such as p53, c-Myc, Sp1, and NF-κB (
      • Ruan H.B.
      • Singh J.P.
      • Li M.D.
      • Wu J.
      • Yang X.
      Cracking the O-GlcNAc code in metabolism.
      ,
      • Slawson C.
      • Hart G.W.
      O-GlcNAc signalling: implications for cancer cell biology.
      ). Many proteins involved in cell proliferation, apoptosis, and adhesion, such as p53, Δ-lactoferrin, Snail1, β-catenin, FoxM1, and Sp1, are regulated by O-GlcNAcylation and ubiquitination (Table I). A better understanding of the regulation of the UPS by O-GlcNAcylation will provide crucial insight into cancer diagnosis and therapy.

      Acknowledgments

      We thank all members of the Yang laboratory for stimulating discussions.

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