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ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze

ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze. ADAPTED FROM. “Dynamic Interplay Between O-GlcNAc and O-Phosphate: Roles in Transcription, Signaling and in Cellular Response to Stress.”. Gerald W. Hart Johns Hopkins University School of Medicine. O-GlcNAc.

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ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze

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  1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze

  2. ADAPTED FROM “Dynamic Interplay Between O-GlcNAc and O-Phosphate: Roles in Transcription, Signaling and in Cellular Response to Stress.” Gerald W. Hart Johns Hopkins University School of Medicine

  3. O-GlcNAc O- Linked N-Acetylglucosamine O-GlcNAc Transferase O-GlcNAcase A dynamic post-translational modification OH OH HO HO O O O O HO HO AcNH AcNH Tyr-Ser-Pro-Thr-Ser-Pro-Ser

  4. Key Features of O-GlcNAc : • NOT elongated to more complex structures. • Localized to the cytoplasm and nucleus. • Present in all higher eukaryotes studied. • As abundant as phosphorylation; UDP-GlcNAc is Nearly as abundant as ATP. • O-GlcNAc proteins are also Phosphoproteins • O-GlcNAc and Phosphorylation are often reciprocal. • Highly dynamic modification - a regulatory role.

  5. Proteosomal Proteins

  6. sWGA bound--GlcNAc Eluted Unbound -110- MW -20- pI 4 pI 9 pI 4 pI 9 Enrichment of O-GlcNAc Modified Proteins using sWGA: Silver stain of 80 ug total protein from each fraction (starting material 30 mg nucleocytoplamic lysate from HeLa cells)

  7. CTD 110.6 MAb Immunopurifies Many Glycoproteins From HeLa Cell Cytoplasmic and Nuclear Extract: 2nd Dimension: 10% SDS-PAGE Visualization: Silver Stain pI: 3 --> 10

  8. Some Identified O-GlcNAc-Modified Proteins: Nuclear Pore Proteins - p62, Nup54, 155, 180, 153, 214, 358; Chromatin Proteins - Many Transcription Factors - SP1, cfos, cJun, CTF, HNF1, v-ErbA, Pancreas Specific TF, SRF, c-Myc, p53, Estrogen Receptors, ß-catenin, NFKB, ELF-1, PAX-6, Enhancer Factor D, Human C1, Oct1, plakoglobin, YY1, PDX-1, CREB, Rb, p107, RNA Polymerase II. RNA Binding Proteins - HnRNP-G, Ewing Sarcoma RNA binding Protein, EF4A1, EF1alpha, 40S ribosomal s24, many ribosomal proteins. Phosphatases/Kinases/Adapters nuclear Tyr phos’ase p65, CKII, IRS-1,2, GSK3ß, PI3-kinase Cytoskeletal Proteins - cytokeratins 8, 13, 18, Neurofilaments H, M, L, Band 4.1, talin, vinculin, ankyrin, synapsin I, myosin, E-cadherin, cofilin, tau, Maps 2, 4, Dynein, alpha tubulin, AP3, AP180, ß-APP, ß-synuclein, piccolo. Chaperones - HSP27, alpha crystallins, HsC70, HsP70, HsP90 Metabolic Enzymes - eNOS, Enolase, Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, UDP-glucose synthase, glycogen synthase Other Regulatory Proteins - Eukaryotic peptide chain initiation factor 2 p67, OGT, CRMP-2, Ubiquitin carboxy hydrolase (UCH), Glut 1, Annexin 1, Nucleophosmin, proteasome components C2, 5/9 and 9/14 of 26S proteasome regulatory & catalytic subunits, respectively, Q04323 UCH homolog, Sec23, Ran, peptidyl prolylisomerase, Rho GDP-dissociation inhibitor, GABA Receptor interacting protein 1, Viral Proteins - adenovirus fiber, SV40 Large T Ag;, Baculovirus Teg protein,

  9. Some Clues To O-GlcNAc Functions: (>~200 Papers Since 1984 Directly Concerned with O-GlcNAc) • Required for Life at Single Cell Level - (OGT -/+ still emb. lethal.) • Regulates Transcriptional Activation or Suppression, Dep. upon Metabolism - SP1, ERs, Stat5, NFkB, p53, c-Myc; Regulates Degradation. • Regulates Protein Synthesis (via p67-EIF2 kinase);(Gupta & Datta) • Regulates ß-catenin and E-cadherin trafficking (Andrews et al.) • Neurodegenerative Disease - Tau, ß-APP, NFs, O-GlcNAcase maps to late-AD locus; OGT Maps to Parkinson Dystonia Locus; O-GlcNAc is Reduced in Human AD. • YY1 Transcription Factor is Regulated by O-GlcNAc - Prevents binding to Rb. • O-GlcNAc on Glycogen Synthase Prevents its Activation by Insulin (Parker; McClain). • O-GlcNAc on eNOS prevents activation by Akt (Brownlee) • Retinoblastoma (Rb) is O-GlcNAc Modified in G1 & O-GlcNAc-Rb binds E2F. • O-GlcNAcylation of 26S Proteosome Inhibits Degradation; 5/19 and 9/14 of Catalytic Core and Reg. Core subunits, respectively, are modified (Cell 115, 715; BBRC 312,1284) • Regulator of Gibberellic Acid Signaling in Plants-OGT=Spy; Secret Agent • Blocks Insulin Signaling and OGT Over-Expression in Muscle or Adipose Causes Diabetes in Mice (McClain & Hanover)

  10. Complex Interplay Between O-Phosphate & O-GlcNAc:

  11. OPTIONS FOR O-GlcNAc MODIFICATION

  12. O-GlcNAc Transferase Summary: • OGT has been highly conserved throughout evolution. • OGT is a unique glycosyltransferase. • Localized near centromere on X-chromosome. • 1 Normal Allele is Required for ES Cell Viability (Marth et al.)

  13. OGT structure catalytic • Single gene encoding • 103 kDa peptide • migrates at 110 kDa TPR domains - (tetratricopeptide repeats) Generates O-GlcNAc linkage on peptides Inexact peptide sequence motif for glycosylation Associates with self and other proteins in complex Located in nucleus and cytoplasm Expressed in all mammalian tissues studied Modified by O-GlcNAc and tyrosine-phosphate

  14. Identified OGT-TPR Binding Proteins: (Proteins That Target OGT and Control Specificity) • Co-Repressor mSin3A binds OGT and Suppresses SP1 Driven Transcription (Kudlow) • GRIF-1 - Targets to GABA Receptor (Anne Stephenson) and Regulates its signaling; OGT binding protein (Sai Iyer). • Milton (Drosophila GRIF-1 analog) required for kinesin-mediated axonal transport of mitochondria to Synapses (Neuron 36, 1063 (Schwarz). • OIP106 - GRIF-1-Like Protein Targets OGT to RNA polymerase II - binds via TPRs. • Many more yet to be identified - pulled out by yeast 2H.

  15. y = 0.6802 + 27.714x R= 0.99833 1400 1200 1000 800 600 400 200 0 0 10 20 30 40 50 Effect of [UDP-GlcNAc] On OGT Activity In the Presence of Alkaline Phosphatase 50 mM!! pmol incorporated Physiological Range [UDP-GlcNAc] mM

  16. O-GlcNAc’ase Structure Single gene encodes 916 amino acid polypeptide of 103 kDa migrates at 130 kDa OGA OGA peptide cleaves GlcNAc from glycopeptides Predominantly expressed in the cytoplasm Expressed in all human tissues studied Highly conserved in mammals and found in C. Elegans Located on Chromosome 10 in humans

  17. Functional aspects of O-GlcNAcase • Maps exactly to 10q23.1 - late onset Alzheimer’s Disease Locus. • Ogase has Histone Acetyltransferases activity. • Inhibition BLOCKS insulin signaling. 53 nM Ki inhibitor of O-GlcNAcase.

  18. Identification of O-GlcNAc Modified Proteins by Mass Spectrometry Why Did O-GlcNAc Remain Undetected? • Generally Not Affect Gel Electrophoresis • Not Easily Labeled - No 32P! • Very Labile - both Chemically and Enzymatically- Falls Off in MS. • Stoichiometry Similar to O-Phosphate.

  19. b ions - 164 251 348 449 536 633 720 848 Y--S--P--T--S--P--S--K 866 703 616 519 418 331 234 147 - y ions O-GlcNAc (204) Fragmentation of GlcNAc-CTD by CID-MS/MS Parent ion 535 ([M+2H+] + 1 GlcNAc) NL 7.62e6 y8 866.3 100 95 Poor Fragmentation No Site Information. 90 85 80 75 70 Peptide 65 60 Relative Abundance 55 50 sugar 45 40 35 GlcNAc 30 y6 410.3 819.2 25 203.8 616.2 b2 20 b8 433.8 15 250.9 10 848.4 476.8 5 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

  20. Replacement of O-GlcNAc with DTT Using b-elimination/michael addition Strategy for O-GlcNAc/O-Phosphate site mapping 100 H DTT O-GlcNAc BAP PSVPVSerGSAPGR PSVPVSerGSAPGR PSVPVSerGSAPGR Alkaline induced b-Elimination 0 1314.3 GlcNAc O (dehydroalanine) CH2 CH2 1650 1100 m/z (Serine-O-GlcNAc) C HSCH2CHOHCHOHCH2SH N H C 100 (DTT) 1247.4 C N H C O O 0 1650 1100 m/z 100 1421.7 DTT (or BAP) 0 H CH2 1650 1100 m/z C N H C MALDI-TOF O Relative intensity Relative intensity Michael Addition 1. Provides tag for Affinity enrichment 2. Tag is stable in mass spectrometer Relative intensity

  21. BEMAD Useful for Simultaneous Mapping of O-GlcNAc and O-Phosphate: • O-GlcNAc much more sensitive to ß-Elimination. • Treatment with Phosphatase & O-GlcNAcase. • Density-Labeled DTT is Cheap & Available. • Same Approach work for ‘classical’ O-glycans. • Phosphorylation Mapping studies Must Account for Abundance of O-GlcNAc.

  22. Another way to recognize O-GlcNAc proteins Make GlcNAc derivative with a N3-reactive azide instead of Ac UDP-GlcN -->-->Protein with O-GlcN Probe with FLAG or biotin Detect Probe Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21

  23. Reciprocal O-GlcNAcylation & Phosphorylation of c-Myc: OH O HO Thr58 is Mutation Hot Spot In Human Lymphomas. O HO AcNH -Leu-Leu-Pro-Thr58-Pro-Pro-Leu- O -O -P-O- O -Leu-Leu-Pro-Thr58-Pro-Pro-Leu-

  24. Mutations of c-Myc in Lymphomas Major O-GlcNAc Site. Site-Specific mAB - Thr58-O-GlcNAc: • Prevention of Phosphorylation at Ser 62 Elevates O-GlcNAc at Thr 58. • Stim. Of Growth Reduces O-GlcNAc at T58; Increases Phos. & vice versa. • Inhibition of GSK3ß increases O-GlcNAc at T58. • Regulates c-Myc association with Tumor Suppressor Rb p107. GSK3 Erk Transactivation Domain

  25. O-GlcNAc O-GlcNAc X X Heat Shock UV Osmotic Stress Free Radicals Reductive Stress Heat Shock Proteins Glutathione SOD Catalase The rapid increase in O-GlcNAc and the effect of increased HSP70 in the presence of increased O-GlcNAc suggests that O-GlcNAc forms an integral component of the cell’s stress signaling pathways. Natasha Zachara

  26. The Addition of O-GlcNAc toProteins in Response to Stress is Dynamic Cont Time post-heat shock Cont 1 1 3 6 9 24 48 48 h WB: O-GlcNAc 177 114 80 64 50 Cos-7 cells were treated with heat shock (45oC, 1h) and allowed to recover at 37oC. 37 O-GlcNAc Precedes HSP70 WB: HSC/HSP70

  27. O-GlcNAc is A Sensor of Cellular Stress: • O-GlcNAc and OGT are rapidly elevated in response to many forms of cellular stress • This is dose dependent; does not require mRNA or Protein Synthesis • Increased levels of O-GlcNAc results in increased thermotolerance • In part a result of more rapid increased HSP70 production/stabilization. • HSP70 as an O-GlcNAc lectin - 2 French Groups. • Decreased protein aggregation? • Decreased O-GlcNAc Results in decreased thermotolerance

  28. UDP-GlcNAc as a Metabolic Sensor: Glucose Metabolism Overall Energy Fatty Acid Metabolism UDP-GlcNAc Nucleotide Metabolism Nitrogen Metabolism

  29. REVIEW OF METABOLIC PATHWAYS

  30. Elevation of O-GlcNAc Blocks Insulin Signaling: • Huge Literature - Diabetes Requires Glc to GlcNAc. • GlcN 10X more potent than Glc in inducing Insulin-Resistance. • O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.

  31. Elevation of O-GlcNAc Blocks Insulin Signaling: • Huge Literature - Diabetes Requires Glc to GlcNAc. • GlcN 10X more potent than Glc in inducing Insulin-Resistance. • O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals. Insulin receptor glucose P-Y P-Y IRS1/2 p85 Glut4 vesicle p110 HSP PKCz,d PI 3-kinase PDK1 [UDP-GlcNAc] Thr 308-P OGT ! Insulin resistance AKT1/2 O-GlcNAc PUGNAc x-Ser/thr-x-x x-Ser/thr-x-x Ser 9-P O-GlcNAcase GSK3b Glycogen synthesis * *? * * Does Specific Elevation of O-GlcNAc Cause Insulin Resistance? Yes! *

  32. 20000 10000 0 51% Insulin Resistance induced through the HBP correlates with increased O-GlcNAc in 3T3-L1 Adipocytes Deoxyglucose uptake (insulin dependent 14[C] DPM) Requires Both GlcN & Insulin for Insulin-Resistance & for Elevated O-GlcNAc. Glucosamine (mM) 0 0 5 5 chronic insulin (nM) 0 1 1 0 WB: anti-O-GlcNAc

  33. PUGNAc Elevates O-GlcNAc and Induces Insulin Resistance in 3T3-L1 adipocytes: Acute insulin (nM) 0 0 1 1 10 10 PUGNAc - + - + - + WB:anti-O-GlcNAc 110.6 antibody Deoxyglucose uptake (insulin dependent 14[C] DPM) PUGNAc - + - + - + - + - + Acute Insulin (nM) 0.1 0.33 1 3.3 10 (A) (B) 39% 53 nM Ki inhibitor of O-GlcNAcase. 23% Insulin concentration (nM)

  34. Insulin Signaling Pathway for Glut4 Translocation to Plasma Membrane - Where is O-GlcNAc Blocking? -O-GlcNAc -O-GlcNAc Many O-GlcNAc Including Munc18 -O-GlcNAc --> Glycogen Synthase

  35. PUGNAc does not effect the protein levels or insulin stimulated tyrosine phosphorylation of the insulin receptor or IRS-2. PUGNAC Blocks Insulin-Stimulated Phosphorylation of Threonine 308 on AKT/PkB*: AKT Activation is Blocked! PUGNAc Blocks Insulin-Stimulated Phosphorylation of GSK3ß at Ser9, But Not Thr202/204 MAPK or Ser473 on Akt/PKB: GSK3ß Activation is Blocked!

  36. Elevation of O-GlcNAc Blocks Insulin Signaling: • Blocks AKT phos. at T308 and S9 on GSK3ß • Inhib. OGase greatly increases OG on ß-catenin and IRS1. Insulin receptor glucose P-Y P-Y IRS1/2 p85 Glut4 vesicle p110 HSP PKCz,d PI 3-kinase PDK1 [UDP-GlcNAc] Thr 308-P OGT ! Insulin resistance AKT1/2 O-GlcNAc PUGNAc x-Ser/thr-x-x x-Ser/thr-x-x Ser 9-P O-GlcNAcase GSK3b Glycogen synthesis ? *? * * Transgenic Mice with Overexpressed OGT in Muscle or Adipose - Become Diabetic. (McClain & Hanover) *

  37. Insulin Stimulates (10 min)Tyr-P Of O-GlcNAc Transferase: Insulin (nM) 0 1 100 IP: OGT WB: OGT OGT OGT IP: OGT WB:PY Note: activity of these immunoprecip Was tested with no differences against CKII peptide. Post-translational modification may effect localization, substrate specificity, binding Partners, ect. (SH2 domain containing protein ?) Phospho-tyrosine response would place OGT in Signaling pathway downstream of insulin Receptor. time point coincides with start of glucose Uptake assay. Fig. Insulin stimulated tyrosine phosphorylation of O-GlcNAc transferase (OGT). 3T3L1 adipocytes starved of growth factors For 16 hours were stimulated 10 minutes with insulin at suboptimal (1 nM) or optimal (100 nM) concentrations, and OGT Immunoprecipitates were western blotted for either OGT levels or phosphotyrosine. OGT is Directly in the IR Signaling Pathway.

  38. Proteomics Approach to O-GlcNAc & Insulin Signaling: Spot # response to insulin 1 decreased O-GlcNAc 2 decreased O-GlcNAc 3 likely phosphorylation shift 4 decreased O-GlcNAc 5 likely phosphorylation shift 6 decreased O-GlcNAc 7 decreased O-GlcNAc 4 5 6 3 2 7 unstimulated 1 pI 5 pI 4.5 4 5 6 3 2 Insulin stimulated (100 nM for 10 min.) 7 1 (Small Portion of Gel Shown) Insulin Rapidly Decreases O-GlcNAc on Many Proteins: Insulin Causes Major Changes in O-GlcNAc in 10 min. Fig. Insulin stimulates dynamic O-GlcNAcylation in 3T3L1 adipocytes. 3T3L1 adipocytes starved of growth factors for 16 hours Were stimulated with 100 nm insulin for 10 minutes, and whole cell lysates were separated by 2D gel electrophoresis and western blotted With an anti-O-GlcNAc specific antibody.

  39. Conclusions: • O-GlcNAc is a Major Regulatory PTM in all multicellular eukaryotes - Plants & Animals. • O-GlcNAc Accounts for Many of the Biological Affects Attributed Hexosamine Biosynthetic Pathway - Diabetes & Glucose Toxicity. • O-GlcNAc is Required for Life at the Single Cell Level. • O-GlcNAc is as abundant as Phosphorylation and Often Competes with it. • O-GlcNAc - “Metabolic Sensor” to Modulate Signaling & Transcription in Response to Cellular Status. • Many Toxic Effects of Hyperglycemia Likely Result From Dysregulation of the Balance Between O-GlcNAc and Phosphorylation.

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