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Bi Y, Deng Z, Ni W, Shrestha R, Savage D, Hartwig T, Patil S, Hong SH, Zhang Z, Oses-Prieto JA, Li KH, Quail PH, Burlingame AL, Xu SL, Wang ZY. Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions. Nature communications 2021 12(1) 33574257
Abstract:
O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS's O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.
O-GlcNAc proteins:
A0A384LHA9
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Wang J, Wang Z, Yuan J, Wang J, Shen X. The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma. Aging 2020 12(9) 32357142
Abstract:
Acyl-CoA ligase 4 (ACSL4) has been reported to be overexpressed in hepatocellular carcinoma (HCC) and to enhance cell proliferation. However, the molecular mechanisms underlying the role of ACSL4 in HCC progression remain largely unclear. Here, we aimed to investigate whether and how O-GlcNAcylation and ACSL4 regulate each other and HCC progression. The clinical significance of ACSL4, O-GlcNAc and GLUT1 in HCC was determined by Pearson chi-squared test and Kaplan-Meier analysis. CCK-8, flow cytometry and in vivo tumour formation assays were performed to detect cell proliferation, apoptosis and tumorigenesis. IP technology was used to evaluate the relationship between ACSL4 and O-GlcNAc. ACSL4, GLUT1 and O-GlcNAc levels were elevated in HCC tissues and predicted poor prognosis in HCC patients. ACSL4 overexpression significantly promoted cell proliferation and tumorigenesis and inhibited cell apoptosis, whereas these effects were all obviously impaired when mTOR signalling was repressed or GLUT1 was downregulated. ACSL4 could be O-GlcNAcylated, and silencing of ACSL4 abolished the effects of O-GlcNAcylation on cell growth promotion and apoptosis inhibition. Collectively, this study demonstrates that ACSL4 contributes to the growth and survival of HCC by enhancing GLUT1-mediated O-GlcNAcylation. In turn, O-GlcNAcylation promotes HCC growth partially by increasing ACSL4 expression.
O-GlcNAc proteins:
ACSL4
Species: Homo sapiens
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Wang Z, Qin J, Zhao J, Li J, Li D, Popp M, Popp F, Alakus H, Kong B, Dong Q, Nelson PJ, Zhao Y, Bruns CJ. Inflammatory IFIT3 renders chemotherapy resistance by regulating post-translational modification of VDAC2 in pancreatic cancer. Theranostics 2020 10(16) 32641986
Abstract:
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and effective therapy remains a challenge. IFIT3 is an interferon-stimulated gene with antiviral and pro-inflammatory functions. Our previous work has shown that high expression of IFIT3 is correlated with poor survival in PDAC patients who receive chemotherapy suggesting a link between IFIT3 and chemotherapy resistance in PDAC. However, the exact role and molecular mechanism of IFIT3 in chemotherapy resistance in PDAC has been unclear. Methods: A group of transcriptome datasets were downloaded and analyzed for the characterization of IFIT3 in PDAC. Highly metastatic PDAC cell line L3.6pl and patient-derived primary cell TBO368 were used and IFIT3 knockdown and the corresponding knockin cells were established for in vitro studies. Chemotherapy-induced apoptosis, ROS production, confocal immunofluorescence, subcellular fractionation, chromatin-immunoprecipitation, co-immunoprecipitation and mass spectrometry analysis were determined to further explore the biological role of IFIT3 in chemotherapy resistance of PDAC. Results: Based on PDAC transcriptome data, we show that IFIT3 expression is associated with the squamous molecular subtype of PDAC and an increase in inflammatory response and apoptosis pathways. We further identify a crucial role for IFIT3 in the regulation of mitochondria-associated apoptosis during chemotherapy. Knockdown of IFIT3 attenuates the chemotherapy resistance of PDAC cells to gemcitabine, paclitaxel, and FOLFIRINOX regimen treatments, independent of individual chemotherapy regimens. While IFIT3 overexpression was found to promote drug resistance. Co-immunoprecipitation identified a direct interaction between IFIT3 and the mitochondrial channel protein VDAC2, an important regulator of mitochondria-associated apoptosis. It was subsequently found that IFIT3 regulates the post-translational modification-O-GlcNAcylation of VDAC2 by stabilizing the interaction of VDAC2 with O-GlcNAc transferase. Increased O-GlcNAcylation of VDAC2 protected PDAC cells from chemotherapy induced apoptosis. Conclusions: These results effectively demonstrate a central mechanism by which IFIT3 expression can affect chemotherapy resistance in PDAC. Targeting IFIT3/VDAC2 may represent a novel strategy to sensitize aggressive forms of pancreatic cancer to conventional chemotherapy regimens.
O-GlcNAc proteins:
VDAC2
Species: Homo sapiens
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Xu SL, Chalkley RJ, Maynard JC, Wang W, Ni W, Jiang X, Shin K, Cheng L, Savage D, Hühmer AF, Burlingame AL, Wang ZY. Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 2017 114(8) 28154133
Abstract:
Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.
O-GlcNAc proteins:
A0A178U7T2, A0A178U808, A0A178U8C4, A0A178U9U2, A0A178UA87, A0A178UAK4, A0A178UBS4, A0A178UEJ0, A0A178UET6, A0A178UG83, A0A178UGM3, A0A178UJQ0, A0A178UKB4, A0A178ULB6, A0A178UM22, A0A178UNF9, A0A178UTT8, A0A178UVW2, A0A178UW70, A0A178UXN3, A0A178UZJ4, A0A178V3S5, A0A178V4Y0, A0A178V7N5, A0A178V7S6, A0A178V9D9, A0A178VCA9, A0A178VF25, A0A178VGI0, A0A178VGK7, A0A178VIB4, A0A178VL84, A0A178VMH9, A0A178VN08, A0A178VNI5, A0A178VQ37, A0A178VQ53, A0A178VQY8, A0A178VS21, A0A178VTI3, A0A178VTN0, A0A178VUZ4, A0A178VWA9, A0A178VXV2, A0A178VZ34, A0A178W0U1, A0A178W4Q8, A0A178W585, A0A178W9L4, A0A178WAQ6, A0A178WCT2, A0A178WD70, A0A178WK45, A0A178WKM1, A0A1I9LM16, A0A1I9LM89, A0A1I9LNN0, A0A1I9LPG3, A0A1I9LPZ1, A0A1I9LQ18, A0A1I9LQ49, A0A1I9LQK5, A0A1I9LR14, A0A1I9LR16, A0A1I9LRR1, A0A1I9LRS8, A0A1I9LT31, A0A1I9LT54, A0A1I9LT69, A0A1I9LTD0, A0A1I9LTD1, A0A1I9LTL7, A0A1I9LTL8, A0A1I9LTL9, A0A1P8AM87, A0A1P8ANR5, A0A1P8APV6, A0A1P8APZ6, A0A1P8AQ08, A0A1P8AQI9, A0A1P8AQR8, A0A1P8ARB7, A0A1P8ARJ2, A0A1P8ARU4, A0A1P8ARV7, A0A1P8ARV8, A0A1P8AS00, ECT4, A0A1P8AS28, A0A1P8ASD0, A0A1P8ASK1, A0A1P8ASQ6, A0A1P8AT85, A0A1P8ATA1, A0A1P8ATG6, A0A1P8AUP4, A0A1P8AUP7, A0A1P8AWC8, A0A1P8AXG2, A0A1P8AXN6, A0A1P8AXY9, A0A1P8AYH5, A0A1P8AYN4, A0A1P8B0K6, A0A1P8B0M2, A0A1P8B1D0, A0A1P8B1H7, A0A1P8B1J3, A0A1P8B1N0, A0A1P8B1N9, A0A1P8B1P6, A0A1P8B1P9, A0A1P8B2G0, A0A1P8B2Y3, A0A1P8B4Z4, A0A1P8B569, A0A1P8B6K1, A0A1P8B6K2, A0A1P8B739, A0A1P8B753, A0A1P8B770, A0A1P8B7E4, A0A1P8B7F4, A0A1P8B889, A0A1P8B895, A0A1P8B8G6, A0A1P8B9E0, A0A1P8B9Q7, A0A1P8B9R0, A0A1P8BAL0, A0A1P8BBS5, A0A1P8BBU0, A0A1P8BBV1, A0A1P8BBW1, A0A1P8BCJ2, A0A1P8BCM2, A0A1P8BCS5, A0A1P8BDJ5, A0A1P8BDM9, A0A1P8BES5, A0A1P8BET4, A0A1P8BF20, A0A1P8BF26, A0A1P8BF50, A0A1P8BFA1, A0A1P8BFW7, A0A1P8BGW8, A0A2H1ZEI5, A0A2H1ZEK0, A0A384KDE2, A0A384KK08, A0A384KLV2, A0A384KRL7, A0A384L4P3, A0A384LCJ3, A0A384LD93, A0A384LHA9, A0A384LIC3, A0A384LL64, NSRA, GIP1L, A4FVS4, A8MPR6, A8MQK8, A8MQL9, A8MR17, A8MR45, A8MR97, SMG7, B3H4M3, NEDD1, B3H6D1, B3H7F6, B3H7M2, B6DT55, B9DH05, SP13A, C0Z2N6, C0Z387, SUV2, F4HNU1, F4HPG4, F4HSW8, F4HVV6, F4HVV7, F4HWS5, F4HXP0, F4HY32, F4I0C1, F4I0C2, F4I1G1, NP214, F4I2D0, RB47A, F4I4I8, F4I982, F4I983, F4IC79, PHL, F4IGJ9, F4IHK9, SYD, F4IIR1, FLX, F4IMY0, RSA1, F4IWD6, F4J043, PATH1, F4J0L7, F4J0P2, F4J7C7, VIR, F4J912, SAC3B, F4JB30, F4JDC2, F4JDC3, F4JDC5, F4JFN7, F4JKV2, F4JLR7, F4JP43, F4JPL0, F4JPL1, F4JPL2, F4JRD9, F4JUD2, F4JV21, F4JWJ6, F4JWJ7, F4JWP8, F4JXH8, PIAL2, F4K3D6, F4K3Y7, F4K402, F4K465, F4K4Y6, F4K5M5, STKLU, F4K9A6, CHR4, F4KDJ9, F4KDM9, HEN4, F4KDN1, SRC2, FCA, PABP4, AHL10, O23146, IF4E1, NFYB3, O48697, O48807, LUH, AHL2, O64768, CID7, CAB25, TOL6, O81015, O82263, NGA4, NGA1, KNAT3, DRMH1, ARFG, C3H30, RBG7, QKIL1, Q0WP31, PAT1H, Q0WQD5, Q0WTU8, Q0WUK0, Q0WUY5, TBA4, Q0WVJ1, SLK1, Q1G3K2, Q1JPL5, TPR4, LUMI, AGL11, Q3EDL2, DOF37, MED8, Q56W68, Q56WR5, Q56WT6, Q56X31, Q56YP1, Q56YR0, C3H67, Q66GK1, EPN2, BRM, Q6ID24, FY, Q6NM13, Q6RF52, WOX4, IDD10, IF4G, MED25, DOF18, Q84TH3, C3H32, Q84WZ4, CTF77, SPL8, TGH, C3H33, Q8GYC9, QKIL4, Q8GYU8, Q8GZ14, KTN84, Q8H1P8, CID3, AHL11, Q8L9Z3, DOF46, RL51, RLA11, DOF54, Q8LFT9, CPL3, Q8LPI8, FPA, LRP1B, Q8RWV9, AGD14, ARFS, GEM, AML5, Q8VY17, Q8VYH3, AHL1, Q8VYZ1, Q8VZL1, Q8VZM2, GIP1, RNP1, SEUSS, AML1, Q8W4K6, Q8W569, IF5A2, WRK20, Q93XY1, EPN3, Q93YU3, TCP14, Q93ZW3, TA12B, AHL13, Q940N4, Q940N7, C3H37, Q949Z3, TPL, SLK2, PATH2, C3H55, LRP1C, TIC, SPY, TCP8, Q9C584, MPK18, HAC1, Q9C7A7, Q9C7W1, TRO, Q9C9H8, Y3857, NUP1, Y1385, Q9FF08, ARFH, Q9FH07, FLXL4, DCP5L, Q9FHN1, Q9FIA3, ZHD10, Q9FJ56, Q9FJC2, Q9FKL3, ZHD1, QKIL2, SUMO2, Q9FM47, Q9FM71, EXA1, Q9FNB9, C3H51, Q9FPE7, VIP2, XRN4, ZHD5, SPT, LEUNG, HAC12, PABP8, PEX14, SPL11, EIF3A, Q9LDZ8, HAC5, BPA1, ZHD9, C3H38, ECT2, PUM5, UBA2C, Q9LM78, Q9LM88, Q9LNA8, BH013, Q9LP92, Q9LQ83, C3H12, IDD11, TPR2, Q9LSD7, Q9LSK7, VCS, VCR, Q9LVK1, IDD1, ZHD8, C3H56, AML4, Q9LZQ7, BIG2, Q9M0M3, DNMT2, Q9M0Y0, Q9M141, Q9M1E4, RH52, AHL15, Q9M369, AI5L6, Q9M9Z1, TCP3, NGA3, EIN2, SPL2, IF4B2, MOS1, Q9SCK9, C3H44, Q9SD86, Q9SD87, RH45, Q9SFD3, WRKY1, BZP30, C3H19, AML2, Q9SK04, Q9SK05, Q9SKR5, PUR, Q9SN77, RH40, PUM4, PRP8A, C3H43, SPT51, Q9STX4, Q9SU22, Q9SU23, Q9SU99, Q9SUE8, C3H46, NINJA, BLH2, R47CP, Q9SZ51, TCX5, PEP, PCKA1, FB230, ARFF, ARFD, Q9ZU48, IDD5, FRS3, QKIL3, PUM2, PUM1
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Ma YT, Luo H, Guan WJ, Zhang H, Chen C, Wang Z, Li JD. O-GlcNAcylation of BMAL1 regulates circadian rhythms in NIH3T3 fibroblasts. Biochemical and biophysical research communications 2013 431(3) 23337503
Abstract:
Various physiological processes and behaviors show a circadian rhythm of approximately 24 h, which is crucial in coordinating internal metabolic processes and environmental signals. Post-translational modifications play an important role in regulating circadian core proteins. In this study, we demonstrated that BMAL1 was modified with an O-linked β-N-acetylglucosamine (O-GlcNAc), which stabilized BMAL1 and enhanced its transcriptional activity. Conversely, inhibition of O-GlcNAcylation resulted in inhibition of circadian rhythms of clock gene expression. Because O-GlcNAcylation is sensitive to the glucose level, such a modification may provide a new mechanism connecting metabolism with circadian rhythms.
O-GlcNAc proteins:
BMAL1, BMAL1
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Alfaro JF, Gong CX, Monroe ME, Aldrich JT, Clauss TR, Purvine SO, Wang Z, Camp DG 2nd, Shabanowitz J, Stanley P, Hart GW, Hunt DF, Yang F, Smith RD. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proceedings of the National Academy of Sciences of the United States of America 2012 109(19) 22517741
Abstract:
O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.
O-GlcNAc proteins:
ZEP3, CAMP1, FRPD1, SKT, DLGP4, DPYL2, STXB1, MAP2, NUMBL, M3K5, NOTC2, CTND2, CSK22, ACK1, SYUA, ATX2, ZFR, BSN, GCR, EGR1, NFL, NFM, RC3H2, MAMD1, ATX1L, DERPC, NCAM1, MAP1B, G3P, ATF2, MAP4, KCC2B, AIMP1, FOXK1, STAT3, AINX, NEDD4, RP3A, DVL1, GOGA3, FOXP1, TB182, GMEB2, PI5PA, MRTFB, DOCK4, ABI2, KCNJ3, NCOA1, RGRF2, TNIK, WNK1, G3BP2, MPRIP, XRN1, RLA2, S30BP, NFIA, MARK3, ENAH, PGBM, CDK12, MA6D1, PHAR1, PSD3, NELL1, PRC2C, YETS2, FOXK2, WNK2, LIMC1, TNR6C, AGAP2, ZEP2, AAK1, TNR6A, CAMKV, PKHA7, GRIN1, FCHO2, GARL3, STOX2, UBN1, ABL2, CDV3, PHAR4, TAB3, NUFP2, UNKL, OSBP2, RBM27, CYFP2, TM1L2, ANR40, NACAD, SIN3A, NCOR1, LAMA5, NCOA2, AP180, RAI1, M3K7, TAF6, SRBS1, SH3G1, TLE4, MINT, ZYX, SF01, SYN2, TBR1, SBNO1, CRTC1, GIT1, SLAI1, PKP4, CDK13, RHG23, SH3R1, JHD2C, HECD1, ABLM3, ARMX2, LAR4B, RHG21, FBX41, RPRD2, WWC2, ZN532, BCR, DLGP3, NYAP1, GMIP, NFRKB, MAGI1, CNOT1, NU188, PICAL, SMAP2, SPAG7, PRC2B, ATX2L, MAP6, MCAF1, PHF24, NAV3, AUXI, RERE, RIMB2, PUM1, NU214, KCMF1, EPN1, AGFG2, UBP2L, C2C2L, CNKR2, ZN598, SHAN2, MAST4, RHG32, MYPT2, TB10B, FRM4A, SP130, DLGP2, ZNT6, ABLM2, EMSY, CLAP2, CNOT4, PAMR1, CREST, IFFO1, OSBL6, YTHD3, TM266, SI1L1, SH3R3, RBM14, CNOT2, ANK2, DIDO1, SYNPO, VCIP1, TAB1, SCYL2, ASPP2, F193A, OGT1, NAV1, SYNJ1, RPGF2, EP400, P66A, PDLI5, SCAM1, HS12A, AGFG1, I2BPL, PO121, ABLM1, SPART, RFIP5, CS047, SIR2, AMOT, CCG8, ZCH14, WDR13, UBAP2, NCOA5, FRS3, ZFN2B, BASP1, DCP1A, SRGP2, SRGP1, SYUB, CLIP1, UBXN1, GORS2, EPN4, RB6I2, ANR17, RTN4, TXD12, NECP1, DLGP1, FIP1, F135B, TM263, PLIN3, MYPT1, CRIP2, TSC1, NBEA, RIMS2, ZN704, RBP2, RTN3, 4ET, ELF2, NUDT3, FMN2, NCOA6, SRCN1, ASAP1, RAD1, SON, PLEC, ULK2, ADDA, PCLO, HIPK2, SH2D3, YLPM1, RHG07, TEN1, NCOR2, COR1B, TNIP1, DEMA, E41L3, SYUG, APCL, MECP2, E41L1
Species: Mus musculus
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Sakabe K, Wang Z, Hart GW. Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code. Proceedings of the National Academy of Sciences of the United States of America 2010 107(46) 21045127
Abstract:
Dynamic posttranslational modification of serine and threonine residues of nucleocytoplasmic proteins by β-N-acetylglucosamine (O-GlcNAc) is a regulator of cellular processes such as transcription, signaling, and protein-protein interactions. Like phosphorylation, O-GlcNAc cycles in response to a wide variety of stimuli. Although cycling of O-GlcNAc is catalyzed by only two highly conserved enzymes, O-GlcNAc transferase (OGT), which adds the sugar, and β-N-acetylglucosaminidase (O-GlcNAcase), which hydrolyzes it, the targeting of these enzymes is highly specific and is controlled by myriad interacting subunits. Here, we demonstrate by multiple specific immunological and enzymatic approaches that histones, the proteins that package DNA within the nucleus, are O-GlcNAcylated in vivo. Histones also are substrates for OGT in vitro. We identify O-GlcNAc sites on histones H2A, H2B, and H4 using mass spectrometry. Finally, we show that histone O-GlcNAcylation changes during mitosis and with heat shock. Taken together, these data show that O-GlcNAc cycles dynamically on histones and can be considered part of the histone code.
O-GlcNAc proteins:
H2A1B, H2B1B, H4
Species: Homo sapiens
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Zeidan Q, Wang Z, De Maio A, Hart GW. O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins. Molecular biology of the cell 2010 21(12) 20410138
Abstract:
Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.
O-GlcNAc proteins:
RS6
Species: Homo sapiens
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Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Science signaling 2010 3(104) 20068230
Abstract:
Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
Species: Homo sapiens
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Wang Z, Udeshi ND, O'Malley M, Shabanowitz J, Hunt DF, Hart GW. Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Molecular & cellular proteomics : MCP 2010 9(1) 19692427
Abstract:
Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.
O-GlcNAc proteins:
D3ZN95, TAU, SYUA, MECP2, SPTB2, SYUG, SYUB
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Dias WB, Cheung WD, Wang Z, Hart GW. Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification. The Journal of biological chemistry 2009 284(32) 19506079
Abstract:
Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk.
O-GlcNAc proteins:
KCC4
Species: Homo sapiens
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Wang Z, Park K, Comer F, Hsieh-Wilson LC, Saudek CD, Hart GW. Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes. Diabetes 2009 58(2) 18984734
Abstract:
O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals.
Species: Homo sapiens
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Ramirez-Correa GA, Jin W, Wang Z, Zhong X, Gao WD, Dias WB, Vecoli C, Hart GW, Murphy AM. O-linked GlcNAc modification of cardiac myofilament proteins: a novel regulator of myocardial contractile function. Circulation research 2008 103(12) 18988896
Abstract:
In addition to O-phosphorylation, O-linked modifications of serine and threonine by beta-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force-[Ca(2+)] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (F(max)) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.
O-GlcNAc proteins:
MYH6, MLRV, MYL3, TNNI3, ACTC
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Wang Z, Pandey A, Hart GW. Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. Molecular & cellular proteomics : MCP 2007 6(8) 17507370
Abstract:
O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glycoproteomics analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had an apparent increase of O-GlcNAcylation upon GSK-3 inhibition by lithium, whereas surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and beta-elimination followed by Michael addition with DTT, we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed an apparent increase of O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor-1, a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on host cell factor-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.
O-GlcNAc proteins:
VIME
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