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Huynh VN, Wang S, Ouyang X, Wani WY, Johnson MS, Chacko BK, Jegga AG, Qian WJ, Chatham JC, Darley-Usmar VM, Zhang J. Defining the Dynamic Regulation of O-GlcNAc Proteome in the Mouse Cortex---the O-GlcNAcylation of Synaptic and Trafficking Proteins Related to Neurodegenerative Diseases. Frontiers in aging 2021 2 35822049
Abstract:
O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer's disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson's disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.
O-GlcNAc proteins:
TANC2, AMRA1, CAMP1, SKT, AGRIN, KANL3, TTLL3, NHSL2, CTTB2, CCDC6, SHAN1, SYGP1, DPYL2, STXB1, CLOCK, NOTC2, VIAAT, CTND2, TPD53, REPS1, NLK, ACK1, SYUA, ATX2, PDLI1, ZFR, HCN1, BSN, TOM1, SYN1, GCR, EGR1, NFL, NFM, ATX1L, DERPC, KCC2A, CNTN1, HSPB1, MAP1B, G3P, ATF2, MTAP2, RS2, FOXK1, STAT3, AINX, EPB41, RFX1, LMNA, INPP, VATA, DVL1, CNBP, ATX1, NCAN, GOGA3, PTPA, GCP3, TB182, GMEB2, YTHD1, PI5PA, MRTFB, LIPA3, NACAM, TNIK, WNK1, NPTN, NEO1, S30BP, ZEP1, APOC2, EMAL1, RELCH, PRC2C, YETS2, FUBP2, QRIC1, LIMC1, DAB2P, ZEP2, AAK1, TNR6A, FCHO2, DRC1, SRBS2, GRM5, PACS2, OXR1, PHAR4, LIN54, MLIP, UNKL, SMG7, RBM27, CYFP2, SYNRG, SRC8, SKIL, NCOR1, LAMA5, HCFC1, P3C2A, SAP, APC, TOB1, AP180, FXR1, HS71A, LASP1, MAFK, M3K7, TAF6, ASPP1, SRBS1, DBNL, SH3G1, TLE4, IF4G2, MINT, ZYX, NUP62, OMGP, TFE3, SYN2, TBR1, RBL2, SBNO1, SLAI1, PKP4, SH3R1, JHD2C, ABLM3, ARMX2, LAR4B, HELZ, S23IP, RBM26, BCR, AHDC1, PAPD7, MFF, KMT2D, ERC2, NFRKB, WDFY3, GGYF2, TEX2, CNOT1, IF2A, PICAL, PLPR3, PRC2B, C2CD5, TPPP, ATX2L, MAP6, NAV3, AUXI, RIMB2, AVL9, NU214, AP4E1, UBP2L, C2C2L, IF4G3, ZN598, SHAN2, LPP, MYPT2, PHIPL, TB10B, CCD40, ZC3HE, DLGP2, ZC21A, BAIP2, EMSY, CLAP2, LIPA2, SRRM2, PAMR1, GEPH, YTHD3, POGZ, EPC2, SI1L1, RBM14, F126B, ANK2, CDAN1, SYNPO, VCIP1, TAB1, MEF2C, F193A, OGT1, EP400, EPN2, P66A, PDLI5, GTPBA, ZBT20, RTN1, BRD3, AGFG1, ABLM1, MRTFA, DC1L1, SPART, RFIP5, NUP35, WASF1, SC6A8, SGIP1, AGAP3, P66B, TAF9, WDR13, LRP5, UBAP2, BASP1, DCP1A, SYUB, TRFE, TRIM7, CIC, S12A6, GORS2, TAB2, EPN4, RNF34, ANR17, NECP1, FLIP1, ROA0, RBM33, TPD54, ODO2, DLGP1, FIP1, TM263, PLIN3, LNEBL, KC1D, NBEA, INP4A, RIMS2, RBP2, RTN3, NUDT3, ATR, ADRM1, FMN2, NCOA6, SON, ULK2, ADDA, MAGD1, MAP1A, GRM3, PCLO, GAB1, FBX6, NPAS3, GUAD, NCOR2, ATRN, NFAT5, DEMA, E41L3, SLIT3, CARM1, DYR1B, MECP2, E41L1, HDAC6
Species: Mus musculus
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White CW 3rd, Fan X, Maynard JC, Wheatley EG, Bieri G, Couthouis J, Burlingame AL, Villeda SA. Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation. Proceedings of the National Academy of Sciences of the United States of America 2020 117(36) 32848054
Abstract:
Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which division-coupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in the mature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNA-sequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc-specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directed mutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch.
O-GlcNAc proteins:
STAT3
Species: Mus musculus
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Zaro BW, Batt AR, Chuh KN, Navarro MX, Pratt MR. The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase. ACS chemical biology 2017 12(3) 28135057
Abstract:
Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.
O-GlcNAc proteins:
A2A5R8, A2A6U3, A2AF81, A2AG39, A2AIW9, A2AJ72, A2AJI1, A2AKV2, A2AL12, A2AMW0, A2AUR3, LAS1L, TRM1L, A5A4Y9, A6PWC3, B0QZF8, B1AU76, UPP, B7ZC19, B7ZP47, B8JJC1, D3YWF6, D3YWK1, D3YWS3, D3YYP4, E9PX53, E9Q066, I2BP2, E9Q4Q2, E9Q5L7, E9Q7W0, E9QP59, F8WGW3, G3UX26, G3UYZ0, G3UZ44, G3X972, H3BKW0, H7BWX9, GTPB1, AIP, ATOX1, HDAC1, GSH0, DHX15, IKBE, AKAP2, SLK, IMPCT, IF6, ACOT1, NMT1, DHB12, SRPK1, ZN326, KLC1, RPP30, IDHC, CASP8, GCR, TYSY, RIR1, S10AA, LEG1, G3P, TPIS, PRDX3, CBX3, TISD, CATA, IMDH2, NFKB1, MAP4, CEBPB, CDK4, FKBP4, HMGB2, KAP3, MP2K1, RANG, PTN11, FBRL, PTN12, FMR1, HMGCL, DYN1, CAP1, STAT1, STAT3, PURA, ALD2, SIPA1, PURA2, GSHR, FOSL2, FOSL1, GSTM5, PCY1A, VATA, HDGF, UBP10, RHOX5, HMGA2, CCHL, NUB1, FAF1, ZNRD2, TB182, PCBP1, ARL1, PFD3, TCTP, HMGB1, DYL1, UB2L3, HDAC2, ELAV1, 4EBP2, PYRG1, TCPB, SPTC2, PSME2, BOP1, WBP2, XDH, HMMR, E2AK2, CO6A1, FABP5, LARP7, CNN2, PP4R2, RM10, Q3TFP0, GUAA, FUBP2, TRADD, CTU2, Q3U4W8, SNX27, BABA1, EDC4, COBL1, SKAP2, ARH40, CSTOS, LRRF1, ZMAT1, Q45VK5, JIP4, MDC1, Q5SUW3, SRC8, SAMH1, KHDR1, SPB6, CAPR1, PAPS1, TS101, PA1B2, FNTA, IGBP1, FSCN1, FXR1, CBX5, HS105, RAI1, MELK, FOXC2, DBNL, CYTB, NDRG1, RALY, GPDM, PUR2, RAB3I, F120A, NOP58, Q6DFZ1, TPM4, Q6NXL1, Q6NZD2, TNPO3, SMHD1, UGGG1, UBXN7, TXLNA, DC1L2, KI18B, JUPI2, LARP1, CAND2, ACAP2, HNRPQ, SPAG7, ATX2L, MAP6, ELP1, PJA2, PGRC2, KCMF1, Q80VB6, FA98B, WDTC1, CPPED, LPP, PEF1, IF4B, ATG4B, Q8BGJ5, FTO, Q8BH80, PRUN1, AHSA1, RCC2, NCEH1, LSS, FBLN3, PPR18, SRRM2, MSRB3, PPME1, RL1D1, TBCD4, NHLC2, MAP1S, TLK1, CND2, RAE1L, SEP10, ZFP57, UBA6, UBA3, STON1, PPM1F, GNL3, Q8CIH9, HMCS1, Q8K0C7, PDXK, ANGE2, LRC41, SDE2, DNM1L, ANLN, MATR3, CBR3, MEPCE, ERF3A, DC1L1, SPART, TDIF2, HEXI1, SNP47, UBP15, MAVS, UBXN4, ACSF2, MICU1, CBWD1, BACH, ISOC1, IPYR2, CSDE1, PIP30, GCSH, Q91X76, DUS3L, BAG2, KCC1A, TTC1, HNRLL, RIN1, PP6R3, MARC2, DBR1, ATAD3, PSIP1, NXF1, NONO, PLST, RRAGC, VMA5A, TARA, DDAH2, TADA1, GRPE1, ABD12, NU155, OGFR, NPM3, GLOD4, COPRS, DPOE4, MIEN1, TRAP1, VATG1, CHSP1, OCAD1, RANB3, MFR1L, NDUF7, TBC15, PPIL4, MPPB, CYBP, ZCHC8, CD37L, MMS19, ARPIN, HNRPM, NXP20, SPF27, TOE1, Q9D4G5, ATAD1, CF226, IPYR, ORN, CNN3, KAP0, PLIN3, AKAP8, EIF3F, IFG15, LIMA1, NEK7, RTN3, STK3, NUP50, SYSM, HSPB8, BAG3, CUL3, RABX5, CAF1A, DREB, TOM40, DNJC7, NFU1, FBX6, NUBP1, DEST, TEBP, ACOT9, NFKB2, KAD2, SKP1, PDC6I, VAPA, CARM1, RAD9A, IF2G, SAE2, TRIP6, MBD2, HNRPF
Species: Mus musculus
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Li X, Zhang Z, Li L, Gong W, Lazenby AJ, Swanson BJ, Herring LE, Asara JM, Singer JD, Wen H. Myeloid-derived cullin 3 promotes STAT3 phosphorylation by inhibiting OGT expression and protects against intestinal inflammation. The Journal of experimental medicine 2017 214(4) 28280036
Abstract:
Signal transducer and activator of transcription 3 (STAT3) is a key mediator of intestinal inflammation and tumorigenesis. However, the molecular mechanism that modulates STAT3 phosphorylation and activation is not fully understood. Here, we demonstrate that modification of STAT3 with O-linked β-N-acetylglucosamine (O-GlcNAc) on threonine 717 (T717) negatively regulates its phosphorylation and targets gene expression in macrophages. We further found that cullin 3 (CUL3), a cullin family E3 ubiquitin ligase, down-regulates the expression of the O-GlcNAc transferase (OGT) and inhibits STAT3 O-GlcNAcylation. The inhibitory effect of CUL3 on OGT expression is dependent on nuclear factor E2-related factor-2 (Nrf2), which binds to the Ogt promoter region and increases gene transcription. Myeloid deletion of Cul3 led to defective STAT3 phosphorylation in colon macrophages, which was accompanied by exacerbated colonic inflammation and inflammation-driven tumorigenesis. Thus, this study identifies a new form of posttranslational modification of STAT3, modulating its phosphorylation, and suggests the importance of immunometabolism on colonic inflammation and tumorigenesis.
O-GlcNAc proteins:
STAT3
Species: Mus musculus
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Chuh KN, Batt AR, Zaro BW, Darabedian N, Marotta NP, Brennan CK, Amirhekmat A, Pratt MR. The New Chemical Reporter 6-Alkynyl-6-deoxy-GlcNAc Reveals O-GlcNAc Modification of the Apoptotic Caspases That Can Block the Cleavage/Activation of Caspase-8. Journal of the American Chemical Society 2017 139(23) 28528544
Abstract:
O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.
O-GlcNAc proteins:
A2A4A6, A2A5R8, GPTC8, SPD2B, A2ACG7, A2AFQ9, A2AFW6, A2AG46, CKAP5, A2AH75, A2AJ72, MA7D1, A2AL12, A2AMW0, A2AMY5, TPX2, PPIG, LAS1L, A5A4Y9, A6PWC3, A6PWK7, UBP36, B1AT03, B1AT82, B1AU75, B2RQG2, OTUD4, B7ZCP4, B7ZP47, D3YUW8, D3YWF6, D3YWK1, D3YX62, SAFB1, D3YXM7, D3YZ06, D3YZP6, D3Z069, D3Z158, D3Z3F8, D3Z6W2, E0CYM1, E9PUH7, E9PVM7, E9PWG6, E9PWV3, E9PWW9, E9PY48, E9PYT3, E9PZM7, E9Q066, E9Q2X6, E9Q3G8, E9Q450, E9Q4K7, E9Q4Q2, KIF23, BD1L1, NUMA1, E9Q7M2, E9Q986, E9Q9E1, E9Q9H2, E9QKG3, E9QKG6, E9QKZ2, E9QLA5, E9QP49, E9QP59, E9QPI5, F2Z3X7, F6S5I0, F7AA26, F7BQE4, FARP1, F8VQ93, F8VQC7, F8VQE9, F8VQK5, F8WI30, G3UZ44, G3UZX6, G3X8R0, G3X8Y3, G3X928, G3X963, G3X972, G3X9V0, G5E896, G5E8E1, H3BJU7, H3BK31, H3BKK2, H7BX26, I1E4X0, I7HIK9, J3QNW0, DPYL2, GTPB1, AKAP1, TCOF, AIP, HDAC1, RL21, GSH0, KIF1C, DHX15, SC6A6, IF6, ILK, ATX2, NMT1, E41L2, DHB12, SRPK1, ZN326, ZFR, PARG, SPD2A, SP1, CASP8, HPRT, LDHA, G6PI, TYSY, RIR1, GNAI2, ITB1, 4F2, H2B1F, MAP1B, HMOX1, LEG1, G3P, KS6A3, COF1, GNAO, IFRD1, VIME, TPM3, UBL4A, CBX3, CXA1, CATA, IMDH2, IL1RA, MCM3, CDK4, NKTR, FKBP4, CBX2, HMGB2, AIMP1, KAP3, MP2K1, SYWC, KIF4, NEDD1, DPOLA, RANG, UBP4, PTN11, RAB18, PTN1, PTN12, LDLR, DNLI1, CAP1, STAT3, STA5B, PURA, ALD2, RAGP1, NEDD4, STT3A, ALDH2, GSHR, GFPT1, PCY1A, MCM4, ICAL, PLCB3, CDN2A, HDGF, UBP10, KPYM, CCHL, IDHP, DDX6, GOGA3, COX17, ACTN4, GCP3, TB182, EIF3E, ABCE1, PFD3, HNRPK, 1433E, RAP1A, RS25, TCTP, DNJA1, HMGB1, IF5A1, RS17, RS12, UB2L3, HXD13, HDAC2, ELAV1, TP53B, CASP3, PYRG1, TCPB, STIM2, SRSF3, CSRP2, SPTC2, BOP1, SMAD4, M4K4, HNRL2, MARK3, LARP7, CNN2, PP4R2, PEPD, CDCA2, Q3TFP0, GUAA, PDE12, Q3TL72, PRC2C, NOL9, FUBP2, TRADD, CTU2, ZN865, Q3U4W8, Q3UG37, NAT9, NOL8, Q3UJQ9, SC31A, NCBP1, LRRF1, DDX17, LRC47, JIP4, EHMT1, CA050, AAPK1, NSRP1, Q5RL57, Q5SQB0, TENS3, PUR4, Q5UE59, SRC8, SAMH1, KHDR1, GRB10, HELLS, SPB6, RIPK1, CAPR1, ASNS, LAP2A, CDC37, TS101, SNTB2, FNTA, BAP31, PLPP1, FSCN1, FXR1, DDX5, ATRX, HS105, DDX3Y, DDX3X, TGFI1, DBNL, SH3G1, CYTB, SMAD2, NDRG1, ZYX, SQSTM, TPP2, ZN512, LAR4B, F120A, CNDG2, NOP58, LTV1, Q6NV52, Q6NXL1, Q6NZD2, ANKL2, Q6P5B5, XPO1, KIF15, FHOD1, TXLNA, PTN23, JUPI2, NUDC1, TACC1, UBE2O, LARP1, ACAP2, 2AAA, MTCH2, ZN503, CYFP1, HNRPQ, SPAG7, DEK, ACTN1, ATX2L, CKP2L, ZN516, ERBIN, SEPT9, PGRC2, Q80VB6, UBP2L, PI42B, ZN598, SAFB2, Q80ZX0, DLG1, LPP, PEF1, IF4B, Q8BGJ5, FTO, TIPRL, Q8BH80, MISSL, ERC6L, CARF, PRUN1, NUP93, FBX30, HBAP1, AHSA1, RCC2, IPO5, SYLC, CKAP4, MAP11, PALM2, CPNE3, SENP7, CSN7B, NSD2, DPP9, Q8BWW3, KANK2, PXK, PIGT, ITPK1, NHLC2, MAP1S, GWL, PKHH2, CND2, THOP1, SEP11, SKA3, CA198, SEP10, AROS, UBA6, LIPB1, SMAG1, Q8CCM0, ZN276, NAA30, SNX8, SYEP, OGT1, GNL3, PDLI5, FERM2, AGO2, HMCS1, AMERL, SCNM1, DNM1L, NEK9, ANLN, EDC3, MATR3, CHAP1, MEPCE, ERF3A, CC137, TDIF2, VPS18, RFC3, MCMBP, HEXI1, LUZP1, SNP47, TMX1, MAVS, UBXN4, Q8VCQ8, ACSF2, PARN, VIGLN, PSMD2, NAA40, F1142, CBWD1, PAXI, SFPQ, CPIN1, RAB14, IPYR2, PUS7, CSDE1, PIP30, RABE2, CISD1, Q91X76, DUS3L, KCC1A, TTC1, SRGP2, SNX18, RISC, HNRLL, Q921K2, PP6R3, LRC59, UBXN1, DBR1, KCC2G, Q924B0, WAC, SMC6, PAWR, SIAS, STML2, PSIP1, NXF1, PDXD1, NONO, PLST, RRAGC, VMA5A, MAOM, DCTN2, ZN281, CT2NL, GRPE1, ABD12, RTN4, NU155, OGFR, NPM3, NOP16, GLOD4, Q9CQ43, MTAP, IFM3, CYB5B, PAF15, PSMD9, WIPI3, SKA2, VATG1, CHSP1, LRC40, RANB3, SMC1A, MFR1L, ARHGP, DDX47, TBC15, PPIL4, MPPB, CYBP, TECR, PAIRB, ZCHC8, SPCS2, Q9CZP3, CD37L, SSBP3, MMS19, MGRN1, ARPIN, HNRPM, SYRC, MCES, Q9D4G5, ATAD1, F162A, TRIR, IPYR, PHF10, ARFG3, ORN, BOLA1, CNN3, KAP0, PLIN3, AKAP8, XRN2, GNAI3, PUR6, RAI14, SENP3, ARFG1, SIL1, VPS35, DGCR8, SYCC, ELP4, LIMA1, XPO2, RBP2, RTN3, PALLD, TMOD3, STK3, COPB, NUP50, DDX21, SH3L1, DDX20, MBNL1, BAG3, GKAP1, ZN207, TRXR1, PPCE, CAF1A, LIMD1, NDRG3, DNJC7, NFU1, COPG1, NUBP1, SMAP, DEST, ACOT9, PR40A, FOXO1, FIZ1, NFKB2, KAD2, AKA12, PRKRA, PDC6I, CHIP, COR1C, VAPA, NDKM, E41L3, TAGL2, CARM1, MTNB, BCL10, IF2G, P5CS, COG1, MD2L1, EIF3G, SAE2, ILF3, TRIP6, USO1, BAZ1B, HNRPF, KEAP1
Species: Mus musculus
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Tong QH, Tao T, Xie LQ, Lu HJ. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications. Biosensors & bioelectronics 2016 80 26866564
Abstract:
Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.
O-GlcNAc proteins:
FOS, AKT1, STAT3, CREB1
Species: Mus musculus
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Gurel Z, Zaro BW, Pratt MR, Sheibani N. Identification of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic retinopathy. PloS one 2014 9(5) 24788674
Abstract:
Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.
Species: Mus musculus
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Chuh KN, Zaro BW, Piller F, Piller V, Pratt MR. Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification. Journal of the American Chemical Society 2014 136(35) 25153642
Abstract:
Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.