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Li F, Yang G, Tachikawa H, Shao K, Yang Y, Gao XD, Nakanishi H. Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT. The Journal of general and applied microbiology 2021 67(1) 33229814
Abstract:
O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.
O-GlcNAc proteins:
MBF1, HIS2, TRP, SYDC, CDC25, ERF3, NOT3, SC160, SIR3, GCR1, PBS2, PT111, MET3, SYIC, SWI1, CDC16, SPS4, HSF, CDC24, FUS1, SIR4, ERF1, WHI2, RT13, EI2BD, KIN1, KIN2, DPOA, ACOX, YAK1, NSP1, CYC8, STI1, UBC5, DPOG, TUP1, SNF5, GLN3, MED15, SFL1, KICH, NUP1, ACE2, VPS1, SNF2, XRN1, NPR1, RM20, SAN1, VPS34, SIN3, NAA38, SPA2, KC11, MBR1, GRR1, SWI4, RBSK, PUF4, STE50, SNT1, BUD3, SRO9, MRC1, SYP1, PAT1, SRB8, MSH1, PHO91, URK1, SPT5, MIG1, IDH2, KIP2, HIBCH, IDH1, HRR25, MCM4, PUS3, PSK1, HS104, MLF3, GLPK, MED16, TREA, SMY1, SFP1, APE2, ISF1, NUP2, PAN1, MED22, SWI3, PT127, YEF3, PMD1, CHD1, NRP1, SLA1, SLX5, NGR1, SEC8, UGPA1, RGT1, BEM3, HFA1, SKG6, SMY2, VPS17, BCK2, BUD2, SEC3, PRP8, PACC, NIP80, MSN4, DCA13, STE13, MKS1, YBZ4, EDE1, PIN4, SEF1, LST4, MTC2, SEG2, FAB1, SCD5, WHI3, NOT4, GCS1, MPE1, DOA1, EAP1, RRN3, YKH1, YK03, DBP7, SA190, SET3, YK20, PET10, PXL1, PAM1, VRP1, BOB1, CDC27, AP2A, RIB1, AKL1, TAF5, ISW1, SEA4, RTG3, ECM21, STU1, MUM2, AIM3, YSW1, AMN1, MED8, CENPU, SDS24, YB75, BIT2, SAF1, TPS3, TSL1, SIC1, KSP1, KIC1, YHR2, RIM4, RRF1, TRM5, YHP7, YHS7, RT107, KEL1, 2A5D, SSN2, SEC31, HAL5, UME6, MPT5, TCPD, RGA1, CAJ1, PIK1, CAT8, YM11, PHO90, MBP1, NUP60, SEN34, ECM1, NPR2, PTI1, IF4F1, IF4F2, PAC2, RSP5, UBP5, BEM2, TOG1, KC13, BOI2, YEI6, GEA2, EDC3, MIT1, RSMB, PCL6, DOT6, GLE2, PEA2, WRIP1, YM24, MED3, SEC9, NAGS, 6P21, YIP2, MLP2, VHS2, POG1, NU159, PRK1, SDS3, SPO22, YIF5, AGE2, ACA2, VID28, YIU0, RLP7, MKT1, GTS1, AZF1, DIM1, BNI1, REF2, VNX1, STB1, GZF3, LAM5, MOB2, RIM15, WWM1, LSB3, IRC5, RMD8, STU2, SUM1, TAF1, AP3B, TRS85, FYV8, ALY2, NET1, BBC1, YJ12A, YJ12B, CDT1, PTK2, ACF4, JSN1, MET7, CENPK, IML1, BCA2, ASK10, SEC16, BUL1, YPT11, CLA4, BRO1, NUP42, NU145, YG51, PSP2, ATP22, SPT20, PSP1, PHO23, NBP1, SCY1, PAN2, PSD2, VID30, ARO8, MCM6, MDS3, INO80, ITC1, PRP43, SLD3, DUO1, YGD4, PIB2, YG3A, PBP1, RT102, SOK2, THO2, MSO1, SNF12, ZRG17, BRE5, CA120, FOL1, CWC25, ELOA1, YNS4, BNI5, INN1, DMA2, NST1, VAC7, SLM2, ARK1, GPD1, PTA1, BCK1, UBP3, MDM1, SKO1, UTP6, MLP1, NU116, LGE1, LEE1, OAZ, ROD1, MUK1, SYH1, INA17, HOS3, YP113, GDE1, GIP3, DIG1, SVL3, RLI1, HOT1, PKH3, TRS31, DIG2, RMD1, TR732, STE20, TDA1, WAR1, TCB3, SKY1, PLB2, NAB6, TAF12, GIR2, YD239, NUP53, MSS11, GIS1, SPO71, YAP6, LIC4, POLH, EIF3G, MUS81, TR120, SIZ1, SEG1, SPO20, PUF6, EUC1, UTP14, TDA9, RSE1, IRC21, RCO1, MFB1, GLU2B, RTN1, NTE1, TAF9, EIS1, HEL2, MSC3, YL225, RGC1, YL177, SKG3, EAF1, RGA2, DCK1, IWS1, IRC20, NDE1, BSP1, RSC3, ECM30, IRC3, VIP1, IMH1, NVJ2, SYT1, EXP1, HBT1, WHI4, YD23B, PUF3, FRA1, YL032, ENT4, YL076, YO036, SGT1, GYP1, YO186, ESA1, HRK1, SOG2, VTS1, NDD1, TCO89, RSA1, AP3D, NEW1, TAF10, RCN2, GGPPS, RTK1, MED2, USV1, YP150, BBP, TIP41, PUF2, KPR5, SMC4, INP53, HER1, GLE1, HSP42, MMP1, STB3, EAF3, RTN2, LAS17, OSH2, CEX1, KCS1, YO22B, LDB19, PAR32, SRF1, DFM1, ADF1, YI31B, YE030, VPS5, YO11A, PNO1, SEY1, PET20
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Fan Z, Li J, Liu T, Zhang Z, Qin W, Qian X. A new tandem enrichment strategy for the simultaneous profiling of O-GlcNAcylation and phosphorylation in RNA-binding proteome. The Analyst 2021 146(4) 33465208
Abstract:
RNA-protein interactions play important roles in almost every step of the lifetime of RNAs, such as RNA splicing, transporting, localization, translation and degradation. Post-translational modifications, such as O-GlcNAcylation and phosphorylation, and their "cross-talk" (OPCT) are essential to the activity and function regulation of RNA-binding proteins (RBPs). However, due to the extremely low abundance of O-GlcNAcylation and the lack of RBP-targeted enrichment strategies, large-scale simultaneous profiling of O-GlcNAcylation and phosphorylation on RBPs is still a challenging task. In the present study, we developed a tandem enrichment strategy combining metabolic labeling-based RNA tagging for selective purification of RBPs and HILIC-based enrichment for simultaneous O-GlcNAcylation and phosphorylation profiling. Benefiting from the sequence-independent RNA tagging by ethynyluridine (EU) labeling, 1115 RBPs binding to different types of RNAs were successfully enriched and identified by quantitative mass spectrometry (MS) analysis. Further HILIC enrichment on the tryptic-digested RBPs and MS analysis led to the first large-scale identification of O-GlcNAcylation and phosphorylation in the RNA-binding proteome, with 461 O-GlcNAc peptides corresponding to 300 RBPs and 671 phosphopeptides corresponding to 389 RBPs. Interestingly, ∼25% RBPs modified by two PTMs were found to be related to multiple metabolism pathways. This strategy has the advantage of high compatibility with MS and provides peptide-level evidence for the identification of O-GlcNAcylated RBPs. We expect it will support simultaneous mapping of O-GlcNAcylation and phosphorylation on RBPs and facilitate further elucidation of the crucial roles of OPCT in the function regulation of RBPs.
O-GlcNAc proteins:
NACAM, SAP18, PLOD2, NOP56, DDX3X, PLXB2, RRP8, SERA, PSMD3, MCA3, PRPF3, TPD54, TIM44, ACTN4, ACSL4, PLOD3, IF2P, ZC11A, SC22B, PR40A, MPPB, CSDE1, U520, NU155, EIF3G, SPF27, RL1D1, CLPX, RTN3, LC7L3, VAPB, SMC2, AP2A1, WIZ, BAG2, TOM40, ACL6A, EGFR, LMNA, TFR1, FRIH, RPN1, RPN2, ITB1, SYEP, HNRPC, SRPRA, VIME, GNAI3, ANXA5, LAMP1, ACADM, TOP1, TOP2A, PABP1, ADT3, TPR, EF2, PDIA4, FPPS, ENPL, ALDR, NDKA, RS2, UBF1, ARF4, NUCL, RAB6A, PSB1, FLNA, SDHB, UBA1, NDKB, ITA6, SFPQ, AT2B4, THIL, RS12, PSA4, SYVC, 1433T, MAP4, PSA5, PSB4, NDUS1, ECHM, KCY, AMRP, SDHA, METK2, CPSM, PUR9, HNRH1, 1433S, STIP1, P5CR1, MCM4, HSP74, CTNA1, MYH9, DEK, RL4, SPB5, NUP62, RBMX, TCPZ, ECE1, PRS6B, KI67, RAGP1, ATRX, SYQ, LMAN1, NASP, FAS, AL7A1, SYSC, MCM2, ACADV, NU153, RBP2, DNLI3, MRE11, CPT1A, F10A1, TCPD, RAB7A, IDH3G, HCFC1, DHB4, HDGF, ROA3, 6PGD, NUP98, ACLY, TCP4, SYYC, UBP14, SNAA, IF5, TERA, DSRAD, TPD52, EIF3B, NU107, EPIPL, SC61B, SRP54, B2MG, SMD2, RL23A, YBOX1, NOP14, IF4G2, GTF2I, NUCB2, RT22, HMGN5, RBM10, TFAM, CLH1, SPTB2, SET, CAP1, EXOSX, EWS, ODO1, RL18A, NUCB1, M2OM, LMNB2, SRS11, CALD1, RL18, C1QBP, CKAP4, KHDR1, DHX9, GOGA2, SSRP1, AHNK, AIMP1, ILF3, SRSF5, SRSF6, TIF1B, TCOF, PICAL, SNW1, TRI29, EIF3A, MLEC, CAPR1, SMC1A, RRP1B, GANAB, NUMA1, U5S1, RRS1, ACOX1, PLEC, RNPS1, PUM3, RB11B, SEPT7, DDB1, CDC37, SRSF7, PCKGM, HNRL2, INF2, PDS5A, PREP, RRP12, TOIP1, HP1B3, RBM26, BRE1A, CDKAL, PRP8, ZC3HE, QSOX2, IKIP, TM10C, EIF3M, PABP2, KTN1, CAND1, THOC6, P66A, MISP, CCAR1, PELP1, NDUF2, RM50, PAF1, TXND5, TOIP2, THOC2, TM263, NU133, PDC6I, SCFD1, LMO7, ELYS, RT27, HS105, NU205, RAD50, SMRC1, TNPO1, FUBP1, P5CR2, DNJA3, PTCD3, DDX27, EFGM, IWS1, NIBA2, YMEL1, PSMD1, EIF3C, ROAA, CMS1, MBB1A, GNL3, PDIP3, PININ, ACAD9, SFXN1, CYBP, RM47, RTN4, DDX21, AAAS, CARF, AATF, BCLF1, MYOF, SYLC, NXF1, SEC63, LIMA1, SEPT9, KAD3, RCOR1, ACINU, TMCO1, PPIE, PA2G4, RUVB2, TR150, RT23, CHTOP, TLN1, HYOU1, SAM50, SP16H, UTP18, SRPRB
Species: Homo sapiens
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Ge Y, Ramirez DH, Yang B, D'Souza AK, Aonbangkhen C, Wong S, Woo CM. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nature chemical biology 2021 17(5) 33686291
Abstract:
O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.
O-GlcNAc proteins:
JUN, SP1, JUNB, NUP62
Species: Homo sapiens
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Petrus P, Lecoutre S, Dollet L, Wiel C, Sulen A, Gao H, Tavira B, Laurencikiene J, Rooyackers O, Checa A, Douagi I, Wheelock CE, Arner P, McCarthy M, Bergo MO, Edgar L, Choudhury RP, Aouadi M, Krook A, Rydén M. Glutamine Links Obesity to Inflammation in Human White Adipose Tissue. Cell metabolism 2020 31(2) 31866443
Abstract:
While obesity and associated metabolic complications are linked to inflammation of white adipose tissue (WAT), the causal factors remain unclear. We hypothesized that the local metabolic environment could be an important determinant. To this end, we compared metabolites released from WAT of 81 obese and non-obese women. This identified glutamine to be downregulated in obesity and inversely associated with a pernicious WAT phenotype. Glutamine administration in vitro and in vivo attenuated both pro-inflammatory gene and protein levels in adipocytes and WAT and macrophage infiltration in WAT. Metabolomic and bioenergetic analyses in human adipocytes suggested that glutamine attenuated glycolysis and reduced uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) levels. UDP-GlcNAc is the substrate for the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme O-GlcNAc transferase. Functional studies in human adipocytes established a mechanistic link between reduced glutamine, O-GlcNAcylation of nuclear proteins, and a pro-inflammatory transcriptional response. Altogether, glutamine metabolism is linked to WAT inflammation in obesity.
O-GlcNAc proteins:
SP1
Species: Homo sapiens
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Ramirez DH, Aonbangkhen C, Wu HY, Naftaly JA, Tang S, O'Meara TR, Woo CM. Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells. ACS chemical biology 2020 15(4) 32119511
Abstract:
O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.
O-GlcNAc proteins:
SBNO1, CNOT1, P121C, DX39A, GTPB1, AP3B1, PGRC1, TAF4, EIF3F, IPO5, IF2B3, NOP56, DDX3X, ARI1A, IRS4, ANM5, TCRG1, PSA7, HGS, MYPT1, HNRDL, XPO1, SET1A, PUR4, NPC1, TIF1A, NKRF, OGT1, PPM1G, EIF3D, EIF3H, DHX15, SERA, HNRPR, IF4G3, E41L2, ZN207, BUB3, ACTN4, HTSF1, AP1G1, SYNC, AKAP8, CALU, SMCA5, JIP4, OGA, HNRPQ, DIAP1, TSN3, SNX2, DKC1, CLAP2, CPNE3, PHF2, ANR17, H2AY, FLNB, NCOR1, CISY, PR40A, SF3B1, CSDE1, U520, EIF3G, PRAF3, SRP72, MTA2, TOX4, SC24D, SC31A, SCAF4, ZRAB2, LC7L3, VAPB, IPO7, SC24B, ACSL3, AP2A1, AIFM1, LDHA, COX2, HPRT, AATM, PGK1, LMNA, TFR1, ALDOA, OAT, G3P, RPN1, RPN2, AT1A1, ADT2, PCCA, IF2A, RLA0, ITB1, ATPB, ENOA, PYGL, G6PI, NPM, LDHB, PDIA1, H10, TBB5, HEXB, PROF1, SYEP, HS90A, HNRPC, 4F2, HS90B, ASNS, ODPA, RU17, RSSA, SNRPA, GSTP1, HMGB1, DLDH, ROA1, PARP1, LKHA4, HS71B, H14, ODP2, THIO, CH60, BIP, HSP7C, EPB41, ODPB, LAMP1, ACADM, TOP1, TOP2A, PYC, C1TC, MPRI, PRPS2, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, XRCC6, XRCC5, EF2, PDIA4, PLST, GLU2B, KPYM, ENPL, PO2F1, HNRPL, SYDC, PLAK, ALDR, EZRI, GNS, RS2, CREB1, H12, AT2A2, JUNB, PYRG1, DDX5, PRS6A, TCPA, RL35A, RL7, VINC, SON, RCC1, NUCL, HXK1, E2AK2, SPEE, IF2B, ANXA7, LMNB1, FLNA, VDAC1, FBRL, PUR2, PUR6, UBA1, NDKB, ROA2, RFX1, TCEA1, SFPQ, PPIB, RS3, NFYA, SAHH, COF1, IF4B, EF1B, MCM3, BRD2, ATPA, PSA1, PSA3, PSA4, PAX6, U2AF2, RL13, PTBP1, SYTC, SYVC, EF1G, RFA1, APEX1, PYR1, CALR, MAP4, CALX, PSB5, TKT, PRDX6, PRDX5, PRDX3, RL12, PEBP1, PDIA3, 2AAA, CDC27, AMRP, SDHA, QCR1, PUR9, HNRH1, STIP1, PRDX2, RL9, CSTF2, MCM4, MCM5, MCM7, GLYM, HSP74, PHB, MYH9, COPB2, ADDA, BASI, FUS, NU214, DEK, MP2K2, ATPG, RL4, SRP14, NUP62, RBMX, GRP75, IF4A3, RS19, RL3, TXLNA, TCPZ, MDHC, MDHM, ECHA, IF2G, GARS, SYIC, LAP2A, LAP2B, MTREX, RS27, LPPRC, MATR3, RANG, VDAC2, UBP5, KI67, RAGP1, NOP2, CRKL, BAG6, RL27A, RL5, RL21, RL28, RS9, STT3A, COPD, PRC2A, TCPE, AL9A1, RL34, NASP, FAS, TCPG, EFTU, SYAC, SYSC, PSB3, MCM2, YLPM1, TMEDA, RBM25, NU153, RBP2, GSK3A, TAF6, GUAA, MRE11, GDIB, EMD, F10A1, LRBA, RL14, TCPQ, TCPD, ANX11, PAPOA, RAB7A, SMCA4, HCFC1, DHB4, ROA3, 6PGD, HNRPM, IMA1, AGFG1, HNRPF, MSH6, RBM5, NUP98, ACLY, COPB, COPA, MOT1, SC24C, SYRC, SYYC, AT1B3, RD23B, P5CS, IF5, XPO2, TERA, AFAD, DSRAD, PSA, SYMC, CTBP2, NU107, TPIS, ACTB, IF4A1, PSA6, ARF3, ABCE1, RAP1B, RS3A, RL26, RL15, S61A1, HNRPK, RS7, RS8, 1433E, RS14, RS23, RS11, SMD1, RL7A, RS4X, H4, RAN, RL23, GBB2, RL10A, RL11, RL8, PPIA, RL40, TRA2B, AP2B1, IF5A1, RACK1, YBOX1, EF1A1, TBB4B, CSK21, IF4G2, GTF2I, TCPB, PRKDC, RL24, RL19, SRSF3, FOXK1, RBM10, MPCP, CLH1, HNRPU, SPTB2, FOXK2, CAP1, LAT1, EXOSX, EWS, RL18A, FKBP4, RL6, TOP2B, KMT2A, LMNB2, TF65, IF4G1, TLE3, SRS11, PUR1, SUH, GABPA, PRDX1, RL18, C1QBP, KHDR1, SRSF1, DHX9, CD47, SSRP1, RBBP4, AHNK, AP1B1, NU160, BPTF, TP53B, AIMP1, ILF2, ILF3, LMAN2, TRAP1, PP1R8, ACACA, ROA0, PRDX4, CBX3, PSMD2, SRSF6, TIF1B, PTK7, PABP4, EIF3I, TCOF, SF3B2, TMED1, PICAL, RIPK1, HDAC1, CUL4B, CD166, IDI1, NFYC, CKAP5, HNRPD, SCRB2, DSG2, EIF3A, UBP2L, SCRIB, TTL12, DCTN1, DYHC1, SRC8, CAPR1, RBM39, MCM6, MDC1, EPN4, SMC1A, RRP1B, UBP10, GANAB, LBR, ZN638, IMB1, NUMA1, SEPT2, SART3, U5S1, SYK, BRD3, PDIA6, IPYR, TEBP, NONO, PWP2, RCN1, PCBP1, PCBP2, SF3B3, SAFB1, SF3A1, NCOA2, SF01, MARE1, NSDHL, TAB1, AAAT, VAS1, ZYX, CCDC6, PKN2, DDB1, CDC37, SRSF7, CPSF6, NRF1, H31T, QSER1, QRIC1, P3H1, TB10B, AMOT, DHB12, PRC2B, H2B2F, HP1B3, CE170, ZC3HD, RBM26, RIF1, RPRD2, ZN318, ECM29, ZMYM4, MAP1S, LIN54, EDC4, PRP8, SCYL2, NFRKB, ZC3HE, LARP1, FIP1, MCAF1, GGYF2, SPT6H, SND1, DHX30, KDM3B, ZCCHV, NUP54, POGZ, NUFP2, MAVS, I2BP2, RBBP6, HUWE1, YTHD3, CENPV, LYRIC, ZN598, GP180, CAND1, CARM1, DDX42, P66A, ARI3B, MGAP, PHF6, CHERP, ANKH1, SUGP1, CCAR1, SPB1, PHAR4, SPART, CCAR2, NUP93, S11IP, FNBP4, CPSF7, ARFG1, ENAH, AFG2H, TXND5, LS14A, Z280C, TNR6A, SMRC2, TBC15, PNPT1, HM13, PO210, GEMI5, ZN384, SMAP2, NU133, PDC6I, PCNP, CKAP2, ATX2L, P66B, ELYS, DDX1, GBF1, NICA, UBXN4, HS105, LAR4B, NU205, AKAP1, TFG, CBP, DDX17, CELF1, RENT1, SMRC1, FUBP2, TNPO1, UBP7, NCLN, FUBP1, FKB10, KBP, PDLI5, FUBP3, CHAP1, Z512B, ZFR, PRRC1, DOCK7, RBM14, VPS35, CIC, EFGM, SIN3A, MINT, CDC5L, PSMD1, EYA3, ATX2, HCD2, ACON, TS101, TCPH, ANM1, SH3G1, COR1B, DIDO1, HNRL1, DDX23, TMED9, NUP58, RBM4, NAA15, B2L13, YTHD1, UNK, ILKAP, SP130, BRD8, I2BPL, SLK, S6A15, PININ, NELFA, PTN23, WNK1, AMPB, GORS2, CYBP, TAF9B, GLOD4, CBX8, NCOA5, CHD8, APMAP, DCP1A, RTN4, ANLN, GEPH, PDLI7, DDX21, SYFB, SYIM, SMC4, RBM12, DDX18, CARF, UGGG1, CDK12, TECR, IF2B1, HPBP1, ITSN2, CNOT2, HACD3, RCC2, SYLC, SUCB1, UBQL2, PCYOX, S30BP, PUF60, NRBP, DACH1, BAZ2A, BAZ1B, CDC23, TASOR, ACINU, CDV3, MRTFB, YETS2, HECD1, PKCB1, DD19B, PRP19, MAGD2, FAF1, TRI33, SRRM2, PA2G4, RUVB2, RUVB1, VDAC3, E41L3, TR150, NOP58, SHLB1, LC7L2, TMED7, STRAP, RTCB, HBS1L, TLN1, HYOU1, PRC2C, SP16H, COPG1, DC1L1, S23IP
Species: Homo sapiens
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Darabedian N, Yang B, Ding R, Cutolo G, Zaro BW, Woo CM, Pratt MR. O-Acetylated Chemical Reporters of Glycosylation Can Display Metabolism-Dependent Background Labeling of Proteins but Are Generally Reliable Tools for the Identification of Glycoproteins. Frontiers in chemistry 2020 8 32411667
Abstract:
Monosaccharide analogs bearing bioorthogonal functionalities, or metabolic chemical reporters (MCRs) of glycosylation, have been used for approximately two decades for the visualization and identification of different glycoproteins. More recently, proteomics analyses have shown that per-O-acetylated MCRs can directly and chemically react with cysteine residues in lysates and potentially cells, drawing into question the physiological relevance of the labeling. Here, we report robust metabolism-dependent labeling by Ac42AzMan but not the structurally similar Ac44AzGal. However, the levels of background chemical-labeling of cell lysates by both reporters are low and identical. We then characterized Ac42AzMan labeling and found that the vast majority of the labeling occurs on intracellular proteins but that this MCR is not converted to previously characterized reporters of intracellular O-GlcNAc modification. Additionally, we used isotope targeted glycoproteomics (IsoTaG) proteomics to show that essentially all of the Ac42AzMan labeling is on cysteine residues. Given the implications this result has for the identification of intracellular O-GlcNAc modifications using MCRs, we then performed a meta-analysis of the potential O-GlcNAcylated proteins identified by different techniques. We found that many of the proteins identified by MCRs have also been found by other methods. Finally, we randomly selected four proteins that had only been identified as O-GlcNAcylated by MCRs and showed that half of them were indeed modified. Together, these data indicate that the selective metabolism of certain MCRs is responsible for S-glycosylation of proteins in the cytosol and nucleus. However, these results also show that MCRs are still good tools for unbiased identification of glycosylated proteins, as long as complementary methods are employed for confirmation.
O-GlcNAc proteins:
CALR, UBP10, TRADD, CYLD
Species: Homo sapiens
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Xu S, Sun F, Wu R. A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation. Analytical chemistry 2020 92(14) 32574038
Abstract:
O-GlcNAcylation has gradually been recognized as a critically important protein post-translational modification in mammalian cells. Besides regulation of gene expression, its crosstalk with protein phosphorylation is vital for cell signaling. Despite its importance, comprehensive analysis of O-GlcNAcylation is extraordinarily challenging due to the low abundances of many O-GlcNAcylated proteins and the complexity of biological samples. Here, we developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective as compared to the engineered enzyme and click chemistry reagents. Biological triplicate experiments were performed to validate the effectiveness and the reproducibility of this method, and the results are comparable with the previous chemoenzymatic method using the engineered enzyme and click chemistry. Moreover, because of the promiscuity of the galactosyltransferase, 18 unique O-glucosylated peptides were identified on the EGF domain from nine proteins. Considering that effective and approachable methods are critical to advance glycoscience research, the current method without any sample restrictions can be widely applied for global analysis of protein O-GlcNAcylation in different samples.
O-GlcNAc proteins:
SBNO1, CNOT1, SWAHB, P121C, PDLI1, TAF4, RNT2, PODXL, KMT2D, MYPT1, ZN609, SC16A, SET1A, ZN185, TNC18, PRPF3, TPD54, SYNJ1, PLIN3, MAFK, BRD4, N4BP1, ICOSL, ANR17, ZN217, NCOR1, ATRN, TOX4, ERLN2, AGFG2, VAPB, SC24A, SC24B, CNOT4, BAG3, LMNA, GCR, HSPB1, IF2A, K1C18, K2C8, K1C19, ROA1, TACD2, ATX1L, LYAG, PPAL, TPR, K1C13, ZEP1, SDC1, ATF1, CBL, GATA3, ARNT, MAP4, CLIP1, HXC9, NU214, MP2K2, CUX1, PBX2, MLH1, STAT3, LAP2A, KI67, RFX5, SOX2, NU153, RBP2, TAF6, HCFC1, AFF3, AGFG1, ATX1, AF17, DSRAD, FOXA1, NU107, FOXK1, SPTB2, TFAP4, EWS, SP2, KMT2A, IF4G1, NOTC2, TLE3, TLE4, REL, ACK1, LG3BP, AHNK, ARHG5, FOXO1, BPTF, RIPK1, NFYC, CDK13, UBP2L, LAGE3, MDC1, EPN4, RRP1B, NCOA6, GSE1, MEF2D, NUMA1, R3HD1, JHD2C, TRIP6, ELF2, TAB1, ZFHX3, ZYX, ADRM1, TAF9, RFX7, QSER1, QRIC1, TB10B, CRTC2, PRC2B, ZN362, UBAP2, RPRD2, ZN318, TASO2, ARID2, ANR40, BICRL, ABLM2, GRHL2, NIPBL, LIN54, TET2, NFRKB, KCD18, MDEAS, ZC3HE, FIP1, SAS6, MCAF1, BCOR, HAKAI, SPT6H, KDM3B, POGZ, MAVS, EMSY, RAI1, SRGP1, SH3R1, YTHD3, CASZ1, P66A, I2BP1, RB6I2, FOXP4, NAV2, GID4, MGAP, CDAN1, SUGP1, MILK2, NUP93, ZN687, FNBP4, ARFG1, ENAH, PHC3, SP20H, KMT2C, STT3B, DLG5, WIPF2, ZFN2B, LMO7, ATX2L, CSKI2, P66B, SMG7, CBP, SEM4D, FUBP2, LPP, PF21A, INT12, CERS2, GWL, PDLI5, CHAP1, ANCHR, Z512B, ZFR, EP400, RBM14, CIC, MINT, S29A1, DPH2, WAC, DIDO1, HNRL1, YTHD1, CEP44, SP130, I2BPL, FOXP1, WNK1, E41L1, ZHX3, GORS2, PKHA5, RC3H2, TAF9B, NCOA5, TANC2, CELR2, UBN1, PDLI7, RBM12, CARF, TAB2, CNOT2, KANL3, STAP2, TCF20, UBQL2, S30BP, SIX4, TASOR, GMEB2, ZHX1, YETS2, PKCB1, NOTC3, TRI33, SRRM2, CHM2B, SCML2, POLH, R3HD2, ZN281, WNK2, PRC2C, NCOR2, GMEB1, ZHX2
Species: Homo sapiens
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Zhu Y, Willems LI, Salas D, Cecioni S, Wu WB, Foster LJ, Vocadlo DJ. Tandem Bioorthogonal Labeling Uncovers Endogenous Cotranslationally O-GlcNAc Modified Nascent Proteins. Journal of the American Chemical Society 2020 142(37) 32870666
Abstract:
Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide N-acetylglucosamine (GlcNAc). This modification, known as O-GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O-GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O-GlcNAc modification can occur cotranslationally and that this process prevents premature degradation of such nascent polypeptide chains. Here, we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O-propargyl-puromycin (OPP) and target the specific subset of nascent chains that are cotranslationally glycosylated with O-GlcNAc by metabolic saccharide engineering using tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-d-galactopyranose (Ac4GalNAz). Using various combinations of sequential chemoselective ligation strategies, we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous cotranslationally O-GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their cotranslational O-GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O-GlcNAc and other cotranslational protein modifications and should stimulate an improved understanding of the role of O-GlcNAc in cytoplasmic protein quality control and proteostasis.
Species: Homo sapiens
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Liu Y, Chen Q, Zhang N, Zhang K, Dou T, Cao Y, Liu Y, Li K, Hao X, Xie X, Li W, Ren Y, Zhang J. Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response. Nature communications 2020 11(1) 33214551
Abstract:
O-GlcNAc modification plays critical roles in regulating the stress response program and cellular homeostasis. However, systematic and multi-omics studies on the O-GlcNAc regulated mechanism have been limited. Here, comprehensive data are obtained by a chemical reporter-based method to survey O-GlcNAc function in human breast cancer cells stimulated with the genotoxic agent adriamycin. We identify 875 genotoxic stress-induced O-GlcNAc chromatin-associated proteins (OCPs), including 88 O-GlcNAc chromatin-associated transcription factors and cofactors (OCTFs), subsequently map their genomic loci, and construct a comprehensive transcriptional reprogramming network. Notably, genotoxicity-induced O-GlcNAc enhances the genome-wide interactions of OCPs with chromatin. The dynamic binding switch of hundreds of OCPs from enhancers to promoters is identified as a crucial feature in the specific transcriptional activation of genes involved in the adaptation of cancer cells to genotoxic stress. The OCTF nuclear factor erythroid 2-related factor-1 (NRF1) is found to be a key response regulator in O-GlcNAc-modulated cellular homeostasis. These results provide a valuable clue suggesting that OCPs act as stress sensors by regulating the expression of various genes to protect cancer cells from genotoxic stress.
O-GlcNAc proteins:
RBM47, SBNO1, CNOT1, RGPD3, P121C, PDLI1, MYO1C, AIP, PSD11, PGRC1, TAF4, CLIC1, IPO5, IF2B3, AGRIN, PLOD2, HMGN4, IMA4, PESC, NOP56, DDX3X, PODXL, IMA3, NFIB, ARI1A, G3PT, PDCD5, TCRG1, PSA7, SCAM3, HGS, MYPT1, HNRDL, XPO1, ZN609, SC16A, SR140, SET1A, NPC1, TBX3, ARC1B, TIF1A, PGRC2, PFD6, NKRF, ZN185, OGT1, HMGB3, PPM1G, EIF3D, IPO8, RPA34, NUP42, DHX15, PRP4, SERA, PSMD3, RFOX2, PAPS1, MCA3, HNRPR, PRPF3, TPD54, IF4G3, KLF4, E41L2, DENR, XPOT, PRC1, ZN207, GET3, BUB3, ACTN4, BUD23, SYNC, KRT86, CPSF5, U3IP2, CALU, SAHH2, MED14, SMCA5, ZN862, GANP, KDM1A, ACSL4, SNX3, OGA, HNRPQ, PLOD3, MAFK, IMA7, UGDH, PQBP1, DKC1, IF2P, EDF1, DNJA2, BRD4, PFD1, WDR1, CPNE3, ZC11A, CLU, T22D2, PP6R2, CREST, ANR17, PDCD6, TBCA, H2AY, FLNB, NCOR1, SC22B, PR40A, PSIP1, SRS10, SF3B1, CSDE1, NPM3, U520, NU155, WDHD1, CRTAP, IDHC, CCNK, PIAS2, SPF27, DNJC8, RL1D1, SRP72, MTA2, TOM70, TOX4, SC24D, SUN1, NFAT5, AP2A2, SC31A, SEM3D, AGFG2, ZRAB2, LC7L3, LYPD3, FKBP9, SMC2, IPO7, AHSA1, PSMG1, SC24A, SC24B, CNOT4, OXSR1, HS74L, AP2A1, BAG3, CLPT1, ACL6A, LDHA, AATM, EGFR, PGK1, ASSY, LDLR, K1C14, LMNA, APOA2, FINC, ALBU, TFR1, PROC, ALDOA, CYTB, ANXA1, GCR, KITH, THY1, K2C1, G3P, HSPB1, RPN1, GNAI2, AT1A1, AT1B1, ADT2, IF2A, HMGN2, ICAM1, RLA2, JUN, LA, ITB1, K1C18, K2C8, CDK1, ATPB, S10A6, ENOA, PYGL, G6PI, NPM, TPM3, ITAV, ACBP, LDHB, PDIA1, H10, CATD, ANXA2, TBB5, PROF1, SYEP, HS90A, HNRPC, TSP1, SP1, ANXA6, RHOC, DAF, MDR1, 4F2, HS90B, SRPRA, ASNS, CY1, RU17, ITA5, NFIC, VIME, RS17, K2C7, ANXA5, K1C16, RSSA, SNRPA, GSTP1, LEG1, HMGB1, TPM1, ROA1, RU2A, PARP1, PPBI, UCHL1, ALDOC, ATX1L, HS71B, CALM3, RO60, H14, PTPRF, THIO, ESTD, CH60, BIP, HSP7C, LAMP1, TOP1, TOP2A, PYC, C1TC, MPRI, ADHX, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, ACTN1, XRCC6, XRCC5, RINI, EF2, K1C10, K2C5, PDIA4, P4HA1, PLST, T2FB, CD59, MIF, GLU2B, CBPM, AK1A1, KPYM, ENPL, CCNB1, PO2F1, HNRPL, SYDC, PLAK, ALDR, AMPN, ERF3A, EZRI, FOSL1, FOSL2, MCP, NQO1, GNS, ZEP1, RS2, DESP, MUC1, CD44, CBR1, CREB1, H15, H13, H12, NCPR, AT2A2, CD36, STMN1, HSP76, HMGA1, JUNB, UBF1, JUND, ATF7, CEBPB, PYRG1, DDX5, PFKAL, LEG3, TCPA, PTN1, RL35A, RL7, VINC, SON, RL17, PGAM1, RCC1, ATF1, ML12A, NUCL, SPEE, RXRA, NFKB1, IF2B, ANXA7, BTF3, PSB1, MPRD, LMNB1, CSRP1, FLNA, 5NTD, VDAC1, CD9, TGM2, PIMT, FBRL, PUR2, PUR6, UBA1, NDKB, ROA2, RFX1, CBL, TCEA1, ITA6, SFPQ, PPIB, SYWC, RS3, NFYA, SAHH, COF1, IF4B, KTHY, EF1B, PPAC, CDK2, MCM3, RS12, BRD2, DNJB1, ATPA, PSA1, PSA3, PSA4, S100P, ITA3, MOES, DDX6, DNMT1, PAX6, U2AF2, RL13, S10A4, HMGB2, PTBP1, SYTC, SYVC, EF1G, STOM, 1433T, ARNT, RL10, RFA1, APEX1, PYR1, CALR, MAP4, CALX, TEAD1, GRN, EPHA2, 3MG, TKT, RBMS1, PML, EF1D, ERP29, PRDX6, RL12, KCY, PEBP1, PDIA3, 2AAA, NMT1, PURA2, UFO, SORCN, ILEU, RPB2, METK2, TIA1, ZEP2, DNJA1, PUR9, HNRH3, HNRH1, 1433B, 1433S, STIP1, S10AB, L1CAM, PRDX2, CDD, ELF1, RL9, CD70, KINH, CSTF2, MCM4, MCM5, MCM7, GLYM, HSP74, PROF2, PHB, SPB6, RFC4, RL22, K1C9, MYH9, MYH10, COPB2, BASI, FUS, NU214, DEK, K22E, PRS7, ATPG, RL4, PP1G, GNL1, SRP14, NUP62, TAGL2, TALDO, RBMX, VKGC, GRP75, IF4A3, RS19, RL3, OST48, FEN1, CAPG, TXLNA, TCPZ, RL13A, STAT3, MDHC, MDHM, IF2G, GARS, SYIC, LAP2A, LAP2B, STAT1, MTREX, RS27, LPPRC, RL35, CDN2A, ECE1, LIS1, MUC18, MATR3, MSH2, SSRA, RANG, VDAC2, CBX5, UBP5, KI67, RAGP1, RECQ1, NOP2, BAG6, NOTC1, RL27A, RL5, RL21, RL28, RS9, RS5, RS10, IQGA1, CAPZB, IF1AX, RL29, SOX9, COPD, GSH0, PSMD8, PRC2A, TCPE, PTSS1, K2C6C, AGRE5, PAXI, RL34, LMAN1, NASP, FAS, CDK8, TCPG, EFTU, SYAC, SYSC, MCM2, ACADV, YLPM1, TMEDA, RBM25, HINT1, NU153, RBP2, TAF6, GUAA, CRIP1, GDIB, EMD, SERPH, F10A1, MAP2, RL14, TCPQ, TCPD, ANX11, PAPOA, FXR1, FXR2, RAB7A, SMCA4, SSRD, HCFC1, HDGF, ROA3, 6PGD, HNRPM, IMA1, GDIR1, AGFG1, HNRPF, MSH6, CAZA1, CRIP2, NUP98, ACLY, COPA, SC24C, TCP4, SYRC, ATX1, ATN1, SYYC, UBP14, AT1B3, RD23B, SNAA, IF5, PSMD4, XPO2, TERA, AF10, AF17, NP1L1, ADK, DSRAD, SEC13, NH2L1, PSA, EIF3B, SYMC, IF6, CTBP2, TMM33, NU107, EPIPL, TPIS, EIF3E, SC61B, MYL6, ACTB, IF4A1, RS20, PRPS1, PSA6, S10AA, CDC42, DEST, RAB10, UBC12, UBE2N, ARP3, ABCE1, RS3A, RL26, PSME3, RL15, RL27, RL37A, S61A1, PFD3, B2MG, DAD1, SUMO2, WDR5, NTF2, HNRPK, 1433G, RS7, PP1A, PP1B, RS8, RS15A, RS16, 1433E, RS14, RS23, RS18, RS29, RS13, RS11, RUXE, SMD1, SMD2, SMD3, PRS10, RL7A, ERF1, CNBP, RS4X, RL23A, RS6, H4, RAN, RL23, RAP1A, RS24, RS25, RS26, RS30, GBB2, RL30, RL31, RL10A, RL32, RL11, RL8, PPIA, FKB1A, RS27A, TRA2B, AP2B1, 1433Z, RSMN, SUMO1, DYL1, RL38, RS21, RACK1, UBC9, YBOX1, CSK2B, TPM4, EF1A1, ACTS, TBA1B, TBA4A, TBB4B, CSK21, PA1B2, HBB, HBA, PITX1, GTF2I, PHC1, TCPB, RAE1L, PRKDC, SARNP, RL24, ARF1, ERH, RL19, SRSF3, FOXK1, DAB2, EFNB1, RBM10, RBM3, CYC, MPCP, VIGLN, CLH1, FKBP3, HNRPU, U2AF1, SPTB2, TIAR, SRSF2, FOXK2, RUNX1, FABP5, LAT1, TFAP4, OTUD4, PFKAP, XPC, EWS, MEF2A, SP3, H11, RL18A, FKBP4, PLOD1, RL6, M2OM, DYST, KMT2A, LMNB2, TF65, UBXN1, GLGB, IF4G1, K1C17, TLE3, REL, 1433F, PLP2, CSTF1, SRS11, EF1A2, SUH, GABPA, PAX8, FMR1, PRDX1, RL18, CKAP4, KHDR1, LRP1, SRSF1, DHX9, LG3BP, PPID, SSRP1, NSUN2, RBBP4, EP300, AHNK, HSP7E, GALT2, BST2, NU160, TBL3, ASPH, TROAP, BPTF, NFIA, SF3A3, AIMP1, ILF2, ILF3, LMAN2, TRAP1, FOXC1, MYO1E, CSTF3, ECH1, ACACA, CAF1B, RED, MTAP, TADBP, ROA0, PRDX4, CBX3, PSMD2, GPS2, SRSF9, SRSF5, SRSF6, TIF1B, G3BP1, PTK7, PABP4, EIF3I, TCOF, SF3B2, HAP28, FKBP5, SMAD4, PICAL, TBB3, PRP4B, PIN1, RIPK1, HDAC1, DCTN2, SNW1, TRA2A, CUL4B, DYR1A, TPBG, FHL1, MOGS, CD166, SPTN1, DX39B, TBB2A, KLF5, BYST, RUNX2, CDK13, CKAP5, CIRBP, HNRPD, SCRB2, DAG1, VEZF1, DSG2, EIF3A, UBP2L, SCRIB, TTL12, FHL2, DPYL3, DYHC1, IF4A2, SRC8, TRI25, FLNC, FA50A, CAPR1, RBM39, MCM6, ITPR1, PUM1, MDC1, EPN4, SMC1A, RRP1B, NCOA6, GSE1, UBP10, GANAB, LBR, MEF2D, CHD4, LASP1, ZN638, IMB1, NOLC1, NUMA1, SEPT2, SART3, CND1, ACAP1, U5S1, SYK, IF4H, PDIA6, PLEC, NOMO1, PON2, IPYR, TEBP, NONO, PWP2, RNPS1, PCBP1, PCBP2, SF3B3, KS6A1, SAFB1, SF3A2, RBMS2, SC23A, SC23B, SF3A1, SSXT, NCOA2, TRAM1, SF01, MED1, HMGN3, JHD2C, TRIP6, MARE1, ELAV1, ELF2, TAB1, AAAT, TOM34, UB2V2, NEDD8, ZYX, SEPT7, ADRM1, UAP1, PSMD5, DDB1, CDC37, DPYL2, RBBP7, TAF9, SRSF7, CPSF6, NRF1, FSCN1, IF16, KYNU, H2A2C, H2B2E, TRXR1, HNRL2, PDS5A, QSER1, TSR1, SMU1</