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PB2-K627-FLAG infection PB2-E627-FLAG infection a 28 28 28 23 28 ) 24 2 26 26 26 26 21 22 24 24 24 24 19 Relative protein abundance (log PB2 PA NP 20 PB2 PA NP 22 0 0.45 1.4 4.1 36 0 0.45 1.4 4.1 36 0 0.45 1.4 4.1 36 0 0.05 0.15 4.1 36 0 0.05 0.15 4.1 36 0 0.05 0.15 4.1 36 Competing α-FLAG Ab (µg) Competing α-FLAG Ab (µg) CSNK1A1 NCOR1 UBE2V2 RAB6B SND1 PB2 interactor Node sizes for p-values: PPA2 influenza target MZT2B < 0.01 post-translational protein modification RAB1B RPN1 0.10 RARA RNF4 0.50 p = 5.68e-13 b novel RXRA MZT2A PSMD11 PSMC6 CCDC47 PSMD12 PSMC1 CD209 ENSP00- 000358622 TUBGCP2 COPS3 SDC2 interleukin-1-mediated signaling pathway TMEM173 UBE2D1 RAVER1 PSMB6 histone H2A acetylation p = 2.14e-07 p = 2.07e-07 DCP2 SIGLEC9 PSMD1 PSMB7 COPS6 MECR IL1RAP EPB41L1 ING3 IRF3 NCOR2 TRRAP TRAF6 LGALS3BP SNUPN RUVBL1 FOXR2 RBM34 PPARA PLXNB2 DMAP1 IKBKB IL1RN viral process UBQLN4 ribonucleoprotein complex biogenesis AGAP2 p = 4.63e-05 p = 2.02e-11 IRAK1 VPS72 DDX24 ANP32A ENSP00- SARS C14orf166 AHCYL1 PLAC1 000233946 NIF3L1 HECTD1 C9orf78 TSG101 NOP2 NCOA1 H2AFZ XPO1 GNL3L STK3 TRIM25 TACC1 STK24 PALMD FTSJ3 DNAJA4 PSMB9 ITGA5 TBL3 RIOK2 RPS5 RPS11 HIST1H4B EIF4H VPS36 NUP214 EDC3 TRMT61A TP53 JUN CCT6B ANAPC2 CDC20 MRTO4 SF3B2 cellular protein localization DDX1 RSL1D1 cell surface receptor signaling pathway SMCHD1 TIMM13 p = 1.46e-06 p = 5.05e-09 G3BP1 EWSR1 RPS8 ATG16L1 ANAPC4 FCGR2A SF3B1 B2M EIF2S1 RPS7 KPNA2 IGLL1 PSMG1 NCBP2 ATP6AP1 NIFK RPL31 TRIM28 VPS25 ICAM1 ANXA11 IPO8 USP42 ENSP00- ALDH18A1 WARS 000264018 CCT7 TRMT6 ATP6V1B2 IL4 ITGA2B IL31RA MAPK8 ITGB1 COPB2 BUB1B CD8B PSMA1 RPS15A ROCK1 APP RAB7A ARNT RAE1 ANAPC7 PPWD1 CCT3 FAS HLA-B KPNA4 PLAU NONO ARCN1 IGLL5 VEGFB LEPR GPD2 SLU7 TRIM21 ATP6V0D1 ENSP00- 000396505 CHMP6 KPNA3 CLU MINK1 ZNF526 FADD ZFAND6 CCT2 GAK ATP6AP2 ADRB2 HLA-A RAB8B oxidative phosphorylation PPP2R2C COPB1 p = 9.72e-10 COPZ1 ATP5B KPNB1 COPA NCBP1 NDUFB7 PPIL3 DCLK2 GLOD4 MYH8 MPP4 MTA1 MT-CO2 ATP5F1 HSPD1 SSR4 ATP5C1 COPE HSP90AA1 mRNA metabolic process HDAC1 p = 7.99e-32 GINS4 NDUFB8 CCS SMARCA5 COX4I1 SART1 SF3B3 SNRPD3 SRP54 CHD5 TLR10 SHROOM2 RBM4 SMC2 GINS2 YTHDF2 NXF1 SAFB2 BCAS2 MAPK14 DBT SSR1 HNRNPH1 HSPA8 nucleobase-containing compound metabolic process SF3A2 SNRPA1 p = 5.93e-07 SNRPC SRSF2 DMD DGKZ TNPO1 SNRPE SNRPG PRCC ATOX1 HNRNPUL1 GMPS GTF2E2 POLD3 SUMO2 ERLIN2 TBP IMPDH2 C12orf57 PTBP1 DNMT1 mRNA export from nucleus HNRNPD ATP7A PTMA CIRBP EFTUD2 DEF6 SNRPB p = 2.17e-21 SRSF9 BUB3 vesicle fusion SNTG1 FUS DCLRE1C HNRNPM BABAM1 p = 1.46e-06 RAB11B USP7 NUP205 NUP88 CTNNBL1 SNRNP70 SNRPF SEH1L POLA1 SAFB PQBP1 STX5 NUP43 PRIM1 SAR1A SNRPD1 SF3A1 GOSR1 LGALS9 AHCY STX6 FLNC DDX17 NUP37 ADAR DTNA TCEAL1 SNAP23 GLRX TPR ACIN1 STX4 NUP107 SNX6 PRKAG2 U2AF2 UBE2I RPS27A TARBP2 PCNA NDUFB9 NUP98 STX10 U2AF1 NDC1 NUP153 SNRPD2 PRPF8 PSAP TRAP1 SRRM2 NCOR1 RAB5A KCTD13 PB2 interactor Node sizes for p-values: influenza target < 0.01 c 0.10 novel 0.50 IGF2BP2 d HSPA1L PPA2 AHCYL1 UBE2A RARA PCK2 PB2 interactor RXRA FSCN1 ZFYVE19 UBXN1 XIAP influenza target UBE2L3 TYK2 USPL1 IL1RAP MAP2K3 RPS8 PGD ZYX CDKN1B FANCC UBE2V2 AXIN1 MAP2K6 novel UCHL3 TNFRSF1A IBTK TARBP2 MAP3K7 EIF2AK2 DHX30 ENSP00- TSR1 USP9X LRRC49 000286332 TRAF6 UBE4B ATP6V1F CDK2 IRAK1 ATP6V1C2 IFNAR1 IL1RN ANAPC2 TNF CFLAR STAU1 CDK1 RELA LTA Node sizes for EIF2S1 CASP8 ATP6V1B2 UBE2C CHUK MAP2K7 HSP90AA1 MECR CST9 p-values: NCOR2 UBR4 TP53 CASP3 ANAPC7 VIM CKS1B BUB1B HSPD1 < 0.01 RAX 0.10 PPARA CDC20 0.50 NLRC4 ENSP00- 000233946 SNRPB SNRPE SNRPF PRPF4 UBL5 C14orf166 CRNKL1 TXNL4A PRPF31 SF3A3 UBC EFTUD2 SART1 SNRPD3 SNRNP200 TRIM25 TIA1 IK RBCK1 SNRPC LSM2 SNRPD2 XAB2 PRKCA SF3B1 BCAS2 SNRPG PRPF8 SF3B3 ERLIN2 NCOA1 SF3A1 SNRNP70 PITPNB RIPK2 STAT1 IRF3 TICAM1 FLOT2 DDX58 PRKRA DHX58 CSNK1E USP15 TRIM14 MAVS CYLD IFIT3 VPS36 FADD GRP TBK1 GOLGB1 TRMT61A UBE2D2 NUP35 ENSP00- 000358622 PRKCB CALCOCO2 ATG5 PEX14 NFKBIA OTUB1 cell surface receptor signaling pathway RPS27A ESYT1 p = 5.05e-09 IKBKB LMNA PRKCD FLNC NFKB1 B2M FCGR2A ATG16L1 IGLL1 H2AFV NUP133 ATP6AP1 RRP1B DHX8 POLA1 VPS25 TCF20 APC ICAM1 PRIM1 NUP107 RBM4 LASP1 PMPCA ATP6V1B2 SON TRMT6 SF3B2 RBM7 IL4 IL31RA ITGA2B RAI1 MAPK8 U2AF1 NXT2 NXF1 CD8B NUP153 IZUMO4 FOS NUP205 SAE1 USP6 RPL13A RPL35 ROCK1 PSMD4 RAB7A ARNT UBE2E1 NUTF2 APP FYTTD1 EXOSC4 SEH1L NDC1 TSTA3 RPS12 RPS5 FAS PSMC1 MAT2A PLAU HLA-B EXOSC3 RPL18A RPS16 TBL3 PSMC6 EXOSC5 IGLL5 NUP98 PSMD11 NCBP1 VEGFB PNPT1 PSMA3 LEPR PSMB6 MAT2B TRIM21 IPO5 RPS11 RPS20 ENSP00- SUMO1 PSMC5 CHMP6 KPNA5 TNPO1 000396505 PSMA1 KHSRP KIF17 MINK1 EXOSC7 PRKDC FADD EXOSC8 GAK ATP6AP2 RPS24 NCBP2 ADRB2 HLA-A RAB8B TNPO3 UPF3A TUFM EXOSC6 RPS3 RPL31 RPS15A UBE2I PSMA2 EEF1A1 RPS18 RPS10 TOP2A RPS14 CEBPD FHOD1 PSMD1 PSMD2 SUMO2 PSMD12 RPS4X SNRPA1 CWC22 G3BP1 DCLK2 ENSP00- MYH8 000337313 RAVER1 NXF3 CAD SNRPD1 COL23A1 CSE1L SUMO3 FUS VPS26A RIOK2 SNUPN UBQLN4 PKIA GTF2E2 TDG VPS16 TBP XPO1 ENSP00- NUP62 000451560 KPNB1 NUP85 UBR1 NUP88 NUP214 STRADA VPS33A TUBA4A TGS1 G6PD TLR10 EPRS SHROOM2 PRKACA HECTD1 BRAF RPS6KA2 RUVBL1 TUBB S100A6 MAP2K1 (Previous page) Supp. Fig. 1: Validation of ICC-MS and network analysis. a, Relative protein abundance of PB2 (red), PA (cyan), and NP (yellow) in PB2 ICC-MS samples shows decreasing capture with increasing competition antibody. Data shown are in biological triplicate. b, Fully annotated and zoomable version of the diagram in Fig 1d. Minimum-cost flow simulations connect top PB2 interactors identified by ICC-MS (red) to influenza host factors (gray) through novel host proteins (white). Modules comprising different PB2 interactors with enriched GO terms and P values indicated. Node sizes indicate empirical P values based on the control flow simulation. c, Enlarged view of MECR-containing module. d, Validation shows that flow simulation networks of protein-protein interactions reflect biochemical pathways that regulate viral replication. Flow simulation captured PKR interactions with RNA binding proteins DHX30, IGF2BP2, and TARBP, and linked RIG-I with TRIM14 through MAVS, and also IFIT3, LGP2, and USP15. Additionally, export pathways were faithfully reconstructed with NXF1 connecting with NXT2 and CRM1 connecting with NXF3. Our networks linked EXOSC3 to the exosome components EXOSC4, EXOSC5, and EXOSC8 and with the NEXT accessory complex (RBM7), both of which are important for influenza transcription (Rialdi et al., 2017). .
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  • Gain of UBE2D1 Facilitates Hepatocellular Carcinoma

    Gain of UBE2D1 Facilitates Hepatocellular Carcinoma

    Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:290 https://doi.org/10.1186/s13046-018-0951-8 RESEARCH Open Access Gain of UBE2D1 facilitates hepatocellular carcinoma progression and is associated with DNA damage caused by continuous IL-6 Chuanchuan Zhou1,2, Fengrui Bi1, Jihang Yuan1, Fu Yang1 and Shuhan Sun1* Abstract Background: Hepatocellular carcinoma (HCC) is the most common type of liver cancer with increasing incidence and poor prognosis. Ubiquitination regulators are reported to play crucial roles in HCC carcinogenesis. UBE2D1, one of family member of E2 ubiquitin conjugating enzyme, mediates the ubiquitination and degradation of tumor suppressor protein p53. However, the expression and functional roles of UBE2D1 in HCC was unknown. Methods: Immunohistochemistry (IHC), western blotting, and real-time PCR were used to detect the protein, transcription and genomic levels of UBE2D1 in HCC tissues with paired nontumor tissues, precancerous lesions and hepatitis liver tissues. Four HCC cell lines and two immortalized hepatic cell lines were used to evaluate the functional roles and underlying mechanisms of UBE2D1 in HCC initiation and progression in vitro and in vivo. The contributors to UBE2D1 genomic amplification were first evaluated by performing a correlation analysis between UBE2D1 genomic levels with clinical data of HCC patients, and then evaluated in HCC and hepatic cell lines. Results: Expression of UBE2D1 was significantly increased in HCC tissues and precancerous lesions and was associated with reduced survival of HCC patients. Upregulation of UBE2D1 promoted HCC growth in vitro and in vivo by decreasing the p53 in ubiquitination-dependent pathway. High expression of UBE2D1 was attributed to the recurrent genomic copy number gain, which was associated with high serum IL-6 level of HCC patients.
  • The Ubiquitination Enzymes of Leishmania Mexicana

    The Ubiquitination Enzymes of Leishmania Mexicana

    The ubiquitination enzymes of Leishmania mexicana Rebecca Jayne Burge Doctor of Philosophy University of York Biology October 2020 Abstract Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. Although 20 deubiquitinating enzymes (DUBs) have been partially characterised in Leishmania mexicana, little is known about the role of E1 ubiquitin-activating (E1), E2 ubiquitin- conjugating (E2) and E3 ubiquitin ligase (E3) enzymes in this parasite. Using bioinformatic methods, 2 E1, 13 E2 and 79 E3 genes were identified in the L. mexicana genome. Subsequently, bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed that the E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for successful promastigote to amastigote differentiation and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, revealed a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a was found to load ubiquitin onto UBC2, allowing UBC2- UEV1 to form K63-linked di-ubiquitin chains in vitro. UBC2 was also shown to cooperate with human E3s RNF8 and BIRC2 in vitro to form non-K63-linked polyubiquitin chains, but association of UBC2 with UEV1 inhibits this ability.