EMBO/soda-nel huggingface dataset:
Nf1 Loss Promotes Kras-Driven Lung Adenocarcinoma and Results in Psat1-Mediated Glutamate Dependence (2019)
Figure 3

Figure 3. Nf1-Silenced Cells are Sensitive to Reduced Glutamine Levels

(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates).

(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates).

(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).

(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).

(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.

(F, G) Quantification of the expression of the six key altered genes in cultured PDKN1 and PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR.

(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.

(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).

(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).

P-values are reported in Appendix Table S3. For bar charts, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Nf1 Loss Promotes Kras-Driven Lung Adenocarcinoma and Results in Psat1-Mediated Glutamate Dependence (2019)
Figure 4

Figure 4. Nf1-Silenced Cells Display Strong Sensitivity to Glutaminase and Aminotransferase Inhibition in Vitro

(A) Schematic summarizing glutamine processing in LUAD cells: glutamine uptake by SLC transporters, followed by glutamine-to-glutamate conversion by glutaminase (Gls), followed by glutamate-to-α-ketoglutarate (α-KG) conversion by phosphoserine aminotransferase 1 (Psat1). The chemical inhibitors CB-839 and AOA are indicated.

(B, C) Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) or AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).

(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates).

(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines.

P-values are reported in Appendix Table S3. For bar charts, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Nf1 Loss Promotes Kras-Driven Lung Adenocarcinoma and Results in Psat1-Mediated Glutamate Dependence (2019)
Figure 5

Figure 5. Nf1/NF1-Mutant Autochthonous LUAD Tumors are Sensitive to Glutaminase and Aminotransferase Inhibition in Vivo

(A) Schematic describing KrasLSL-G12D/+; p53fl/fl (KP) mice intratracheally infected with pSECC lentiviruses containing sgNf1.3 or control sgTom. Mouse tumor burden was tracked by micro-computed tomography (micro-CT) post-infection, and treatment was initiated on week 8 post-infection. Lungs were finally harvested on week 21 post-infection.

(B, C) Quantification by micro-CT of mean tumor volumes in (B) CB-839-, (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B) and (C).

(D, E) Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D) and (E).

(F, G) Relative responses of KP mice tumors treated with (F) CB-839, (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle. (H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots.

P-values are reported in Appendix Table S3. For bar charts, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 1

Figure 1. Identification and characterization of sec mutants.

A Schematic genomic structure of the SEC locus. Exons are shown as black boxes, promoter and introns are indicated as lines, and 5' and 3' untranslated regions are shown as grey boxes. T-DNA insertion positions are indicated by triangles. P represents the SEC transcript detected by qRT-PCR.

B Identification of sec homozygotes. LB represents the left border primer of the T-DNA insertion. LP and RP represent the left and right genomic primers, respectively.

C qRT-PCR analysis of SEC mRNA levels in sec-4 and sec-5 mutants. The expression level was normalized to that of TUBULIN, a reference gene for qRT-PCR. Independent biological experiments were repeated three times, and one representative result is shown here. Data shown are means ± standard deviation (s.d.), n = 3.

D The early flowering phenotype of sec-5 mutant under long-day (LD) conditions. Scale bar: 1 cm.

E qRT-PCR analysis of the expression levels of FLC, SOC1, and MAF1-3 in 12-d-old vegetative Col-0, sec-4 and sec-5 plants. Independent biological experiments were repeated three times, and one representative result is shown here. The expression level was normalized to that of TUBULIN. Data are means ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.

F The sec-5 mutant exhibits an early flowering phenotype under short-day (SD) conditions. Scale bar: 1 cm.

G qRT-PCR analysis of FLC and SOC1 expression in Col-0 and sec-5 plants under SD conditions. The expression level was normalized to that of TUBULIN. Experiments were repeated three times, and one representative result is shown here. Data are mean ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.

Source data are available on line for this figure.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 2

Figure 2. Loss of SEC function alters histone modification states on FLC chromatin.

A Schematic of the FLC locus. Exons are indicated with black boxes; promoter and introns are shown as lines. P1, P2 and P3 refer to genomic regions examined by ChIP.

B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin.

C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin.

D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin.

E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin.

Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 3

Figure 3. ATX1 interacts with and is functionally dependent on SEC.

A Yeast two-hybrid assay of the interaction between ATX1 and SEC. The full-length coding sequence (CDS) of ATX1 was cloned into the bait vector pGBKT7. A full-length SEC CDS and a truncated fragment were cloned into the prey vector pGADT7 to express complete or truncated SEC-N (including only TPR domains at the N-terminus), respectively. AD vectors expressing SEC or SEC-N were each cotransformed with BD-ATX1. SD/2, SD/-Leu/-Trp medium; SD/4, SD/-Ade/-His/-Leu/-Trp medium. Cotransformed pGBKT7-53/pGADT7-T was used as a positive control.

B Immunoblot analysis for O-GlcNAcylation of ATX1 by SEC using anti-CTD110.6 antibody. HA-tagged ATX1 was expressed alone or together with Flag-tagged SEC in tobacco leaves. Nuclear proteins were extracted from tobacco mesophyll cells, and expression of FLAG-SEC and ATX1-HA was confirmed by western blotting.

C Flowering phenotype of 35S::ATX1-HA overexpression transgenic lines in Col-0 and sec-5 backgrounds, respectively. Scale bar = 1 cm.

D Immunoblot analysis using anti-HA antibody to confirm ATX1-HA expression in Col-0 and 35S::ATX1-HA-overexpressing lines shown in (C). ACTIN was used as a loading control.

E-G Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines in (C). The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of rosette leaf numbers, visible buds percent and bolting rates. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.

Source data are available on line for this figure.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 4

Figure 4. SEC O-GlcNAcylates and activates ATX1 in vitro and in vivo.

A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis.

B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A).

C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount.

D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization.

E Comparison of ATX1 protein levels in Col-0 and sec-5 plants at stages before and after flowering. Seedlings were collected for protein extraction at 21 days (before flowering) and 35 days (after flowering) after seeds were planted on plates. Anti-ATX1 antibody was used for immunoblot assay.

Source data are available on line for this figure.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 5

Figure 5. S947 of ATX1 is necessary for its O-GlcNAc modification and activation by SEC.

A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O-GlcNAc modification level of ATX1ΔN.

B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC; moreover, mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity.

C Quantitative analysis of HKMT activity of ATX1ΔN, ATX1ΔN-12m, and ATX1ΔN-m and the effect of SECΔN and ΔSEC5m in activating those proteins. Experiments were repeated three times. Error bars are SD. His-ΔSEC5m and His-SECΔN were used as the control.

Source data are available on line for this figure.
Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering (2018)
Figure 6

Figure 6. Mutation of S947A inhibits ATX1 function in Arabidopsis.

A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation.

B Quantitative real-time PCR analysis of ATX1 and FLC transcript levels in Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG plants. The transcript levels were normalized to that of TUBULIN. Data are mean ± s.d. of three independent biological replicates, n=3.

C­-E Flowering time phenotype analysis of lines in (A). The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.

F Working model of SEC activation of ATX1 to regulate FLC transcription by O-GlcNAc modification.

Expanded View Figure Legends
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 1

Figure 1. RBR E3 ligases do not require closed states of E2~Ub for ubiquitin transfer. a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition. b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding. c) CSPs from (b) are mapped onto the structures of UbcH5c (PDB 2fuh) and Ub (PDB 1ubq). Residues that exhibit loss of intensity > 1 stdv upon RING1 binding (intensity < 0. 47) are colored on each structure: UbcH5 residues 5, 6, 7, 16, 20, 22, 56, 62, 74, 87, 90, 91, 96, 97, 99-103, 137, 138 (pink) and Ub residues 48, 50 (yellow). The lack of CSPs on the UbcH5 cross-over helix and on the surface of Ub signifies that HHARI RING1 does not induce closed UbcH5~Ub. d) CLUSTAL OMEGA alignments of the C-terminal sequences of RBR RING1 domains (top) and canonical RING domains (bottom). Each alignment includes the 7th and 8th Zn2+ coordinating positions (gray). The allosteric linchpin position critical for E2~Ub activation by RING-type E3s is highlighted in yellow. RBR RING1 domains lack the allosteric linchpin.
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 2

Figure 2. UbcH7~Ub populates E3-independent closed conformations. a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix. b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7(blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 3

Figure 3. HHARI RING1 disfavors closed E2~Ub conformations. a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7. b) Region of 1H-13C-HSQC spectra of 13C-UbcH7 (blue), 13C-UbcH7-O-Ub (black), and HHARI RING1-bound 13C-UbcH7-O-Ub (red) that includes the methyl (13CH3) resonance of the surface-exposed UbcH7 cross-over helix residue, Ala 110 (black arrow) is shown. The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C-UbcH7-O-Ub (black) and reappears at its position in free UbcH7 in the presence of HHARI RING1, consistent with disruption of closed UbcH7~Ub conformations. Pairwise overlays and larger spectra are provided in Appendix Fig S3. c) Regions of 1H-15N-HSQC-TROSY spectra of UbcH7-O-15N-Ub in the absence (black) and presence (red) of a RING1 domain from the RBR E3s HHARI (left) or RNF144 (middle) or a canonical RING domain of BRCA1/BARD1 (right). The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar, while binding of the canonical RING domain of BRCA1/BARD1 has no observable effect on the Ub spectrum. Gray boxes mark area expanded in a)
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 4

Figure 4. RING1 opening of E2~Ub enforces transfer via the RING2 active site. a) Schematic representation of the two constructs used. LEFT. GST-tagged HHARIRBR with its native RING1 domain that does not induce closed conformations of UbcH5~Ub. RIGHT. GST-tagged HHARI construct in which the RING1 domain was replaced with the Ubox domain of E4BU to generate the UBR-hybrid that promotes closed conformations of UbcH5~Ub. b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 5

Figure 5. The Ub hydrophobic patch plays a role in Ub transfer from E2~Ub onto RING2 of RBR E3 ligases. a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition. b) The hydrophobic patch of Ub (PDB 1ubq) is colored yellow on a surface representation and positions of each mutation are noted. c) UbcH7~Ub conjugates were preformed with either with WT-Ub or I44A-Ub. After the addition of apyrase to quench the charging reaction, UbcH7~UbWT (left) or UbcH7~UbI44A (right) was incubated with ParkinRBR. The disappearance of each UbcH7~Ub species and appearance of auto-ubiquitinated E3 was visualized under non-reducing conditions by western blotting for HA-Ub. Time was recorded post addition of ParkinRBR. E2~Ub conjugated with I44A-Ub does not disappear over the timecourse of the reaction. d) UbcH7 conjugated with WT-Ub, I44A-Ub, or V70A Ub as indicated was incubated with either H887A-HOIPRBR-LDD (left) or H359A-HHARIRBR (right) mutants that allow trapping of the E3~Ub thioester with WT-Ub (Stieglitz et al, 2013;[29]). While a HOIP~Ub thioester species is observed when UbcH7 was charged with WT-Ub, no detectable transfer occurs with the I44A-Ub conjugate (left). Similarly, V70A-Ub shows reduced formation of the HHARI~Ub thioester (right). In addition to blotting for HA-Ub, the blot on the right was also blotted for GST-HHARI.
Molecular Insights into RBR E3 ligase Ubiquitin Transfer Mechanisms (2016)
Figure 6

Figure 6. HHARI RING2 binds to Ub. a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL). b) Residues with CSPs greater than 1 stdv (HHARI residues 333, 337-342, 352-354, 363, 365, 371) are colored red in a cartoon (left) and surface (right) representation of HHARI RING2 (PDB 2m9y). The active site C357 is in shown in yellow. c) Sequence alignment of HHARI and Parkin RING2 domains. HHARI residues perturbed by Ub binding (panel a) are in red. Residues that decrease auto-ubiquitination activity when mutated in HHARIRBR are indicated with black boxes. d) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto-ubiquitination assays. Time points (10min) are visualized by western blotting for the GST-tag on HHARI. Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared. e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).