EMBO/soda-nel huggingface dataset:
Who with whom: Functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation (2020)
Figure 4

Figure 4. Linchpin-mediated E2 stimulation is crucial for priming at Hrd1, but not at Doa10.

A Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2). Data for control strains (Hrd1(400R) wt, Δhrd1, Doa10(94H) wt and Δdoa10) is the same as in Fig. 3D. A side-by-side comparison of protein degradation in all tested strains is shown in Appendix Fig. S1 and S2.

B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7. Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Hrd1, "no ATP" reaction does not contain ATP, but wild-type S-Hrd1(400R). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control are shown. In addition, the reaction containing S-Hrd1(400R) shows significantly more ubiquitylation than those with other E3 variants.

C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2E). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, the reaction for Doa10(94R) is significantly faster than that for Doa10(94H) and (94E). The wild-type E3 is identified by a black frame. The scale of the y-axis was set identical to that of plots from other discharge assays (see Fig. 2E and 3A) for easier comparison.

D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Doa10, "no ATP" reaction does no contain ATP, but wild-type S-Doa10(94H). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown. In addition, the reaction containing Ubc7 and S-Hrd1(400R) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants.
Who with whom: Functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation (2020)
Figure 5

Figure 5. The Ubc6~Ub conjugate populates closed conformations more than other E2~Ub conjugates.

A E2~Ub hydrolysis assays for U7BR/Ubc7 and Ubc6. E3-independent Ub discharge assays for both E2s in PBS (absence of nucleophile = hydrolysis) were performed at 32°C. Left: representative Coomassie gels. Right: Plots of E2~Ub hydrolysis (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as mean ± standard deviation (n = 3).

B HSQC-TROSY experiments comparing free and E2-conjugated (15N)Ub. Top: CSPs of (15N)Ub(G76C) conjugated to indicated E2s via disulfide bond (775 µM each) compared to free (15N)Ub(G76C) are shown; Ub resides known to be involved in the closed conformation interface are underlined in grey; * = number of resonances with perturbations too large to be assigned confidently (shown as off-scale in diagram). Bottom: Spectra of resonances for selected residues in Ub's hydrophobic patch for free Ub(G76C) (gray) and Ub conjugated to Ubc6 (green) and U7BR/Ubc7 (yellow).

C Summary of T1 and T2 relaxation time analysis for (15N)Ub(G76C) in various E2 conjugates. The table shows the mean T1/T2 ratios ± standard deviation over all Ub peaks in a given conjugate (or for the free protein) as well as their molecular weights. Bottom: Cartoons depict open/closed conformation equilibria for different conjugates.
Who with whom: Functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation (2020)
Figure 6

Figure 6. Versatility in ERAD priming E2 usage.

A - F Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA (A and D) and lysine-less CPY*-K0-HA (E) as well as by CHX decay assays for the Doa10 model substrates Deg1-eGFP2 (B) and FLAG-Sbh2 (C and F). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2 and FLAG-Sbh2, respectively). For A, data for control strains (Hrd1(400R) wt, Hrd1(400H) and Δhrd1) are the same as in Fig. 3D and 4A. For D, data for control strains (Doa10(94H) wt and Δdoa10) are the same as in Fig 3D and 4A. A side-by-side comparison of protein degradation in all tested strains is shown in Fig. EV4, EV5 and Appendix Figure S1 and S2.
PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis (2020)
Figure 2

Figure 2: DEN-induced liver tumors upregulate caspase-2 expression without a direct effect of DEN on PIDDosome activation

(A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.

(B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5. N-numbers refer to biological replicates.

(C) Experimental outline: 15 days-old (P15) female and male wt and Pidd1-/- mice were injected with DEN (25 mg/kg) or PBS at t(0). The livers were analyzed after t(8h) and t(48h), or the mice were weaned at P19 to induce polyploidization and harvested 5 days later - t(weaning+5d). t(8h) PBS: wt n=5, Pidd1-/- n=5; DEN: wt n=3, Pidd1-/- n=3. t(48h) PBS: wt n=3, Pidd1-/- n=3; DEN: wt n=5, Pidd1-/- n=3. t(weaning+5d) PBS: wt n=3, Pidd1-/- n=3; DEN: wt n=3, Pidd1-/- n=3.

(D) Immunoblots of the experiment described in (C) at t(8h), t(48h), and t(weaning+5d) for CASP2, RAIDD, p53, γH2AX, and HSP90 as loading control.

(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.

(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.

(G) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n=3 biological replicates.

Data information: Data in (B) are represented as median, (E, G) as mean ± SEM, statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001. Scale bar size in (F) represents 10 µm.
PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis (2020)
Figure 3

Figure 3: Nuclear ploidy and binucleation are reduced in HCC tumor tissue

A, B (A) Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.

C, D (C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red "x" in the quantification shown in (D) were the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5). N-numbers refer to biological replicates.

Data information: Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001; scale bar represents 100 µm.
PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis (2020)
Figure 4

Figure 4: Caspase-2 has no impact on the degree of aneuploidy in HCC. Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately.

A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.
PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis (2020)
Figure 5

Figure 5: Human HCC tumors frequently upregulate CASP2 and PIDD1 expression

(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A.

(B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01.

(C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.
PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis (2020)
Figure 6

Figure 6: Computational analysis of the TCGA Provisional LIHC data set for expression of PIDDosome components reveals correlation with proliferation markers.

(A) Genes co-expressed with the PIDDosome components were examined with respect to the associated Hallmark pathways taking positive (pos) and negative (neg) correlation into account.

(B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes).

(C-E) Correlation (Pearson) of CASP2, RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 1

Figure 1. CCR5 deficiency impairs CD4+ T cell memory responses

A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5-/- lymph node cell suspensions, five weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T cell subtypes is shown (n = 5).

B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5).

C, D. Percentage of CD4+ TEM (C) and TCM (D) in the OT-II WT and CCR5-/- populations (n = 5).

E. IFNγ-producing OT-II WT and CCR5-/- memory cells isolated from mice as in A and restimulated ex vivo with OVA323-339 (1 μM) (n = 4).

F. Immunization scheme for NIP-OVA and NIP-KLH in WT and CCR5-/- mice.

G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).

J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.

Data information: B-E, H-K, data are mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, two-tailed unpaired Student's t-test.
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 2

Figure 2. CCR5 increases the sensitivity of antigen-experienced CD4+ T cells

A, B. Representative histograms and quantification of mean fluorescence intensity (MFI; A) or the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3). The gating strategy is shown in Fig. EV1.

C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).

Data information: * p <0.05, ** p <0.01, *** p <0.001, two-way ANOVA (B) or two-tailed unpaired Student's t-test (C-F).
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 3

Figure 3. CCR5 increases TCR nanoclustering in antigen-experienced CD4+ T cells

A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.

D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5-/- cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE.

F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).

G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.

Data information: A-C, F, G, data are mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, one-tailed unpaired Student's t-test. Scale bar, 50 nm (A-C, G).
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 4

Figure 4. CCR5-induced TCR nanoclustering is specific and independent of its costimulatory activity

A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles).

B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.

C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.

Data information: A-C, data are mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, one-tailed unpaired Student's t-test. Scale bar, 50 nm.
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 5

Figure 5. CCR5 deficiency increases Cer levels by upregulating specific CerS

A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).

B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).

E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5).

F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10).

G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3).

H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).

I. Scheme of a canonical CerS gene to illustrate the in silico strategy used to search for CerS-specific transcription factors.

J, K. Venn diagrams showing the number of transcription factors with putative binding sites in the indicated CerS genes in regions 1 (J) and 2 (K). The red circle highlights the transcription factors shared by CerS2, CerS3 and CerS4 promoters, but not present in the CerS6 promoter.

L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm.

M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).

N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).

Data information: E, G, H, N, Each data point is the average of triplicates in an independent experiment. A-H, M, N, Data shown as mean ± SEM of triplicates; * p <0.05, ** p <0.01, *** p <0.001, two-tailed unpaired Student's t-test.
CCR5 deficiency impairs CD4+ T cell memory responses and antigenic sensitivity through increased ceramide synthesis (2020)
Figure 6

Figure 6. Cer levels determine the grade of TCR nanoclustering

A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.

B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).

C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).

D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).

E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.

F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.

G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).

H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.

I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.

J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).

Data information: A, C-E, G-J, data shown as mean ± SEM. * p <0.05, ** p <0.01, one-tailed (E, I) or two-tailed unpaired Student's t-test. Scale bar, 50 nm.