EMBO/soda-nel huggingface dataset:
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf5

(a–c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG–TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG–TRAF6 were analysed by western blotting. (d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin–HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin–HA, ULK1 and AMBRA1 were analysed by western blotting. (e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies. (f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.03 for LC3 and
P = 0.045 for p62. (g) GFP–LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample;
n = 3 independent experiments. Significance is
P = 0.001 and
P = 0.003 (n = 3). Scale bar, 10 μm. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf6

(a) HEK293 cells were co-transfected with vectors encoding ULK1–HA and ULK1–Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.001. (b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1–HA and ULK1–Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1–HA relative to ULK1–Myc is reported on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.037. (c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.044. (d) HEK293 cells were co-transfected with vectors encoding for ULK1–HA, ULK1–Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.014. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf7

(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG–AMBRA1 and purified FLAG–AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB). (b) HEK293 cells expressing FLAG–RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies. (c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies. (d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.038. (e) GFP–LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample.
n = 3 independent experiments. Significance is
P = 0.001 and
P = 0.003. Scale bar, 10 μm. (f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63–HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63–HA, ULK1, AMBRA1 and actin were analysed by western blotting. (g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below. (h) HeLa cells transfected with ULK1–HA, ULK1–Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions (2021)
Figure 4

Figure 4. The C-rich region is a calcium-binding domain and linker.

(A) Sequence alignment of the C-rich region, including disulfide bonds as brackets, calcium-coordinating acidic residues in red and domain interface residues in green. The consensus symbols are according to Clustal Omega. Pink box highlights the C-rich domain in the four human Teneurins.

(B) The Teneurin4 dimer with one chain colored grey in cartoon and transparent surface representation, and the other chain colored according to Fig 1C with the C-rich domain and C-rich linker in cartoon representation and all other domains in surface representation. Region shown in C indicated with dashed box.

(C) The C-rich domain and C-rich linker straddle the TTR domain, colored purple in surface representation. The calcium ions are shown in green. Coloring as in B. C-rich domain shown in D indicated with dashed box.

(D) C-rich domain in cartoon representation with disulfide bonds in stick representation and labelled. Calcium ions are represented by green spheres.

(E) Stick representation and 2mFobs-DFcalc electron density map at 1σ level of the C-rich domain showing that three calcium ions (I-III) are each ligated in octahedral coordination. Calcium-coordinating residues are labeled.

(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).

Data information: Data are represented as mean ± SEM. Each condition was performed in triplicates.
Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions (2021)
Figure 5

Figure 5. Teneurin4WT adopts the compact dimer conformation in the presence of calcium. (A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.

(B) Pair-distance distribution plots for Teneurin4 proteins colored as in A.

(C) Full-length Teneurin4WT ECD model (inset) and fit (red line) against the Teneurin4WT data in physiological calcium concentrations (black dots).

(D) Distributions of radii of gyration of ensembles of structures selected from the random pool (dotted line), to best represent the SAXS data of each of the Teneurin4 variants, colored as in A.
Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions (2021)
Figure 6

Figure 6. Overexpression of full-length transmembrane Teneurin4WT and Teneurin4Mut induces the formation of filopodia.

(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm.

(B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm.

(C) Average number of filopodia per cell in cells transfected with GFP, GFP-Teneurin4WT (Ten4 WT; red) and GFP- Teneurin4Mut (Ten4Mut; blue).

(D) Fraction of filopodia enriched with GFP per cell in GFP-, GFP-Teneurin4WT (Ten4WT; blue)- and GFP-Teneurin4Mut (Ten4Mut; red)-transfected cells.

(E) Cumulative distributions of the length of individual filopodia in cells transfected with GFP, GFP-Teneurin4WT (Ten4WT) and GFP-Teneurin4Mut (Ten4Mut). The box highlights the zoomed area. n indicates the number of fliopodia analysed per condition.

Data information: Data are represented as mean ± SEM. Each symbol represents an individual cell. Data from 15-17 cells per condition for each independent experiment.
Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions (2021)
Figure 7

Figure 7. Overexpression of full-length Teneurin4WT and Teneurin4Mut induces trans-cellular interactions.

(A) Representative images of non-adherent K562 cells electroporated with GFP (left), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP-Teneurin4Mut (GFP-Ten4Mut; right). Zoomed images show the formation of cellular clusters in cells expressing GFP-Ten4WT and GFP-Ten4Mut, but not in GFP-expressing cells. Scale bar top panel, 50 µm. Scale bar bottom panel, 20 µm.

(B) Cumulative distributions of the cluster size in cells electroporated with GFP (black), GFP-Teneurin4WT (Ten4WT; blue) and GFP-Teneurin4Mut (Ten4Mut; red). Only particles with a cluster size above the average size of a large cell (~560 µm2) are included; n indicates the number of cell clusters/total particles.

(C) Fraction of cell clusters detected per image in GFP- (black), GFP-Teneurin4WT (blue)- and GFP-Teneurin4Mut (red)-electroporated cells.

Data information: Data are represented as mean ± SEM. Each dot represents one image. Data from 9-15 images per condition for each independent experiment.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 1

Fig 1: cGAMP incorporated into Gag-GFP Virus-like particles (VLPs) induces IFN-I in infected cells.

A. Schematic representation of cGAMP- and Empty-VLP production. HEK293T cells were transfected with plasmids encoding HIV-1 Gag-GFP and VSV-G envelope to enable VLP production. Overexpression of cGAS WT in the same cells generated cGAMP that was then incorporated into nascent VLPs (cGAMP-VLPs). As control, Empty-VLPs were produced in cells where a catalytically inactive cGAS (cGAS AA) was overexpressed.

B. cGAMP is incorporated into cGAMP-VLPs. Small molecules were extracted from VLP preparations and the cGAMP concentration was measured using a cGAMP ELISA and a cGAMP bioassay.

C. cGAMP-VLPs induce an IFN-I response in target cells. HEK293 cells were infected with decreasing amounts of cGAMP-VLPs and Empty-VLPs (1/5 serial dilutions starting at 2μL of VLP stocks per well) and the infection was monitored 24 hours later by quantifying GFP+ cells by flow cytometry. Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN-I (ISRE). Luciferase activity measured 24 hours later indicated the presence of IFN-I in the supernatants.

Data information: Data in (B) are pooled from four independent VLP productions. Each symbol corresponds to one VLP production and mean and SD are shown. Data in (C) are pooled from three independent VLP productions tested simultaneously in technical duplicates in infectivity and IFN-I bioassays; mean and SD are shown.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 2

Fig 2: cGAMP loading of VLPs increases the magnitude of the CD4 and CD8 T cell responses elicited after immunisation.

C57BL/6 mice were injected with cGAMP-VLPs, Empty-VLPs, or PBS as a control via the intra-muscular route. 14 days later, VLP-specific T cell responses were evaluated in the spleen.

A-C. Immunisation with cGAMP-VLPs enhances VLP-specific CD4 T cell responses. BMMCs from C57BL/6 mice were pulsed overnight with cGAMP-VLPs and used to stimulate cells from spleens of immunised mice. Cells were co-cultured for six hours prior to evaluation of CD4 T cell responses by ICS. CD4 T cells were gated as live, MHC-II-, CD4+, CD8-. CD4 T cells expressing IL2, IFNγ or TNFα were analysed as shown in (A). The percentage of total CD4 T cells producing each cytokine is shown in (B), and the percentage of CD4 T cells co-producing 1, 2 or 3 cytokines is shown in (C).

D-G. Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Cells from spleens of immunised mice were stimulated with the HIV-SQV peptide. IFNγ-producing cells were enumerated by ELISPOT 24 hours after stimulation with peptide (D). Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E). Panel F shows the percentage of total CD8 T cells upregulating CD107a and/or producing each cytokine, and panel G shows the percentage of CD8 T cells co-producing 1, 2 or 3 cytokines.

Data information: Panels (A) and (E) show representative examples of data from four independent experiments. In panels (B-D), (F) and (G), data are pooled from four independent experiments. A total of 19 mice was analysed per condition. Symbols show data from individual animals, and in (B), (D) and (F) are colour-coded by experiment. Horizontal lines indicate the mean. Statistical analyses were performed using a 2-way ANOVA followed by Tukey's multiple comparisons test. In (B), (D) and (F) data were blocked on experiments. ns p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also Fig EV1 and EV2.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 3

Fig 3: Immunisation with VLPs containing cGAMP increases neutralising antibody responses.

C57BL/6 mice were injected with cGAMP-VLPs, Empty-VLPs, or PBS as a control via the intramuscular route. Serum antibody responses were evaluated 14 days later.

A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.

C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.

D-F. Immunisation with cGAMP-VLPs enhances production of neutralising antibodies. Serial dilutions of serum samples from individual mice were incubated with VSV-G pseudotyped HIV-1-GFP for 90 minutes at 37ºC before infection of HEK293 cells. As a control, serial dilutions of the anti-VSV-G neutralising antibody 8G5F11 were tested in parallel. After two days, infection was measured by quantifying GFP+ cells by flow cytometry (D). Neutralising capacities of serum samples from individual animals were calculated as a percentage of neutralisation (calculated relative to the maximum infection in each experiment) (E) and as the half maximal inhibitory concentration (IC50) (F).

Data information: In (A, B, E and F), data are pooled from three independent experiments and a total of 14 mice was analysed per condition. In (C), data are pooled from two independent experiments and 5-8 mice were analysed per condition. Symbols show data from individual animals, and the means are indicated. Statistical analyses were done using a 2-way ANOVA followed by Tukey's multiple comparisons test (A-C) or a Kruskall-Wallis test followed by Dunn's multiple comparisons test (F). ns p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also Fig EV3.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 4

Fig 4: cGAMP loading of VLPs enhances induction of CD4 Tfh responses.

C57BL/6 mice were injected with cGAMP-VLPs, Empty-VLPs, or PBS as a control via the intra-muscular route. 14 days later, T and B cells in the draining inguinal lymph nodes were characterised by flow cytometry and B cell ELISPOT assays.

A-C. Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). The gating strategy is shown in (A) and the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B). The ratio of Tfh/Tfr is shown in C.

D, E. Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. The gating strategy is shown in (D) and the percentage of germinal centre B cells amongst B220+ cells is shown in (E).

F. Immunisation with cGAMP-VLPs increases production of antibody-secreting cells. Cells from draining lymph nodes and spleens were seeded in ELISPOT plates coated with cGAMP-VLP lysates. After overnight incubation, cells producing VLP-specific IgG antibodies were identified using an anti-IgG Fc antibody.

Data information: In (B), (C) and (E), data were pooled from four independent experiments including a total of 19 mice analysed per condition. Symbols show data from individual animals and are colour-coded by experiment. In (F), symbols show data from 5 mice per group measured in duplicate in one experiment. Horizontal lines indicate the mean. Statistical analyses were done using a 2-way ANOVA followed by Tukey's multiple comparisons test (B, C, E) or a Kruskall-Wallis test followed by Dunn's multiple comparisons test (F). ns p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 5

Fig 5: cGAMP-VLPs pseudotyped with IAV HA induce neutralising antibodies and confer protection following IAV infection.

C57BL/6 mice were immunised with PBS as a control, cGAMP-HA-VLPs or Empty-HA-VLPs via the intra-muscular route.

A, B. VLPs pseudotyped with IAV HA induce neutralising antibodies. Two (A) or three (B) weeks after immunisation with the indicated doses of VLPs, sera were collected, heat-inactivated, and titres of antibodies capable of neutralising an IAV expressing a matched HA protein were determined by micro-neutralisation (MN) assay. The dotted line shows the limit of detection (LOD).

C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).

Data information: In (A), data were pooled from two independent experiments including a total of 12 mice per condition. In (B-D), 6 mice per group were analysed for each VLP dose. In (A) and (B), symbols show data from individual animals. Horizontal lines indicate the mean. Statistical analyses were done using a Kruskall-Wallis test followed by Dunn's multiple comparisons test (A), a 2-way ANOVA followed by Tukey's multiple comparisons test (B) or a survival analysis with the Log-rank (Mantel-Cox) test (D). ns p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also Fig EV4.
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 6

Fig 6: Incorporation of cGAMP within VLPs is essential for its protective effect.

Mice were immunised via the intra-muscular route with 5x104 IU of cGAMP-HA-VLPs, Empty-HA-VLPs, Empty-HA-VLPs + matched cGAMP (3.5ng), Empty-HA-VLPs + 10x cGAMP (35ng), Empty-HA-VLPs + AddaVax (1:1 vol ratio), or PBS as a control.

A, B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (A, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).

C, D. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax induce neutralising antibody responses that accompany protection. Three weeks after immunisation with 5x104 IU of the indicated HA-VLPs, sera were collected and heat-inactivated. Titres of antibodies capable of neutralising an IAV expressing a matched HA protein were determined by micro-neutralisation (MN) assay (C). The dotted line shows the limit of detection (LOD). Correlation between the MN titers at week 3 and the weight loss at day 4.5 post-challenge is shown in (D).

Data information: 6 mice per group were analysed for each HA-VLP and PBS. In (C) and (D), symbols show data from individual animals. In (C), horizontal lines indicate the mean. Statistical analyses were done using a survival analysis with the Log-rank (Mantel-Cox) test (B) or a Kruskall-Wallis test followed by Dunn's multiple comparisons test (C). ns p≥0.05; *p<0.05; **p<0.01; ***p<0.001.