EMBO/soda-nel huggingface dataset:
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 1

Figure 1: Identification of novel category of FFAT motifs regulated by phosphorylation.

A: Schematic representation of STARD3, FIP200, MIGA2, PTPIP51, KCNB1 (Kv2.1) and KCNB2 (Kv2.2) proteins and sequence of their Phospho-FFAT motif (7 core residues and 4 upstream and downstream residues). Note that the 4th position of the FFAT (boxed) of these proteins is occupied by a serine or a threonine residue while conventional FFATs have an aspartic acid. Blue rectangles represent transmembrane helices. TPR: Tetratrico peptide repeat; LDT: lipid droplet targeting domain; BTB: Potassium channel tetramerisation-type BTB domain.

B: Sequence of the peptides used for the pull-down assays. The peptides are composed of an amino-terminal biotin, a linker sequence and the FFAT sequence of STARD3 (residues 200-216), FIP200 (residues 725-741), MIGA2 (residues 286-302), PTPIP51 (residues 151-167), Kv2.1 (residues 584-600), Kv2.2 (residues 599-615), and ORP1 (residues 469-485) either without or with a phosphorylated residue (serine or threonine) at position 4 of the core FFAT motif. The negative control peptide is composed of a random sequence.

C: Western blot analysis of proteins pulled down using the peptides described in (B). The input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti-VAP-A, anti VAP-B and anti-MOSPD2 antibodies. Actin was used as a loading control.

D: Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D478S and D478pS) ORP1 FFAT sequence peptides described in (B). Bound proteins were analyzed using anti VAP-A, anti-VAP-B and anti MOSPD2 antibodies. Actin was used as a loading control.

E: Venn diagram of VAPs and/or MOSPD2 partners with FFAT scores between 0 and 3. Proteins with a Phospho-FFAT and a conventional FFAT are shown in green and red, respectively. A total of 42 proteins possesses both a Phospho and a conventional FFAT. Consensus sequences of the 110 Phospho-FFAT (left) and 94 conventional FFAT motifs (right) identified are shown as sequence logos.

F: List of selected VAP-A, VAP-B or MOSPD2 partners having a potential Phospho-FFAT motif. For a full list, see Table EV2.

Data information: Acidic (D and E) or phosphorylated residues (pS and pT), alcoholic (S and T), and aromatic (F and Y) residues are in red, green and blue, respectively; the other residues are in black.
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 2

Figure 2: STARD3 has a non-conventional FFAT motif which needs to be phosphorylated to form a complex with VAP proteins.

A: Schematic representation of STARD3. Transmembrane helices in the MENTAL domain are in dark blue. The Phospho-FFAT motif of STARD3 and the conventional FFAT motif sequences are aligned and highlighted in pink. Upper numbers correspond to the position of residues in STARD3. Lower numbers correspond to the position of residues in the FFAT sequence as described in (Loewen et al, 2003). Acidic (D and E), alcoholic (S and T) and aromatic (F and Y) residues are in red, green and blue, respectively; the other residues are in black. Phosphorylated serines identified by LC/MS/MS are boxed, and the phosphosite probability determined with PhosphoRS is indicated.

B: Coomassie blue staining of the recombinant wild-type MSP domains of VAP-A and VAP-B, and of the KD/MD mutant of VAP-A, after SDS-PAGE.

C: Recombinant MSP domains pulled down with peptides corresponding to the Phospho-FFAT motif of STARD3 (unphosphorylated; mono-phosphorylated on S209 or S213; bi-phosphorylated on S209 and S213), and with the control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are showed in the input fraction.

D,E: Structure of the MSP domain of VAP-A in complex with a Phospho-FFAT motif. Ribbon diagram (D) and surface representation (E) of the MSP domain of VAP-A in complex with the Phospho-FFAT of STARD3 depicted in stick model. The protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red and blue, respectively; the other atoms are in white (Hagemans et al, 2015).

F,G: Close-up view of the hydrophobic pocket region in the unbound (F) and the Phospho-FFAT-bound (G) VAP-A; residues of VAP-A constituting the pocket are indicated.

H: Close-up view of the structure near the Phospho-FFAT motif highlighting critical residues (in stick model) of human VAP-A present in the binding interface.

I: Close-up view of the structure near the conventional FFAT of ORP1 as described in (PDBID: 1Z9O; Kaiser et al., 2005), highlighting critical residues of rat VAP-A involved in the interaction. The nomenclature for rat VAP-A residues is based on the Uniprot sequence (Q9Z270) and not on the PDB file 1Z9O.

J: Superposition of the two structures shown in (H) and (I) showing the distinct conformations of the C-terminal parts of each peptide.

Data information: Phosphorous, nitrogen, oxygen, and sulfur atoms are colored in orange, blue, red, and yellow, respectively. Carbon atoms are shown in green and grey/pink in the MSP domain and the FFAT motif, respectively. The numbers of the peptide residues are noted in subscript.
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 3

Figure 3: The MSP domain binds the phosphorylated Phospho-FFAT with an affinity in the micromolar range.

A-D: Surface Plasmon Resonance analysis of the MSP domain of VAP-A binding onto immobilized unphosphorylated (A), monophosphorylated pS209 (B) and pS213 (C), and bi-phosphorylated pS209+pS213 (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP-A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady-state analysis of the interaction between pS209 (B right) and pS209+pS213 (D right) STARD3 FFAT peptide, and the MSP domain of VAP-A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP-A concentration, and fitted with a Langmuir binding model.

E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 4

Figure 4: In vivo, a unique phosphorylation of the Phospho-FFAT motif allows STARD3 and VAP-A/VAP-B complex formation and the establishment of ER-endosome contacts.

A,B: Binding assays. Immunoprecipitation (GFP-Trap) experiments between GFP-tagged VAP-A (WT and KD/MD mutant; A), VAP-B (B) and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Please note that the mutations modify the FFAT and Phospho-FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho-FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S209A, mutant has both low Phospho-FFAT and FFAT scores (6.5), the S209D mutant has a high conventional FFAT score (2.5) but a low Phospho-FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho-FFAT score of 5.5. Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.

C-G: Recruitment assays. GFP-VAP-A (C, D, E, G; green) and GFP-VAP-A KD/MD-expressing cells (F; green) were left untransfected (C) or transfected with Flag-STARD3 (D, F), Flag-STARD3 S209A (E), and Flag-STARD3 S209D/P210A (G), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.

H: Pearson's correlation coefficients between VAP-A (WT or KD/MD mutant) and STARD3 (WT or S209A, S209D, S209D/P210A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP-A-STARD3: 22; VAP-A-STARD3 S209A: 25; VAP-A-STARD3 S209D: 27; VAP-A-STARD3 S209D/P210A: 26; VAP-A-STARD3 FA/YA: 20; VAP-A KD/MD-STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (**P < 0.01; ***P < 0.001).
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 5

Figure 5: Phosphorylation of STARD3 in its Phospho-FFAT motif governs membrane attachment and cholesterol transfer in in vitro reconstituted assays.

A: Description of the experimental strategy. LA liposomes (endosome-like) are decorated with cSTD3 or pS209 cSTD3 owing to covalent links with MPB-PE (1.2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide]), and mixed with LB liposomes (ER-like) covered by VAPHIS6 attached to DOGS-NTA-Ni2+ (1.2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl], nickel salt). For dehydroergosterol (DHE) transport experiment, LB liposomes also contain DHE and a dansyl phosphatidylethanolamine (DNS-PE). The transport of DHE from LB to LA liposomes is followed by measuring the change in FRET from DHE to DNS-PE.

B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies. C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars).

D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%).

E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).
FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts (2020)
Figure 6

Figure 6: STARD3-mediated cholesterol transport in endosomes depends on S209.

A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.

F: Pearson's correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated.

G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).
TssA forms a gp6-like ring attached to the type VI secretion sheath (2016)
Figure 1

Figure 1. TssA1 is an essential component of the T6SS apparatus.

A. Maximum likelihood phylogenetic tree generated from 61 aligned TssA sequences belonging to the indicated bacterial species. The three TssA proteins from P. aeruginosa (PaTssA1, PaTssA2 and PaTssA3) and E. coli TssA (EcTssA, GenBank accession number: 284924261) are indicated in bold. B. Graphical representation of the H1-T6SS gene cluster in P. aeruginosa (P.a) (Filloux et al., 2008). The generic tss name and the PA number of corresponding genes are indicated. Genes encoding putative baseplate components are shown in magenta with tssA1 highlighted in bold. C. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (∆A1) (both carrying the pBBR plasmid) and the complemented ∆A1 mutant carrying pBBR-tssA1 (∆A1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.
TssA forms a gp6-like ring attached to the type VI secretion sheath (2016)
Figure 4

Figure 4. TssA binds at one end of TssBC tubules.

A. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution. B. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left. C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively. D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.
TssA forms a gp6-like ring attached to the type VI secretion sheath (2016)
Figure 5

Figure 5. Secondary structure weighted sequence alignment of TssA1 with gp6.

A. Protein sequences were extracted from UniProt accession number BP06_BPT4 for Enterobacteria phage T4 (gp6; Bacteriophage T4) and the Pseudomonas Genome Database (TssA1; reference strain PAO1). Conserved positions are shown in black and grey background. The secondary structural elements corresponding to the 3D structure of gp6 (PDB code 3H2T) are shown above the alignment. Regions with significant sequence conservation are indicated with a corresponding block number. B. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.
TssA forms a gp6-like ring attached to the type VI secretion sheath (2016)
Figure 8

Figure 8. Secondary structure weighted sequence alignment of TssK1 with gp8.

A. Protein sequences were extracted from UniProt accession number BP08_BPT4 for Enterobacteria phage T4 (gp8; Bacteriophage T4) and the Pseudomonas Genome Database (TssK1; reference strain PAO1). Conserved positions are shown in black and grey background. The secondary structural elements corresponding to the 3D structure of gp8 (PDB code 1PDM) are shown above the alignment. Regions with significant sequence conservation are indicated with a corresponding block number. B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 1

Figure 1.

Characterization of endogenous and ectopic expression of OAS1-3 in cell lines with genetically modified OAS expression

Characterization of OAS1-3 expression in parental and OAS knockout PC3 cells, obtained using CRISPR-Cas9 technology. Left: Western blot (WB) analyses of the expression of OAS1, 2, and 3 in clones of OAS single-, double- or triple-knockout PC3 cells (KO, DKO, TKO), treated with IFNβ (500 IU/ml) for 24 h, to increase OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: analysis of the expression of OAS1, 2, and 3 in PC3 DKO cells with increased expression of the remaining OAS, without IFNβ treatment. OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1 (see Fig EV1A), OAS2 and 3 expression was detected by WB. The gene expression data are presented as mean + SD of triplicates.**, p<0.001 in Student's t-test.

Characterization of OAS1-3 expression in parental and OAS knockout HME cells, obtained using CRISPR-Cas9 technology. Left: WB analyses of the expression of OAS1, 2, and 3 in selected clones of HME cells in which OAS1-3 were knocked out, to produce OAS single-, double- or triple-knockout cells (KO, DKO, TKO), treated with 100 IU/ml IFNβ for 24 h to increase the levels of OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: The expression of OAS1, 2, and 3 in HME DKO cells with increased expression of the remaining OAS, without IFNβ treatment. The expression of OAS2 or 3 was determined by WB analysis (asterisk, non-specific band). OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1. The gene expression data are presented as mean + SD of triplicates. **, p<0.001 in Student's t-test.

OAS expression was assessed by WB with anti OAS1-3 in HME cells expressing ectopic OAS1-3 (OAS2hi, OAS3hi, OAS1hi) cells without IFNβ treatment or control HME cells (vec) treated with the indicated concentrations of IFNβ for 2 days (to compare the levels of induced and ectopically expressed OASs). β-actin and Ponceau stain are shown as loading controls (# - transfer artifact); ns, non-specific band.
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 2

Figure 2.

OAS1 protects cells from death in response to oxidizing or alkylating agents

Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS, treated with 75 µM H2O2, 40 μM p-Benzoquinone (pBQ) or 2 mM methyl methane sulfonate (MMS). The number of dead cells (Cytotox Green-permeable) was monitored every 3 h using an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.001; *, p<0.05; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.

Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS, treated with 500 μM H2O2, and KO1-3 and OAS1-3hi cells treated with 750 μM H2O2. Data were obtained and presented as in (A). Means of triplicates ± SD. ***, p<0.001; **, p<0.01; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.

Survival of HME cells, measured by the MTT cell survival assay. Left: control (vec) and KO1-3 cells were treated with 250, 500 or 750 μM of H2O2 and analyzed after 24 h; Right: control, OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates. ***, p<0.001; **, p<0.01; *, p<0.05 in Student's t-test.

Source data are available online for this figure.
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 5

Figure 5.

Intracellular locations of endogenous, IFNβ-stimulated and exogenous OAS proteins

HME control (vec) and OAS single knockout cells (KO1-3), pre-treated with IFNβ for 2 days to induce OAS expression, fixed, and stained with anti-OAS1, 2, or 3. Staining with secondary antibodies is shown as a negative control. Scale bar, 10μm.

HME control cells were treated with IFNβ for 2 days to induce OAS expression, fixed and stained with anti-OAS1, 2, or 3. Scale bar, 10μm.

HME cells expressing Flag-tagged OAS1, 2, or 3 were fixed and stained with anti-Flag. Scale bar, 10μm.

Untreated HME (left) or PC3 (right) cells were stained with anti-OAS1-3 and the nuclei were visualized by staining with DAPI. The intensities of OAS1-3 fluorescence signals in the nuclei are plotted. Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining, respectively. Experiments were repeated at least two times; at least 40 cells per condition were counted. Data are presented as distribution plot with mean value +/- SD; statistical significance was determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.

HME control (vec) and KO1 cells were pre-treated with IFNβ for 2 days, followed by a 10-min treatment with H2O2. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method; staining with anti-AIF and anti-HDAC2 staining was used to characterize membrane and nuclear fractions, respectively. Anti-PAR detects all PAR-modified proteins, which appear as smears.

HME control (v), KO1 and OAS1hi (O1hi) cells were pre-treated with IFNβ for 48 h. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method. Anti-AIF and anti-HDAC2 were used to characterize membrane and nuclear fractions, respectively; * indicates lanes where specific signals are detected.

Source data are available online for this figure.