EMBO/soda-nel huggingface dataset:
Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity (2016)
Figure 1

Figure 1. Mutation in IFITM3 alters protein subcellular localization and anti-HIV activity

(A) A schematic of IFITM3 protein is shown, with gray boxes indicating transmembrane domains and colored bars indicating residues targeted for post-translational modification: ubiquitinated lysines (blue), palmitoylated cysteines (purple), and a phosphorylated tyrosine (red). Residues 17-24 are shown in detail to highlight two adjacent, overlapping motifs that recruit NEDD4 (PPNY) and the AP-2 complex (YEML). The IFITM3 Δ1-21 variant initiates translation at an internal methionine residue (green) and thus lacks these motifs. (B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm. (C) Flow cytometric analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown. (D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies. (E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C). (F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown. (H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody. (I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies. (J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm. (K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown. Western blot images are representative of 3 independent experiments. Unpaired t-test (two-sided), **p<0.005. Statistical comparisons were made between the condition indicated and IFITM3 WT. Normality was determined by the KS test and variance determined by the F test.
Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity (2016)
Figure 2

Figure 2. Mutation of N-terminal regulatory motifs of IFITM3 in T cells enhances the restriction of HIV-1

(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm. (B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry. (C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown. (D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies. (E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.
Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity (2016)
Figure 5

Figure 5. Natural mutations in IFITM3 disrupt regulatory motifs and toggle antiviral activity

(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background. (B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). ‘L’ indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3. (C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin. (D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown. (E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1 NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The ‘Colobus native’ sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus (H1N1 PR/8/34, Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.
Identification and characterization of diverse OTU deubiquitinases in bacteria (2020)
Figure 1

Figure 1. Prediction and validation of OTU DUBs from bacteria

Pfam-generated sequence logo of the regions surrounding the OTU catalytic Cys and general base His (marked with asterisks). The conservation of these regions in the human OTUB1 and predicted bacterial OTUs are shown below, together with their relative order in the sequence topology indicated by the sequence position as well as green and red arrows for the typical and atypical arrangements, respectively.

Bacterial species to which the predicted OTUs belong.

Outcome of interactions between the highlighted bacterial species and their respective eukaryotic hosts.

Percent identity matrix calculated from a PSI-Coffee alignment (Notredame et al, 2000) of the predicted OTU domains. OTUB1 (80-271), EschOTU (184-362), ceg7 (1-298), BurkOTU (186-315), ChlaOTU (193-473), RickOTU (161-356), wPipOTU (66-354), wMelOTU (40-205), and ceg23 (9-277) were used to create the alignment.

Coomassie-stained SDS-PAGE gel showing purified protein from the predicted bacterial OTU constructs.

Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.

Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.

Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.
Identification and characterization of diverse OTU deubiquitinases in bacteria (2020)
Figure 2

Figure 2. Substrate specificity profiling of bacterial OTU DUBs

Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates.

Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.

Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.

Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.

Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.

Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.
Identification and characterization of diverse OTU deubiquitinases in bacteria (2020)
Figure 3

Figure 3. wMelOTU structure reveals novel Ub embrace mechanism

Cartoon representation of the 1.5 Å W. pipientis wMelOTU crystal structure with labeled termini, missing regions, and features of the active site.

Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from human OTUB1 (green, PDB 4DDG) and wMelOTU (purple). Surrounding regions are less-well conserved and shown as semi-transparent.

Enlarged region of the OTUB1:Ub structure (PDB 4DDG) showing entry of the Ub C-terminus (red) into the OTUB1 active site (green). The wMelOTU structure (purple) is overlaid to highlight the structural conflict between the downward position of the His-loop and the Ub C-terminus.

1.8 Å crystal structure of the covalent wMelOTU-Ub complex. wMelOTU (cartoon, pink) is linked to the Ub (surface red) C-terminus through its active site. Primary, secondary, and tertiary regions of the Ub-binding S1 site are indicated.

Structural overlay of the apo (violet) and Ub-bound (pink) wMelOTU structures highlighting the repositioning of the His-loop to accommodate entry of the Ub C-terminus, as well as ordering of two regions in the S1 site that form an embrace around Ub.

Detailed view of the primary and secondary interfaces between wMelOTU (pink) and Ub (red) observed in the wMelOTU-Ub structure. wMelOTU and Ub residues participating in the interface are shown with ball and stick representation.

Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.

Detailed view of the wMelOTU (pink) active site region and its coordination of the Ub C-terminus (red). Residues that coordinate Ub or stabilize the active site are shown with ball and stick representation.
Identification and characterization of diverse OTU deubiquitinases in bacteria (2020)
Figure 4

Figure 4. EschOTU structure shows altered sequence topology

Cartoon representation of the 2.1 Å E. albertii EschOTU-Ub crystal structure with labeled termini and active site. Ub is hidden for this initial view of the OTU fold.

Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from human OTUB1 (green, PDB 4DDG) and EschOTU (orange). Surrounding regions are less-well conserved and shown as semi-transparent.

Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from CCHFV vOTU (blue, PDB 3PHW) and EschOTU (orange). Surrounding regions are less-well conserved and shown as semi-transparent.

Full view of EschOTU (orange) covalently bound to Ub (red) in the S1 site. Primary and secondary interactions with Ub are labeled, as well as the Ub Ile44 hydrophobic patch.

An aligned view as in D, showing S1 site interactions between human OTUB1 (green) and Ub (red) (PDB 4DDG). Ub is rotated 21˚ relative to the EschOTU-Ub structure, but maintains similar primary and secondary contacts.

An aligned view as in D, showing S1 site interactions between CCHFV vOTU (blue) and Ub (red) (PDB 3PHW). Ub is rotated by 95˚ relative to the EschOTU-Ub structure and displays swapped primary and secondary contacts.

Detailed view of the primary and secondary interfaces observed in the EschOTU-Ub structure. EschOTU (orange) and Ub (red) residues participating in the interface are shown with ball and stick representation.

Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.

Detailed view of the EschOTU (orange) active site region and its coordination of the Ub C-terminus (red). Residues that coordinate Ub or stabilize the active site are shown with ball and stick representation.
Identification and characterization of diverse OTU deubiquitinases in bacteria (2020)
Figure 5

Figure 5. Cross-kingdom structural analysis of the OTU fold

Cartoon representation of the EschOTU crystal structure colored in a rainbow gradient from N- to C-terminus. The catalytic triad residues are marked on both the structure and the linear color gradient above, showing their positions with respect to each other and the overall OTU sequence. The black and grey arrows relate how the EschOTU fold is permutated with respect to other OTUs. The black open arrow marks the open N- and C-termini, while the closed grey arrow marks a closed loop. OTU subfamilies that follow this overall sequence topology are listed in the lower right. This arrangement is only observed in EschOTU.

As in A, for the human OTUB1 structure (PDB 4DDG). The closed black arrow marks a closed loop, while the open grey arrow marks the open N- and C-termini. This arrangement is representative of the human Otubain, OTUD, and OTULIN subfamilies, as well as vOTUs.

As in A, for the human Cezanne structure (PDB 5LRW). This arrangement is representative of the human A20 subfamily, viral PLP2, and Legionella ceg23.

Cartoon representation of the CCHFV vOTU crystal structure (PDB 3PHW) colored in a rainbow gradient from N- to C-terminus. The catalytic triad residues are marked on both the structure and the linear color gradient above, showing their positions with respect to each other and the overall OTU sequence. The closed black arrow marks a closed loop, while the open grey arrow marks the open N- and C-termini. A schematic for the permutated vOTUP variant is shown to illustrate how it relates to the native sequence topology.

Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.

Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 1

Figure 1. Impaired endothelial cell-mediated response to shear stress in laminin KO mice.

A) Shear-dilation relations of Tek-Cre::Lama5-/- and Lama4-/- mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek-Cre::Lama5-/- arteries and an enhanced response of Lama4-/- arteries. Data shown are mean changes in vessel diameter (Δ µm) ± s.e.m. from 8 experiments with 16 wild type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P<0.01, paired t-test. The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 2

Figure 2. Laminin composition of laminin KO resistance arteries and topography of the endothelial basement membranes.

A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm. B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm. C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 3

Figure 3. Arterial endothelial cells preferentially adhere to laminin 511 via β1-integrins.

A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411. B) Enhanced fold change (Δ) in COX2 mRNA expression and protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test. C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test. D, E) Inhibition assays performed at 25nM laminin 511 (D), laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 4

Figure 4. Laminin 511 affects the size of endothelial adhesion complexes and cortical stiffness.

(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm. B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test. E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- and Lama4-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%) and increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test. G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 5

Figure 5. Laminin 511 increases cell-cell adhesion strength.

A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch’s correction. C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads. D) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch’s correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.