EMBO/soda-nel huggingface dataset:
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 8

Figure 8 - SKIP recruits non-mitochondrial TBC1D15.

A/B. Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous A. Rab7 or B. CD63 (red). Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).

C-F. Effect of FIS1 on late compartment distribution. C. Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 (white).

D. Plot of CD63 pixel distribution as a function of FIS1 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n siC=14, n siFis1=17 cells analysed from 2 independent experiments.

E. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to FIS1 depletion, n siC=13, n siFis1=9 images (3≥ cells per image) analysed from 2 independent experiments (see also Fig EV2C).

F. Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC, n=3 independent experiments.

G-J. Recruitment of endogenous TBC1D15 to the SKIP compartment. G. Left panels: representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP (G-e1D15, green) and ectopically expressing either empty vector, HA-SKIP or PLEKHM1-HA (red), immunolabeled against HA. Zoom insets (2.7x) highlight select peripheral (PP) and perinuclear (PN) cell regions. Right panels: pixel plots of endogenous GFP-TBC1D15 (green line) and HA signals (red line) corresponding to the dashed white lines in G. H-J. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥5 images, 3≥ cells per image, analysed per condition from 2 independent experiments (see also Fig EV3D and E).

K. Graphical summary of TBC1D15 recruitment to SKIP.

Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines, where applicable, all scale bars: 10µm. Graphs report mean (red line) of sample values (open circles), error bars reflect +/- SD. All significance was assessed using two-tailed Student's t-test: *** p<0.001, ns: not significant.
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 9

Figure 9 - Rab7 interactions influence SKIP-associated HOPS complex assembly.

A-D. Effects of SKIP KMI motif on HOPS complex recruitment. A. Analysis of SKIP/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with Myc-SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP-trap beads. Representative immunoblots against RFP and Myc are shown, IP: immunoprecipitation, TL: total lysate. B. Quantification of SKIP/HOPS interactions, n=3 independent experiments. Top graph: fold change in binding of HOPS subunits to AAA mutant relative to wild type (WT) SKIP (1.0), significance: one-way ANOVA test (relative to VPS41). Bottom graph: Co-IP per HOPS subunit relative to VPS39, significance: one-way ANOVA test (relative to VPS39), * p<0.05, *** p<0.001, ns: not significant. C. Colocalization (Mander's overlap) between SKIP and RFP-VPS39 (images shown in D) or mCherry-VPS41 (images shown in Fig EV4C), n≥10 images (2≥ cells per image) analysed per condition from 2 independent experiments. D. Representative confocal images of fixed HeLa cells ectopically expressing Myc-SKIP, KMI motif mutant (AAA) or RUN domain truncation mutant (∆RUN) (red) together with RFP-VPS39 (green), immunolabeled for endogenous VPS11 (eVPS11, blue).

E-I. Effect of HOPS over-expression on recruitment of TBC1D15 to the SKIP compartment. E. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (Ge1D15, green) and ectopically expressing HA-SKIP (red) together with mCh-VPS18 or RFP-VPS39 (white), immunolabeled for endogenous VPS18 or VPS11 (blue), as indicated (see also Fig EV4D). F-I. Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over-expression. F/H. Mander's overlap between F. SKIP or H. eVPS18 and endogenous TBC1D15, n≥5 images (2≥ cells per image) analysed per condition from 2 independent experiments, significance: one-way ANOVA test (relative to EV), ** p<0.01, *** p<0.001, ns: not significant. G/I. Mander's overlap between G. SKIP or I. eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance, with or without FIS1 depletion (siFIS1), n≥3 images (2≥ cells per image) analysed per condition from 2 or more independent experiments.

Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines, respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, as indicated, all scale bars: 10µm. Graphs report mean (red line) of sample values (open circles), error bars reflect +/- SD. Unless stated otherwise, significance was assessed using two-tailed Student's t-test: * p<0.05, ** p<0.01, *** p<0.001, ns: not significant, nd: not determined.
Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps (2022)
Figure 1

Figure 1. Exoribonuclease activity of EXD2 toward the RNA-DNA hybrid substrate.

A．A schematic showing the design of the smFRET assay. The 3'-5' degradation of the biotin-labeled RNA by EXD2 gives rise to the gradual generation of the Cy3- and Cy5-labeled DNA, causing an increase in FRET efficiency. PS represents phosphorothioate.

B-G. (B, D, and F) E histograms of the RDH substrate in the absence of EXD2 (gray) and after 0- (black), 100- (purple), and 200-s (blue) incubation with 200 (B), 100 (D), and 5 (F) nM EXD2. (C, E, and G) Representative smFRET trajectories showing EXD2 at 200 (C), 100 (E), and 5 (G) nM cleaving RDH in real time. The fluorescence intensities of the donor and acceptor are colored green and red, respectively. The corresponding FRET efficiencies are shown below in black. The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9.

H. The FRET trajectories' percentages show the FRET values ending above 0.9 and between 0.2 and 0.9. The word "Surface" represents the experiment with EXD2 anchored on the glass surface.

I. Statistics of the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9 under indicated experimental conditions. In violin plots, light-blue dots represent mean, and black bars show the interquartile range (IQR) (thick bars) and 1.5 times IQR (thin bars) (n = 122, 91, 78, and 101 from left to right).
Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps (2022)
Figure 4

Figure 4. Kinetic analysis of EXD2-mediated RDH cleavage under various conditions.

A-F. (A and D) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC-RDH substrate under 1 mM MnCl2 (A) or the RDH substrate under 0.2 mM MnCl2 (D). The solid red lines are the step-fitting results from an automated step-finding algorithm. (B and E) The corresponding TDP of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (B) or RDH under 0.2 mM MnCl2 (E). (C and F) The dwell-time histograms of combined pauses (Total) in the process of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (C) or RDH under 0.2 mM MnCl2 (F). The histogram fittings to a double exponential function are shown in red. The R2 of the double exponential function are 0.94 and 0.95, respectively.

G. Two characteristic rates of EXD2 degradation of RDH under the indicated experimental conditions. Fit parameters and 95% confidence intervals are shown.

H. The kinetic pathway of EXD2 cleavage of RDH constitutes two rate-limiting steps: nucleotide excision and duplex unwinding.
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 1

Fig. 1. P. falciparum-infected erythrocytes do not induce inflammatory cytokine secretion in human monocyte-derived macrophages. Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC or (b) fresh iRBCL for 24 h. (c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (a-c). (d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm. (e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=5; (c) n=3; (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC (a-c) or RBCL (e).
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 2

Fig. 2. Exposure to ROS promotes cytokine secretion from human macrophages and from PBMC. Cytokine secretion of macrophages (a-d) or PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO, Hypoxanthine (Hyx), the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a,e) or XO (b-d). Grey asterisks mark comparison with Control (b-d). (a) n=7; (b-e) n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 3

Fig. 3. Prolonged exposure to extracellular ROS induces cytokine secretion. (a) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05). (b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 4

Fig. 4. XO inflammatory activity in malaria patients and relation to cerebral malaria. (a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. (b) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). (c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. Linear regression (a), Mann-Whitney test (b), and unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 5

Fig. 5. ROS and P. falciparum-infected erythrocytes generate synergistic secretion of IL1β and chemokines. (a-c) Cytokine and chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2; (c) n=4. Insets show paired samples by donor in two different conditions.
Malaria Inflammation by Xanthine Oxidase-produced Reactive Oxygen Species (2019)
Figure 6

Fig. 6. ROS prime macrophages for P. falciparum-infected erythrocytes activation of the NLRP3 inflammasome. (a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression. (b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors. (c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors. (d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors. (e) Diagram of macrophage inflammasome activation by XO and iRBC.
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 1

Figure 1: AGR2 dimerization with ERMIT assay, principles and validation of the method. The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. A) Upper panel: NMR structure of the non-covalent dimer of AGR2 (PDB ID: 2LNS). The dimer domain is highlighted with a yellow circle (residue 54-70 of each monomer). Monomer A is colored green and monomer B in pink. The figure was generated in Chimera. Lower panel: a close-up illustration of the dimer domain, showing that the dimer is stabilized through two salt-bridges between E60 and K64 of each monomer. B) Molecular modeling showing root-mean-square deviation (RMSD) plot for the MD simulation of the AGR2 WT and E60A mutant. The interaction-energies are defined as the sum of the short-range Coulomb interactions and the short-range Lennard-Jones potential between monomer A and B. C and D) Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2, anti-ERK1 or anti-calnexin (CANX) antibodies (for loading control). E) Principles of the ERMIT assay. A wild type IRE1 bait is used to report for dimerization, whereas a kinase catalytic mutant (K599A) is used as a control to prove that signal observed with the wild type form is due to IRE1 activation. F) AGR2 dimerization was monitored with ERMIT (wild-type (WT)). The luminescence signal was abrogated when using the kinase dead (KD) constructs and by the constructs exhibiting mutations in the dimerization domain (E60A or C81S or double mutant (DM)). The graph represents average signal normalized on reporter protein expression ± SD (n=5; **: WT/KD (p=0.002), E60/WT (p=0.0034), E60/KD (p=0.001), C81/KD (p=0.004), DM/WT (p=0.0021) and DM/KD (p=0.0017), respectively; *: C81/WT (p=0.0098)). G) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4).
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 3

Figure 3: Validation of AGR2-TMED2 interaction and its impact on AGR2 dimerization. / TMED2 functional relationships with AGR2. A) Co-immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ. *indicates Immunoglobulin heavy and light chains. B) Structural association of TMED2 and AGR2. The six TMED2 orientations (pink, dark pink, purple, blue, yellow and green) best positioned to stabilize the AGR2 dimer (colored tan with N-termini and dimer interface alpha helix in red). C) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2. D) Specific interactions between residues in AGR2 dimer (tan) and best oriented TMED2 structure (green). Glu51, Arg74 and Arg97 of TMED2 form ionic interactions with Lys66, Glu59 and Asp97 of AGR2. His28 of TMED2 interacts with Tyr111 of AGR2 through π-π-interactions. E): Co-immunoprecipitation of AGR2 wild-type (WT, left panel) or AGR2 AA mutant (right panel) with TMED2 in HEK293T cells. Immunoprecipitation was performed with mouse anti-Flag antibody to pull-down the ectopic protein tag. F) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression upon TMED2 overexpression. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control. G) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT as described in Figure 1E (n=3). H) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression in total cell lysate (TCL) upon TMED2 silencing. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control. I) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT as described in Figure 1E (n=3).
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 4

Figure 4: AGR2 status alteration impacts on client protein biogenesis. A) The ERMIT-based validation of the complex formation between AGR2 and LYPD3 or CD59, but not OS9. The monomeric AGR2 E60A was used as bait, while LYPD3, CD59 and OS9 were used as prays (n=6). B-C) Expression and subcellular localization of CD59-GFP WT and C94S. D and E) Co-immunoprecipitation of CD59-GFP WT or CD59 C94S mutant with AGR2 WT (D) or AGR2 AA mutant (E) in HEK293T cells. F-I) FACS analysis of CD59 WT (F and G) or CD59 C94S (H and I) total (F and H) and membrane (G and I) expression in HEK293T cells silenced for AGR2 (siAGR2) and TMED2 (siTMED2) when compared to control (siGL2) cells (F and H) or silenced for TMED2 (siTMED2) and overexpressing AGR2 WT or AGR2 AA mutant (G and I). Data are representative of three to four independent experiments. Membranous CD59 expression was detected with anti-GFP APC. ns: non-statistically significant; for F (*): p=0.0385, (***): p=0.0003 (left panel) and p=0.0002 (right panel), respectively; for G (*): p=0.0334, (**) p=0.0036 and (***) p=0.0003; for H (*): p=0.0171 and (**): p=0.0084; and for I (**) p=0.0086 (left panel) and p=0.0076 (right panel), respectively.
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 5

Figure 5: Cellular mechanisms of AGR2 secretion. A) Formation of GFP-LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP-LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP-LC3 positive cells for each condition (left panel). (**): p=0.0032. B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498. C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control. D) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT as described in Figure 1E in the presence of gradual amounts of chloroquine. E) Western blot analysis of secreted AGR2 upon TMED2 overexpression. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. F) Representative pictures of extracellular vesicles (EVs) heterogeneity purified from conditioned media of HEK293T cells transfected with control (ev) or TMED2 plasmid and analyzed by cryo-electron microscopy. Right upper panel: western blot analysis of CD63 in total medium (left) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells (middle) and western blot analysis of AGR2 levels (right) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells. G) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. H) Quantification of extracellular AGR2 level (eAGR2) in cells silenced for TMED2 (siTMED2), compared to control (siGL2). Data are representative of three independent experiments and are presented as extracellular-to-intracellular AGR2 ratio. (**): p=0.0029. I) Secretion of AGR2 wild-type (wt), E60A and ∆45 mutant as determined by Western blot. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.