EMBO/soda-nel huggingface dataset:
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 4

Figure 4. Ubiquitination sites of LGP2

(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of LGP2 in Riplet-expressing cells. LC-MS/MS data was quantified using LFQ method. The chromatographic profiles for each ubiquitination site are shown in the right panels. Schematic representation of ubiquitination sites of RIG-I and LGP2 are shown in the upper panel.

(B) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. 24 h after transfection WCE were prepared. immunoprecipitation was performed with anti-FLAG antibody. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies. The data is a representative of two independent experiments.

(C) FLAG-tagged LGP2 and FLAG-tagged LGP2-4KR expression vectors were transfected into HEK293FT cells. 24 h after transfection, WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody. The data is a representative of two independent experiments.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 5

Figure 5. Ubiquitination of LGP2 attenuates type I IFN expression

(A) RIG-I, WT LGP2, and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). WCEs were subjected to SDS-PAGE, and the proteins were detected by western blotting (lower panels).

(B) HE293 cells stably expressing GFP (control), LGP2, or LGP2-4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activity was determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05).

(C-J) HEK293 cells stably expressing GFP, LGP2, or LGP2-4KR were transfected with 200 ng/ml of short poly I:C (C-E), infected with SeV at MOI = 5 (F-H), or infected with influenza A virus (Flu) at MOI =10 (I, J). Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05).

(K, L) HEK293 cell clones stably expressing LGP2 or LGP2-4KR at lower levels (Fig EV5A) were transfected with short poly I:C, and the expression of mRNAs were determined by RT-qPCR. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05).

Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 6

Figure 6. Ubiquitination of LGP2 increases the expression of antiviral genes

A) Microarray analysis was performed as described in materials and methods. A heatmap of the microarray data of LGP2 and LGP2-4KR stably expressing cells stimulated with short poly I:C for 12 h.

B, C) LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05).

D­-G) LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05).

H, I) WT and indicated mutant HEK293 cells were stimulated with 200 ng/ml of short poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.

J, K) LGP2 and LGP2-4KR expressing vectors were transected into HeLa cells with Riplet expressing vector. 18 hr after transfection, cells were fixed, and then the PLA signals of LGP2 and endogenous MAVS were detected. Peroxisome was visualized using CellLight Peroxisome-GFP reagent. Colocalization of GFP (peroxisome) and the PLA signals (red) were observed by confocal microscopy. Scale bars represent 10 μm (J). The colocalization area was normalized to the area of the PLA signals (n = 25, t-test, *p < 0.05) (K).

Data information: All data except panel A are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 7

Figure 7. Riplet is required for the antiviral gene expression at a late phase

A, B) WT, LGP2 KO, and Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05).

C, D) WT and Riplet KO A549 (C) and MEF (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005).

E) WT and Riplet KO BMM were infected with influenza A virus (Flu) at MOI = 10. PML and SP100 mRNA expression was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 0.05).

F) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10. WCE was prepared at the indicated time points and subjected to SDS-PAGE. Proteins were detected by western blotting with the indicated antibody.

G, H) LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV (G) or stimulated with 200 ng/ml of short poly I:C (H). IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Right graph in panel G is an enlargement of Y-axis of the left graph (dotted line).

Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
Lysosomal K+ Channel TMEM175 Promotes Apoptosis and Aggravates Symptoms of Parkinson's Disease (2022)
Figure 2

Figure 2. Bcl-2 binds to and regulates TMEM175 through caspase-independent way.

A Representative current traces recorded with the pan-caspase inhibitor Z-VAD-FMK (zVAD) at a concentration of 10 μM in pipette solution. The basal currents were recorded for 20 min, and then 10 μM HA14-1 was added to the bath solution.

B Current amplitudes (at -100 mV) normalized to the basal values (0 min). The red solid line indicates the time frame for HA14-1 application. n = 8.

C Schematic diagram showing the dKO strategy of Bax/BAK1 in HEK293T cells.

D Bax/BAK1 protein levels were detected by western blotting.

E Representative TMEM175 whole-cell currents recorded in Bax/BAK1 dKO cells overexpressing TMEM175 with 10 µM HA14-1 bath application.

F Current amplitudes measured at -100 mV at 20 min after application of HA14-1 normalized to the basal value (0 min).

G, H Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.

I Immublotting results with IP or input (whole-cell lysates) from mouse midbrain, SH-SY5Y, HEK293T or HeLa cells as indicated. The α-ctrl represents that the magnetic beads used in IP were not pre-incubated with any antibody.

J Representative TMEM175 whole-cell currents recorded in HEK293T cells expressing TMEM175 cotransfected or not cotransfected with Bcl-2.

K Statistics of the current densities in (J).

L Representative TMEM175 lysosomal currents recorded in HEK293T cells expressing TMEM175 with or without Bcl-2 application in bath solution.

M Statistics of the current densities in (L).

Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with NS for not significant (P > 0.05), ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.
Lysosomal K+ Channel TMEM175 Promotes Apoptosis and Aggravates Symptoms of Parkinson's Disease (2022)
Figure 3

Figure 3. Bcl-2 regulates TMEM175 through physical contact.

A Predicted binding mode I (left) and mode II (right) for Bcl-2 (PDB ID: 6O0K) bound with TMEM175 (PDB ID: 6WC9). The inserted shows the overhead and lateral views of the bound complexes. The protein backbone is shown as cartoon and colored in cyan for TMEM175 and purple for Bcl-2. Pairs of residues forming hydrogen bond were displayed in sticks and colored by atom type. Hydrogen bonds were displayed in yellow dash lines.

B Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from cells transfected with Bcl-2 and TMEM175 or its mutants TMEM175-V145A, TMEM175-R377V, as indicated.

C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector, TMEM175, TMEM175-V145A or TMEM175-R377V overexpressing HEK293T cells.

D Representative whole-cell currents recorded at different time points after application of the Bcl-2-selective inhibitors HA14-1 in TMEM175, TMEM175-V145A or TMEM175-R377V overexpressing HEK293T cells.

E Normalized currents (at -100 mV) to the basal value (0 min) in the ramp protocol used in (D). n = 5 for TMEM175 group. n = 6 for TMEM175-V145A group. n = 6 for TMEM175-R377V group.

F Current amplitudes of TMEM175 and TMEM175-R377V transfected HEK293T cells measured at -100 mV in the ramp protocol used in (D). n = 5 for TMEM175 group. n = 6 for TMEM175-R377V group.

Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests (C and F) or analyzed using post hoc tests with ANOVA (E), and is indicated with NS for not significant (P > 0.05), ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.
Lysosomal K+ Channel TMEM175 Promotes Apoptosis and Aggravates Symptoms of Parkinson's Disease (2022)
Figure 4

Figure 4. ROS regulate TMEM175 through positive feedback.

A-F Whole-cell recording was performed in HEK293T cells transfected with TMEM175-GFP. (A) (Left) Representative current traces showing the effects of 20 μM EM20-25 in the bath solution. (Right) Statistics of the currents normalized to the basal values (0 min). n = 6. (B) (Left) Representative current traces showing the effects of 200 μM H2O2 in the bath solution. (Right) Statistics of the currents normalized to the basal values (0 min). n = 5. (C) Representative current traces showing the effects of GSH (1 mM in pipette solution) on HA14-1 (1 μM added to bath)-induced activation of TMEM175 currents. (D) Statistics of the currents in (C) normalized to the basal values (0 min). n = 6 for each group. (E) Representative currents recorded with pipette solutions containing 100 μM MPP+ and 10 μM Z-VAD-FMK, 100 μM Z-VAD-FMK or 1 mM GSH, as indicated. The red traces indicate the basal currents. The blue traces indicate the currents recorded 20 min after the initiation of whole-cell mode. (F) Quantification of the current fold changes in (E) normalized to the basal currents.

G Representative TMEM175 lysosomal current traces showing the effects of 500 μM H2O2 in the bath solution. The blue trace indicates basal current. The red trace indicates the current recorded 20 min after application of H2O2.

H Statistic of current fold changes in (G) normalized to basal currents (0 min). n = 7.

I, J ROS levels in HEK293T cells overexpressing TMEM175 or empty vectors pretreated or not pretreated with 500 μM MPP+ for 1 hour. ROS were detected using CM-H2DCFDA dye by flow cytometry (I), and the quantified results are presented in (J).

K (Upper) schematic diagram showing the strategy of TMEM175-KO in HEK293T cells. 92 bp in exon 4 and intron 4 were removed from the tmem175 locus. (Lower) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western-blotting.

L CM-H2DCFDA (green) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre-treatment with vehicle or 10 μM HA14-1 for 20 min. Scale bars = 10 μm.

M Statistics of CM-H2DCFDA fluorescence intensity of (L). The n values represent the cell numbers counted within the same experiment.

Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests (A, B, F, H, J, M) or analyzed using post hoc tests with ANOVA (D), and is indicated with NS for not significant (P > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. In panel M, n value means the number of technical replicates. In other panels, n value means the number of biological replicates made for each data point.
Lysosomal K+ Channel TMEM175 Promotes Apoptosis and Aggravates Symptoms of Parkinson's Disease (2022)
Figure 5

Figure 5. TMEM175 overexpression impairs mitochondrial membrane potential and mitophagy in HEK293T cells.

A Flow cytometry analysis of JC-1 staining in HEK293T cells transfected with mock vector or TMEM175. Horizontal axis and vertical axis represent fluorescence intensities of JC-1 monomers and JC-1 aggregates, respectively.

B Statistics of the ratios of JC-1 aggregates/monomers in (A).

C Flow cytometry analysis of Rhodamine 123 staining in HEK293T cells transfected with mock vector or TMEM175.

D Rhodamine 123 fluorescence of cells in (C) is plotted against SSC.

E Statistics of Rhodamine123 mean fluorescence intensities in (C).

F Fluorescence of G3BP1 excited with 488 nm in control (upper) and TMEM175-overexpressing (lower) cells. Scale bars = 10 μm.

G Statistics of the numbers of puncta per cell (left) and the percentage of cells with puncta in (F). We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment.

H Co-localization of mitochondrial indicator TOM20-mCherry and lysosomal indicator LysoTracker in WT (upper row) or TMEM175 KO (lower row) HEK293T cells. Co-localization of mitochondrion and lysosome is observed as a yellow colour in the merged image (marked by white arrows). Scale bars = 10 μm.

I The number of merged puncta per cell in (H). We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment.

Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels G and I, n value means the number of technical replicates. In other panels, n value means the number of biological replicates made for each data point.
Lysosomal K+ Channel TMEM175 Promotes Apoptosis and Aggravates Symptoms of Parkinson's Disease (2022)
Figure 7

Figure 7. TMEM175 KO mitigates MPTP-induced PD symptoms in mice.

A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages (W: weeks; M: months).

B Statistics of the relative TMEM175 protein levels normalized to GAPDH in (A).

C Schematic diagram showing the KO strategy of TMEM175 in mice. Introns 2-10 were removed from the TMEM175 locus.

D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT-qPCR using SYBR Green PCR Master Mix.

E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues.

F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175-KO mice. Ψ is lysosomal membrane potential (deﬁned as Vcytosol - Vlumen). The currents were elicited with ramp voltage protocols (-100 to +100 mV in 1 s, Vh = 0 mV).

G Statistics of the current amplitudes of (F).

H, I Quantification of rears in the cylinder (H) and the latency to fall from a wire hang apparatus (I) pre- and post-MPTP treatment.

J Swing Duration CV (CV%) (Left panel) and Ataxia Coefficient in Gait tests 7 days post-MPTP.

K Latency to fall in rotarod tests 7 days post-MPTP.

L Immunoblots of the TH levels post-MPTP treatment in WT and TMEM175 KO mouse midbrains.

M Quantification of the relative TH levels normalized to GAPDH post-MPTP.

N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175-KO mice post-MPTP treatment. Scale bars = 100 μm.

O Statistics of the TH-positive neurons in (N).

P An overview model illustrating the presumed roles of TMEM175 in apoptosis and PD. Anti-apoptotic mitochondrial protein Bcl-2 binds directly to and regulates lysosomal potassium channel TMEM175. Bcl-2 inhibitors and mutations of the binding sites release the inhibition and thus activate TMEM175. Activation of TMEM175 inhibits mitophagy, impairs mitochondrial function, and consequently increases ROS production. ROS activates TMEM175 through a positive feedback way to augments apoptosis. TMEM175-mediated apoptosis promotes the death of DA neurons in PD.

Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.

Expanded View Figure Legends
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 1

Figure 1. The ARHGAP45 interactome of Jurkat T cells.

A Jurkat T cells (WT), ARHGAP45-deficient Jurkat T cells (ARHGAP45-/-) and 5 independent clones (C1 to C5) of Jurkat T cells expressing ARHGAP45OST molecules were analyzed for ARHGAP45 protein expression. Immunoblot analysis of equal amounts of proteins from cell lysates that were either directly analyzed (Total lysates), or subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin (Affinity purification), and probed with antibody to ARGHAP45 or anti-HSP60 (loading control). Left margin, molecular size in kilodaltons (kDa).

B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors.

C ARHGAP45-specific interactors highlighted in the text are displayed in a stoichiometry plot (Voisinne et al., 2019) where the ratios of the prey to bait cellular abundances ('abundance stoichiometry') are plotted as a function of their maximal interaction stoichiometries ('interaction stoichiometry'), both using log10 scale (see Datasets EV1 and EV2). The ARHGAP45 bait is shown as a yellow dot. The size of the dots is commensurate to the maximal protein enrichment in ARHGAP45OST samples as compared to WT control samples. The TNKS prey for which it was not possible to determine the cellular abundance is shown at the bottom of the stoichiometry plot. For each time point, three independent biological replicates were performed and each biological replicate was analyzed in triplicate by MS.
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 3

Figure 3. Arhgap45-/- naïve T and B cells showed impeded LN entry.

A CTV-labeled splenocytes (20 x 106 cells) from either Arhgap45-/-or WT mice were mixed with CMTPX-labeled WT splenocytes (20 x 106) and injected intravenously in WT recipient mice. After 4 hours, single cell suspensions were prepared from the spleen and mesenteric LNs (mLN) and the percentages of CTV- and CMTPX-labelled B and T cells determined by flow cytometry.

B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests.

C Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests.
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 4

Figure 4. Reduced chemotaxis of Arhgap45−/− naive T and B cells.

The cells specified below were loaded into the upper chamber of a Transwell migration device without (no) or with the specified chemokines in the lower chamber. The percentage of cells that migrated to the bottom chamber after 2 h (T cells) or 4 h (B cells) is shown.

A Chemotaxis of naïve CD4+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12.

B Chemotaxis of naïve CD8+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12.

C Chemotaxis of naïve B cells from WT and Arghap45−/− mice in response to CCL19

D Adding CCL19 in both the upper and lower chambers (CCL19 UL) prevented migration of WT and Arghap45−/− naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM-1 (CCL19 + ICAM-1) did not change the pattern of chemotaxis observed using 'bare' membrane inserts (CCL19).

E Chemotaxis of naive CD4+ T cells and B cells from WT, Arghap45−/−, and Arhgap45∆T/∆T mice in response to CCL19.

F Chemotaxis of activated CD4+ T cells from WT and Arghap45−/− mice in response to CCL19. Data information: In (A) to (F) each dot represents an individual mouse and as shown CD4+ T cells from 3 to 9 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). Mean and SD are shown. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; A, B, C, D, F multiple unpaired T test; E two-way ANOVA test.