EMBO/soda-nel huggingface dataset:
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 3

The N‐terminal 61 residues of USP45 mediate binding to ERCC1

A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full‐length or indicated fragments of USP45. Thirty‐six hours post‐transfection cells were lysed and GFP immunoprecipitated. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies. GFP empty vector was used as a negative control.
B Sequence alignment of the first 62 residues of USP45 from the indicated species. Conserved residues that were mutated in part (C) are indicated in red. The residues whose mutation in (C) is shown to inhibit binding to ERCC1 are marked with an asterisk. Residue Thr37 (marked with a solid triangle) was found to destabilise expression when mutated to Ala.
C, D HEK293 cells were transiently transfected with constructs expressing the indicated wild‐type or mutant GFP–USP45 [1–62] fragment (C) or GFP full‐length USP45 (D) or empty GFP (C and D) as a control and processed as described in (A). LICOR–Odyssey quantitation of results shown in (C) is shown in Supplementary Fig S4.
E As in (C) except HEK293 cells were transfected with the indicated N‐terminal Znf‐UBP domain‐containing DUBs. Similar results were obtained in at least two separate experiments.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 4

USP45 deubiquitylates ERCC1

HEK293 cells were treated with the proteasome inhibitor lactacystin (5 μM) or bortezomib (10 μM) for the times indicated. Cells were lysed in the presence of 0.1 M N‐ethylmaleimide to inhibit deubiquitylation and lysates were subjected to immunoblotting with indicated antibodies.
Wild‐type (WT) and USP45 knockout (KO) U2OS cells were lysed and subjected to immunoblotting as in (A).
USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG‐ERCC1. Thirty‐six hours post‐transfection cells were treated with lactacystin (5 μM) for 6 h and lysed with a buffer containing 0.1 M N‐ethylmaleimide. Flag‐ERCC1 was immunoprecipitated and treated in the absence or presence of 0.1 μg of recombinant USP45 wild‐type (WT), non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] (AA) or catalytically inactive USP45 [Cys199Ala] (CA) for 1 h. The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining. Similar results were obtained in at least two separate experiments.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 5

USP45 knockout cells are hypersensitive to DNA damage

A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA). The cells were treated with the indicated doses of mitomycin C (MMC) 16 h. Medium was changed and number of colonies quantified after 10 days. Each data point is the average of 3 experiments undertaken with 6 replicates ± SD.
B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines, similar levels of wild‐type and catalytically inactive USP45 [Cys199Ala] were expressed.
C, D As above, except rescue experiments are undertaken using the non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] mutant.
E, F As in (A), except that DNA damage is induced using UV‐C irradiation (E) or 16 h hydroxyurea treatment (F). Similar results were obtained in at two separate experiments.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 6

Defective recruitment of ERCC1 to sites of DNA damage in USP45 knockout cells

Wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA) were used to analyse ERCC1 foci formation. Staining and analysis of endogenous ERCC1 foci formation was undertaken before (left panel), or after DNA damage induction by mitomycin C (40 ng/ml, 16 h, middle panel) or UV (20 J/m2 followed by 3‐h recovery, right panel). Three independent experiments were performed in which 500 cells per experiment were analysed. Scale bar, 10 μm.
Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified. Three independent experiments were performed in which 500 cells per experiment were analysed. Results are the mean of 3 experiments ± SD.
The wild‐type and USP45 knockout U2OS cells were treated with mitomycin C (40 ng/ml, 16 h) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times. Similar results were obtained in two separate experiments. The data are presented as the average number of ERCC1 staining foci per cell.
As in (C), except that γ‐H2AX‐staining foci were analysed. The data are presented as the average number of γ‐H2AX staining foci per cell.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 7

USP45 localises at sites of DNA damage

Representative immunofluorescence images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h). Cells were fixed 10 min after laser micro‐irradiation and stained for localisation of endogenous USP45, phosphorylated γ‐H2AX and DAPI. The right‐hand panel shows magnification of one micro‐irradiated cell. Similar results were obtained in at least three independent experiments. Scale bar, 10 μm.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 8

USP45 is recruited to sites of damage following UV‐C irradiation

U2OS cells were transiently transfected with the GFP–USP45 or GFP alone. Thirty‐six hours post‐transfection cells were irradiated with UV‐C (150 J/m2) through a micropore filter (lower panels) or left untreated (upper panels). Five minutes post‐irradiation, cells were fixed and co‐immunostained with antibodies recognising cyclobutane pyrimidine dimer (CPD) DNA lesions and GFP. The white arrows indicate co‐localisation between GFP–USP45 and CPD. Scale bar, 10 μm.
U2OS wild‐type (WT) and USP45 knockout (KO) cells were irradiated with UV‐C (20 J/m2). At the indicated post‐irradiation times, genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody. Data are shown from a triplicate experiment in which each blot is derived from independent cells.
Same as (B) but using a High Sensitivity CPD/Cyclobutane Pyrimidine Dimer Elisa kit (NM‐MA‐K001) from Cosmo Bio. For the DNA damage detection, the manufacturer protocol was followed. Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times. Calf thymus DNA, UV‐C irradiated (10 J/m2) and not irradiated was used as positive and negative samples, respectively.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 9

USP45 recruitment to sites of DNA damage is rapid and transient

Live‐cell fluorescence analysis of U2OS cells stably expressing either GFPNLS empty, wild‐type (WT) USP45 or catalytically inactive USP45 [Cys199Ala]. Cells were treated with BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h) and images captured at the indicated times after laser micro‐irradiation. The location of the micro‐irradiation stripe in each cell is indicated with a yellow line. Similar results were obtained in at least two independent experiments. Scale bar, 10 μm.
A conserved ATG2-GABARAP family interaction is critical for phagophore formation. (2019)
Figure 1

Figure 1. CRISPR/Cas9 GFP-tagged ATG2A localizes to early autophagy membranes.

U2OS cells modified to expressed endogenous GFP-tagged ATG2A (green) were grown in complete media (CM) or starved (EBSS) for 2 h before fixation and immunostaining with antibodies against ATG2B (red) and LC3B (magenta) and analysed by confocal microscopy. Arrows mark ATG2A/LC3B/ATG2B positive structures. Scale bar 10µm.

(B-D) Cells were treated as in (A) and stained with anti-WIPI2 (red) or (C) anti-ATG16L1 (red) or (D) GABARAP-L1 (red). Arrows mark structures of interest. Scale bar 10µm.

U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.
A conserved ATG2-GABARAP family interaction is critical for phagophore formation. (2019)
Figure 3

Figure 3. ATG2A LIR domain is essential for autophagy flux.

U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.

Quantification of (A) using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).

D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.
A conserved ATG2-GABARAP family interaction is critical for phagophore formation. (2019)
Figure 4

Figure 4. Mutation of ATG2A LIR prevents phagophore closure.

Graphical representation of proteinase K assay (i) showing protection of p62/SQSTM1 inside sealed autophagosomes or proteinase K sensitive p62 present within open phagophores. (ii) Graphical representation of sytnatxin17 (STX17) translocation to completed autophagosomes and not phagophores. Autophagosomes are identified as being both LC3B and STX17 positive vesicles.

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.

Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.
A conserved ATG2-GABARAP family interaction is critical for phagophore formation. (2019)
Figure 5

Figure 5. ATG2A-LIR is essential for phagophore formation.

(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.

Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.

Model of ATG2 function based on the current knowledge. ATG2A localizes to ER membranes and facilitates lipid transfer from the ER to the growing phagophore. ATG2 interaction with GABARAP/GABARAP-L1 is essential for anchoring ATG2 to growing phagophore and mutation of the GABARAP interaction region results in the formation of immature phagophores.

Expanded View Figure Legends
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 1

Figure 1 RNF34 negatively regulates RLR signaling pathways

(A-B) Results of the ELISAs used to determine IFN-β (A) and IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times.

(C-D) Luciferase activity driven by the IFN-β (C) or NF-κB (D) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction.

(E) Plaque assay of VSV loads in supernatants from cells transfected with shCtrl, shRNF34-1, shRNF34-2 or shRNF34-3 and subsequently infected with VSV.

(F) Microscopy images of shCtrl-, shRNF34-1-, shRNF34-2- or shRNF34-3 cells infected with NDV-GFP (MOI = 1.0) for the indicated times. Gray represents cells; green represents NDV. Scale bar, 300 μm.

(G) Immunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.

(H and J) RT-PCR analysis of the expression of the IFN-β (H), IL8, ISG54 and ISG56 (J) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.

(I) Results of the ELISA used to determine IFN-β secretion in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.

Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 2

Figure 2 RNF34 interacts with MAVS

(A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1. Luciferase assays were performed 24 hrs after transfection.

(B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34.

(C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.

(D) GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody are shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control.

(E) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 hrs and then analyzed with anti-Flag antibody.

(F) Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 hrs. Scale bar, 10 μm.

(G) Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control.

(H) In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI =1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 μm.

(I) One hundred cells in Figure 2H were counted, and the quantification of PLA signals per cell is shown.

(J) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.

Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 3

Figure 3 RNF34 enhances the K27/K29-linked ubiquitination of MAVS

(A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. The levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody.

(B) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody.

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody.

(D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody.

(E) Immunoprecipitation analysis of siCtrl- or siRNF34-1-transfected cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody.

Cell-based studies were performed independently at least three times with comparable results.
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 4

Figure 4 RNF34 initiates the K63 to K27-linked polyubiquitination transition on MAVS

Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody.

Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or 4KR mutant together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed by performing immunoblotting with an anti-Flag antibody.

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody.

(D) Co-immunoprecipitation analysis of the ubiquitination of endogenous MAVS in THP-1 cells transfected with siCtrl, siRNF34, or siTRIM31 oligos and infected with VSV for the indicated times.

(E) Co-immunoprecipitation analysis of the endogenous interaction of MAVS-TRIM31 and MAVS-RNF34 in PBMCs infected with VSV for the indicated times.

Cell-based studies were performed independently at least three times with comparable results.