Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry and gRNA-targeted genes were determined. (B) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the Robust Rank Aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) ( Li et al , 2014 ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| > 0.33 and - log 10 (RRA) > 2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue and those enriched in Itgb1 Hi in red. (C) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing miniTurbo-tagged Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P -values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n=3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. (D) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (E, F) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (G-I) Itgb1 surface levels determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates determined by WB ( H ) and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (J) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P -values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n=4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Software, Clone Assay, Comparison, Control, Stable Transfection

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) Itgb1 surface levels in WT and WDR48-KO, WDR20-KO and WDR48/20-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing with WT. Data are shown as Mean±SD, n=3 independent experiments. (B) Itgb1 surface levels in WT and WDR48/20-dKO fibroblasts transiently expressing EGFP, mScarlet, EGFP-WDR48 and/or WDR20-mScarlet determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (C) Itgb1 surface levels in WT and USP12/46-dKO fibroblasts transiently expressing EGFP, EGFP-USP12 WT , EGFP-USP12 1XMUT (E190K, deficient in binding to WDR48), EGFP-USP12 2XMUT (F287A, V279A, deficient in binding to WDR20) or EGFP-USP12 3XMUT (E190K, F287A, V279A, deficient in binding to WDR48 as well as WDR20) determined by flow cytometry. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (D) Itgb1 surface levels in WT and WDR48-KO fibroblasts transiently expressing EGFP-WDR48 WT or EGFP-WDR48 MUT (deficient in binding to USP12 as well as USP46) determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (E) Itgb1 surface levels in WT and WDR20-KO fibroblasts transiently expressing WDR20 WT -EGFP or WDR20 MUT -EGFP (deficient in binding to USP12 as well as USP46) determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Flow Cytometry, Comparison, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A-C) Itgb1 surface levels determined by flow cytometry ( A ) and Itgb1 and SNX17 protein levels in cell lysates determined by WB ( B ) with densitometric quantification ( C ) in WT, USP12/46-dKO, SNX17-KO, and USP12/46/SNX17-tKO fibroblasts. Gapdh served as a loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (D) Itgb1 surface levels in WT, USP12/46-dKO, SNX17-KO, and USP12/46/SNX17-tKO fibroblasts transiently expressing indicated combinations of constructs determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Flow Cytometry, Control, Comparison, Expressing, Construct

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) Itgb1 mRNA levels in WT and USP12/46-dKO fibroblasts determined by qPCR. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (B, C) WB ( B ) and densitometric quantification ( C ) of Itgb1 protein levels in WT and USP12/46-dKO fibroblasts at indicated time points after cycloheximide (CHX) treatment (5 μg/ml). Gapdh served as a loading control. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (D) Quantification of surface Itgb1 degradation kinetics in WT and USP12/46-dKO fibroblasts. The amount of Itgb1 remaining over indicated times were measured by capture-ELISA. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (E) Quantification of surface Itgb1 degradation kinetics in WT fibroblasts stably expressing EGFP, and USP12/46-dKO fibroblasts stably expressing EGFP-USP12 WT or EGFP-USP12 C48S determined by capture-ELISA. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (F, G) The internalization rate ( F ) and the recycling rate ( G) of Itgb1 in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 WT or EGFP-USP12 C48S determined by capture-ELISA. Statistical analysis was carried out by two-sided Welch’s t -test. Data are shown as Mean±SD, n=3 independent experiments. (H, I) WB ( H ) with densitometric quantification ( I ) of Itgb1 protein levels in lysates of WT and USP12/46-dKO fibroblasts treated with DMSO, MG132 (0.5 uM) or BafA1 (40 nM) for 9 hours. Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (J) Itgb1 surface levels in WT, USP12/46-dKO and SNX17-KO fibroblasts treated with DMSO or BafA1 (40 nM) for 9 hours determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (K) Representative immunofluorescence (IF) images of Itgb1 and Lamp1 in WT and USP12/46-dKO fibroblasts treated with DMSO or BafA1 (100 nM) for 3 hours. Arrowheads show the accumulation of Itgb1 in Lamp1-positive endo/lysosomes. Boxes indicate magnified cell regions displayed in the Zoom panel. Sum intensity projections from confocal stacks are presented. Scale bar, 10 µm. (L) Superplots showing the Pearson correlation coefficients (PCC) between Itgb1 and Lamp1 in WT and USP12/46-dKO fibroblasts. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments; 49 cells were analyzed in DMSO-treated WT cells, 43 in BafA1-treated WT cells, 52 in DMSO-treated USP12/46-dKO cells and 45 in BafA1-treated USP12/46-dKO cells.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Comparison, Control, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) WB of Itgb1 protein levels in WT and USP12/46-dKO fibroblasts transfected with control non-targeting siRNA (CTL) or siRNAs targeting jointly HGS and TSG101 (ESCRT-KD). Gapdh served as a loading control. Representative images from 3 independent experiments are shown. (B) IP of denatured Itgb1 from WT, USP12/46-dKO and Itgb1-KO fibroblast lysates with or without ESCRT-KD and analyzed by WB for Ubiquitin (Ub) and Itgb1. Itgb1-KO served as a negative control. Gapdh served as a loading control. Representative images from 3 independent experiments are shown. (C) IP of ubiquitinated proteins in WT and USP12/46-dKO fibroblasts with or without ESCRT-KD and analyzed by WB for Itgb1. Gapdh served as a loading control. Representative images from 3 independent experiments are shown. (D) Amino acid sequence of Itgb1 cytoplasmic tail. The ubiquitin-conjugated lysines detected by MS are colored in red. The underlined regions indicate the NPxY motifs that bind to Talin, Kindlin and SNX17. See also supplementary table S1. (E) IP of denatured Itgb1 from WT and USP12/46-dKO fibroblasts overexpressing HA-tagged Ub WT , Ub K48R or Ub K63R treated with or without ESCRT-KD siRNAs and analyzed by WB for HA and Itgb1. Gapdh served as a loading control. Representative images from 3 independent experiments were shown. (F) In vitro deubiquitination assay using recombinant WDR48-WDR20-USP12 (WT or C48S) complex, UCHL5 or USP7 and ubiquitinated Itgb1 enriched from USP12/46-dKO ESCRT-KD fibroblast cell lysate followed by WB for ubiquitin. BSA served as a negative control. Representative images from 3 independent experiments were shown. (G) Representative Structured Illumination Microscopy (SIM) images of Itgb1 and Ub in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 treated with or without ESCRT-KD siRNAs. Boxes indicate magnified cell regions displayed in the Zoom panel. Arrowheads show the Itgb1-labeled focal adhesion sites and arrows show the direction of line profiles. Scale bar, 10 µm. (H-J) Superplots showing the PCC between Ub and EGFP-USP12 ( H ), Ub and Itgb1 ( I ), and Itgb1 and EGFP-USP12 ( J ) in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 and treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by two-sided Welch’s t -test. Data are shown as Mean±SD, n=3 independent experiments, in total 42 cells per condition.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Transfection, Control, Ubiquitin Proteomics, Negative Control, Sequencing, In Vitro, Recombinant, Microscopy, Stable Transfection, Expressing, Labeling

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) Amino acid sequence alignments of the cytosolic tails of WT α5β1 integrin (α5 WT β1 WT ) and mutant α5 KR β1 KR where lysine residues were substituted for non-ubiquitinable arginine residues. (B) Representative IF images of exogenous human Itga5 (h-Itga5) and Lamp1 in BafA1-treated Itgb1-KO WT and Itgb1-KO USP12/46-dKO fibroblasts. Itgb1-KO cells were retrovirally transduced with human α5 WT β1 WT or α5 KR β1 KR integrins. A human specific Itga5 antibody was used for IF. Boxes indicate magnified cell regions displayed in the Zoom panel. Arrowheads indicates the colocalization of h-Itga5 with Lamp1. Sum intensity projections from confocal stacks are shown. Scale bar, 10 µm. (C) Superplots showing the PCC between Itgb1 and Lamp1 in fibroblasts as described above. Statistical analysis was carried out by ordinary two-way ANOVA with Šidák’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments; 48 cells were analyzed in α5 WT β1 WT -expressing Itgb1-KO WT cells, 44 in α5 KR β1 KR -expressing Itgb1-KO WT cells, 52 in α5 WT β1 WT -expressing Itgb1-KO USP12/46-dKO cells and 55 in α5 KR β1 KR -expressing Itgb1-KO USP12/46-dKO cells . (D) Human Itga5 surface levels in DMSO- or BafA1-treated cells as described above. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments. (E) Human Itga5 surface levels in cells as described above treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test. Data are shown as Mean±SD, n=3 independent experiments.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Sequencing, Mutagenesis, Transduction, Comparison, Expressing

Journal: bioRxiv

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1101/2024.05.14.594138

Figure Lengend Snippet: (A) Quantification of cell adhesion of WT and USP12/46-dKO fibroblasts at indicated time points after seeding on FN-coated plates. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test at the 30-minute time point. Data are shown as Mean±SD, n=4 independent experiments. (B) Cell spreading area of WT and USP12/46-dKO fibroblasts on FN-coated glass surface shown at indicated time points after seeding. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test at the 120-minute time point. Data are shown as Mean±SD, n=4 independent experiments. (C) Normalized wound healing area of WT and USP12/46-dKO fibroblasts on FN-coated plates after 12 hours. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=4 independent experiments. (D, E) Representative images ( D ) and quantification ( E ) of WT and USP12/46-dKO MDA-MB-231 cells upon migration through transwell inserts 16 hours after seeding. Scale bar, 200 µm. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=4 independent experiments. (F, G) Representative images ( F ) and quantification ( G ) of WT and USP12/46-dKO MDA-MB-231 cells upon migration through Matrigel-coated transwell inserts 16 hours after seeding. Scale bar, 200 µm. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as Mean±SD, n=4 independent experiments. (H, I) Kaplan Meier-plot of overall survival ( H ) a nd progression free interval ( I ) of breast cancer patients with high (red line) or low (blue line) combined total USP12 and USP46 gene expression levels. The GDC TCGA dataset obtained from the UCSC Xena project ( Goldman et al , 2020 ) was used. Two-group risk model with cut-off at the median was applied. P -values were calculated by Log-rank test.

Article Snippet: Full-length recombinant WDR20 (1-569aa) N-terminally tagged with His6 was expressed in E.coli., and WDR48 (1-580 aa) N-terminally tagged with Strep-tag II together with untagged USP12 WT and USP12 C48S (both 40-370aa) were expressed in insect cells as reported previously ( Li et al , 2016 ) using the pCoofy expression vectors (a gift from Sabine Suppmann, Addgene plasmid # 43974) and purified to approximately 80% purity followed previously published protocols ( Aretz et al , 2023 ).

Techniques: Comparison, Migration, Gene Expression