e 592 usp47 r d systems  (R&D Systems)

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 1. Yeast two-hybrid screen reveals DAAM1 as a novel USP10-binding partner. A) Yeast 2-hybrid data (Hybrigenics), human placental mRNA library, and human USP10 as bait. B) DAAM1 subdomain schematic and yeast 2-hybrid fragment data. These data suggest that USP10 binds to DAAM1’s FH2 domain.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Two Hybrid Screening, Binding Assay

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 2. USP10 colocalizes with DAAM1. A) 5 nM 488-SNAP-FL-DAAM1 or 5 nM 488-SNAP-DAAM1-FH2-C binds to 5 nM 561-SNAP-USP10 in single-molecule TIRF colocalization assays. This indicates that the FH2 domain of DAAM1 is sufficient for binding to USP10. B) Recombinant FL-DAAM1 is autoinhibited. TIRF assay with1 µM actin in the presence or absence of 5 nM FL-DAAM1. Actin filament nucleation was tested (the total number of filaments present in several (n = 9) fields of view) at 150 s. FL-DAAM1 was strongly autoinhibited in this assay. 3.2 µm RhoA was used to activate FL-DAAM. The difference from FL-DAAM1 was not significant, however, 5 nM DAAM1(FH2-C), a constituently active form significantly nucleated filaments compared to actin alone, or autoinhibited FL-DAAM1.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Binding Assay, Recombinant

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 3. FL-DAAM1 and FH2-C inhibit USP10’s DUB activity. A and C) The kinetics of USP10 (5 nM) DUB activity was measured using a fluorogenic sub strate Ub-AMC in the absence or pres ence of the indicated concentrations of A) FL-DAAM1 or C) FH2-C. In A and C, the symbols are means and the error bars are standard deviations. B and D) The rate constants are derived from non-linear curve fitting and are shown with 95% Confidence Intervals (CI). N = 3 for each condition. Comparisons of the fitted curves were done in GraphPad Prism using both Extra Sum of Squares F test and ΔAICc.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Activity Assay, Derivative Assay

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 4. Proximity Ligation Assay validation of USP10-DAAM1 interaction in HCFs. A) Immunoblot with quantification of HCFs either untreated or treated with 1 ng/ ml for 72 hrs. Quantification is expressed as the mean fold change after normalization to GAPDH. B) Representative images of untreated and TGFβ1 treated HCFs (PLA, green), counterstained for actin (phalloidin, red) and nucleus (DAPI, blue). Stroked portions of the merged channels are magnified (Merge Magnified). C) Quantification of puncta density. Bar= 50 µm. N = 5. Statistical significance was calculated using ordinary one-way ANOVA after a log transformation of the data.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Proximity Ligation Assay, Biomarker Discovery, Western Blot, Transformation Assay

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 5. DAAM1 and USP10 precipitate with actin stress fibers but USP10 does not affect DAAM1’s actin nucleating activity. A) Representative phase-contrast images of TGFβ1 treated HCFs subjected to a series of buffer washes with increasing concentration of detergent. B) Immunoblot analysis of total protein from HCFs transfected with siGLO or siDAAM1. siDAAM1 did not significantly affect total USP10 levels. C) Immunoblot analysis of stress fiber isolates generated from TGFβ1 treated cells transfected with nontargeting siGLO or siDAAM1. DAAM1 knockdown also reduced USP10 association with stress fibers suggesting that it is required for USP10’s association with actin. Statistical significance was calculated using an unpaired t-test. Bar= 200 µm. N = 3 D) TIRF assay was used to test if USP10 had a role in actin assembly. 5 nM USP10 did not alter actin nucleation at 150 s.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Activity Assay, Concentration Assay, Western Blot, Transfection, Generated, Knockdown

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 6. Knockdown of DAAM1 increases integrin and FN recycling. A) HCFs were transfected with siGLO or siDAAM1 and treated with TGFβ for 3 days to increase total integrin expression. siDAAM1 transfection resulted in increased expression of αv-, β1-, β5-integrins. N = 6. Statistical significance was calculated using an unpaired t-test. B) Live cell integrin recycling assay. 48 hrs post-transfection cells were blocked and treated with Ab against α5β1 or αv at 10 ug/ml for 30 min prior to cell surface stripping for 30 s. Cells were incubated for 90 min prior to incubation with 2◦Ab-488 for 30 min. N = 3 with a total of 15 images analyzed for each condition. Statistical significance was calculated using an unpaired t-test. C) HCFs were transfected with siGLO, siDAAM1, or siUSP10. After 3 days cells were lysed and subjected to Ubiquant™ ubiquitin capture ELISA. siDAAM1 reduced, whereas siUSP10 increased ubiquitination of β1 and β5. N = 4. Statistical significance was calculated using an unpaired t-test between conditions. D) Live cell FN recycling assay. HCFs were transfected with siRNA targeting USP10 or DAAM1 or control siGLO siRNA. 24hrs post transfection HCFs were loaded with biotinylated-FN for 3 h. After trypsinization (to separate cells from extracellular, non-internalized FN), HCFs were replated and imaged after 48 h. Prior to imaging by live cell confocal, cells were incubated with streptavidin-488 to detect only recycled biotinylated-FN. (Phillips et al., 2021) N = 3 with a total of 15 images analyzed for each condition. Statistical significance was calculated using one-way ANOVA. Separate exper iments demonstrated that cell architecture highlighted with SiR-actin, was non-variant between conditions E) Image analysis of D was performed with ImageJ’s Analyze Particles function. Statistical significance was calculated using one-way ANOVA.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Knockdown, Transfection, Expressing, Stripping Membranes, Incubation, Ubiquitin Proteomics, Enzyme-linked Immunosorbent Assay, Control, Imaging, Variant Assay

Journal: European journal of cell biology

Article Title: The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

doi: 10.1016/j.ejcb.2023.151347

Figure Lengend Snippet: Fig. 7. Working model. Our previous studies demonstrate that an increase in USP10 activity leads to a net accumu lation of integrins and FN on the cells surface, TGFβ acti vation, and myofibroblast development. (Boumil et al., 2020; Gillespie et al., 2017; Phillips et al., 2021) In this study we found a novel interaction between USP10 and the formin, DAAM1. A) USP10 binding to DAAM1 is anti-fibrotic. DAAM1 sequesters USP10 to stress fibers, inhibiting USP10’s DUB activity (more ubiquitin on integ rins>less cell surface integrin accumulation>less patho logical myofibroblasts). B) When DAAM1 is not bound to USP10, it is free to deubiquitinate integrins, tipping the delicate balance towards cell surface integrin accumulation and fibrotic myofibroblast development. This axis may act as a level of control over USP10 activity to regulate integrin protein levels. Cartoon created with Biorender.com. Structures created with Alphafold.

Article Snippet: Reagents in the assay are recombinant USP10 (R&D, E-592–050), DAAM1 (Origene, TP317675), DAAM1-FH2-C purified in the Bernstein lab, and the DUB inhibitor (10 mM N-ethylmaleimide, Sigma-Aldrich).

Techniques: Activity Assay, Binding Assay, Ubiquitin Proteomics, Control

Journal: Cell chemical biology

Article Title: Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity

doi: 10.1016/j.chembiol.2020.12.007

Figure Lengend Snippet: Key Resources Table

Article Snippet: UbVS labeling of recombinant DUBs Each DUB (1μM) (Boston Biochem, E-519 (USP7),E-320 (USP8), E-326 (USP5), E-325 (UCHL3), E-327 (UCHL5), E-546 (USP25)E-522(USP9X), E-570(USP28), E-574 (OTUD3), E-592 (USP10), E-594 (USP15), E-596 (USP4)) was incubated with saturating amount of UbVS or HA-UbVS as indicated (1 hour at 30°C).

Techniques: Virus, Recombinant, Plasmid Preparation, Software