Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Cdc37 , StressMarq Biosciences , Cat# SPC-142; RRID:AB_2570605.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: Molecular Cancer

Article Title: FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation

doi: 10.1186/1476-4598-13-150

Figure Lengend Snippet: FW-04-806 is identified as an Hsp90 binding medicine. (A) LC/MS spectrum detects the compound eluted from the Hsp90-loaded affinity column. Peak a and b represent elution samples from the control and FW-04-806 bound Hsp90-loaded column, respectively. MS spectrum displays M/z of Peak b. (B) Western blot confirms FW-04-806 binds to NBD of Hsp90. The “–” symbol represents no drug-loaded affinity column, the “+” symbol represents the drug-loaded affinity column. The test proteins are SKBR3 cell lysate, His-tagged yeast Hsp90, and His-tagged NBD, MD, CDD of yeast Hsp90. Human Hsp90 Antibody and His-probe antibody were used respectively. (C) Soluble FW-04-806 was added into SKBR3 cell lysate, His-tagged full-length, NBD, MD and CDD of yeast Hsp90 up to 10 μM before incubation with drug-loaded affinity resin. Picture a and c show Human Hsp90 Antibody; picture b and d show His-probe antibody. (D) Molecular structure of FW-04-806. (E) Geldanamycin (GA) binds to N-terminal Hsp90 (Protein Data Bank [PDB] ID 1YTE) in the ATP binding pocket. Hsp90 is shown in the green ribbon view; GA is shown in stick view. (F) FW-04-806 docks to N-terminal Hsp90 (PDB ID 2K5B). N-Hsp90 is shown in green ribbon view; FW-04-806 is shown in stick view. (G) FW-04-806 binds to the N-Hsp90/Cdc37 complex (PDB ID 1US7). N-Hsp90 is shown in green ribbon view; Cdc37 is shown in yellow ribbon view; FW-04-806 is shown in orange ball view. (H) Stick view of FW-04-806 bound to N-Hsp90/Cdc37 complex. N-Hsp90 is shown in green stick view; FW-04-806 is shown in white stick view; Cdc37 is shown in blue stick view. (I) Stick views of FW-04-806 and N-Hsp90 in white and green, respectively.

Article Snippet: Recombinant human Cdc37 was obtained from Sino Biological Inc. MG132 was obtained from Sigma Aldrich.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Affinity Column, Western Blot, Incubation

Journal: Molecular Cancer

Article Title: FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation

doi: 10.1186/1476-4598-13-150

Figure Lengend Snippet: FW-04-806 inhibits Hsp90/Cdc37 chaperone/co-chaperone interactions. (A) FW-04-806 (10, 20, 40 μM) was added into recombinant NBD Hsp90 protein did not affect ATP binding capacity of Hsp90, while 17AAG (0.5, 1 μM) decreased ATP binding. Western blot was analyzed with His-probe antibody. (B) FW-04-806 had little effect on Hsp90 ATPase activity as determined by the malachite green reagent. The assay used 0.5 μM Hsp90 protein, 1 mM ATP, and FW-04-806 or 17AAG at 25, 50, 100, or 200 μM, or vehicle (DMSO) at 620 nm. Results are presented as means ± SD of three independent experiments. *p < 0.05: significant difference from control by analysis of variance; **p < 0.01: very significant difference from control by analysis of variance. (C) FW-04-806 directly affect Hsp90-Cdc37 interaction in Pull-down assay. 400 ug of purified Hsp90 (His-tag)protein was bound with Ni-NTA resin and divided into four groups evenly with the addition of recombinant Cdc37 protein 50 μg each and FW-04-806 0, 10, 20, 40 μM, respectively. After incubation and wash, The resins were boiled with loading buffer and analyzed by western blotting, His-probe antibody and Cdc37 antibody was used respectively. (D) SKBR3 cells were treated with FW-04-806 or DMSO for 24 h. Cdc37 or Hsp90 were immunoprecipitated from whole-cell lysates (500 μg each) with an anti-Cdc37 or anti-Hsp90 antibody respectively, then analyzed by immunoblotting with antibody against Hsp90, Cdc37 and HER2 (E) SKBR3 cells were treated with FW-04-806 at 10, 20, 40 μM for 24 h; 17AAG was used as a positive control at 1 and 2 μM. Hsp70, Hsp90, and Cdc37 protein level were analyzed with western blotting using relevant antibodies. (F) SKBR3 cells were transiently transfected with control siRNA or Hsp90 siRNA for 48 h. Whole-cell lysates were analyzed with western blotting against HER2, Hsp90, and β-actin.

Article Snippet: Recombinant human Cdc37 was obtained from Sino Biological Inc. MG132 was obtained from Sigma Aldrich.

Techniques: Recombinant, Binding Assay, Western Blot, Activity Assay, Pull Down Assay, Purification, Incubation, Immunoprecipitation, Positive Control, Transfection

Journal: The Journal of biological chemistry

Article Title: Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.

doi: 10.1074/jbc.275.1.279

Figure Lengend Snippet: FIG. 5. Geldanamycin treatment of B4 cells prevents the pro- gression of Cdk9/Hsp70 to Cdk9/Hsp90/Cdc37. A, a proposed path- way for Cdk9 folding/stabilization and the assembly of the Cdk9/cyclin T1 heterodimer. The chaperone proteins clearly identified as Cdk9- associated proteins are diagrammed. However, other chaperones and cofactors required for the folding/assembly process may exist in vivo. In a process separated from this pathway, a major portion of cellular Cdk9, either translated without the protection of Hsp70 or abandoned by the chaperones along the pathway, is rapidly degraded in the cell. B, GA treatment of B4 cells alters the ratio of the two Cdk9 chaperone com- plexes. B4 cells expressing Cdk9-HA were treated for 20 h with increas- ing GA concentrations as indicated. Cdk9-HA and associated proteins were affinity-purified from GA-treated cells and analyzed by SDS- PAGE and silver staining. Control conditions included no treatment (lane 2) and treatment with Me2SO used to dissolve GA (lane 3). A control purification from parental 293 cells was also included (lane 1). The positions of nonspecific bands (asterisk) are indicated. C, the iden- tities of proteins found in B were confirmed by Western blotting with antibodies specific for Hsp90, cyclin T1, Hsp70, Cdc37, and HA tag. D, GA treatment of B4 cells did not significantly affect the cellular levels of Hsp70, Hsp90, and cyclin T1. Whole cell lysates used for the purifi- cation of Cdk9-HA complexes in A and B were analyzed by Western blotting with antibodies specific for Hsp90, cyclin T1, Hsp70, and HA tag as indicated.

Article Snippet: Antibodies and Stable Cell Lines Expressing Cdk9-HA and HACycT1—Anti-Cdk9 and anti-Cdc37 antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and anti-Hsp70 and antiHsp90 were from StressGen Biotechnologies (Victoria, BC, Canada).

Techniques: In Vivo, Expressing, Affinity Purification, SDS Page, Silver Staining, Control, Purification, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.

doi: 10.1074/jbc.275.1.279

Figure Lengend Snippet: FIG. 6. Geldanamycin inhibits Hsp90/Cdc37 function and precludes the generation of the Cdk9/cyclin T1 heterodimer. A, whole cell lysates pre- pared from B4 cells treated with either 0.5 mM GA or Me2SO for 48 h were nor- malized for total protein by SDS-PAGE and Coomassie Blue staining. B, Cdk9-HA and its associated cyclin T1 were affinity-purified from these B4 cell lysates and detected by Western blotting with antibodies specific for cyclin T1 and HA tag. C, HA-tagged cyclin T1 (HA- CycT1) and its associated Cdk9 were af- finity-purified from an equal amount of lysate prepared from G3 cells treated with 0.5 mM GA or Me2SO for 48 h. The purified proteins were analyzed by West- ern blotting with antibodies specific for Cdk9 and HA tag.

Article Snippet: Antibodies and Stable Cell Lines Expressing Cdk9-HA and HACycT1—Anti-Cdk9 and anti-Cdc37 antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and anti-Hsp70 and antiHsp90 were from StressGen Biotechnologies (Victoria, BC, Canada).

Techniques: SDS Page, Staining, Affinity Purification, Western Blot, Purification

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: Reduction of β-amyloid pathology by celastrol in a transgenic mouse model of Alzheimer's disease

doi: 10.1186/1742-2094-7-17

Figure Lengend Snippet: Effect of celastrol and PMA on the formation of the HSP90-cdc37 complex in HEK293 APPsw cells . A) Western-blot depicting the amount of cdc37 recovered in the HSP90 immunoprecipitate following treatment with celastrol and PMA. B) Histogram representing the quantification of cdc37 in the HSP90 immunoprecipitate. No significant effect of PMA was observed but a significant effect of celastrol was detected (P < 0.005) showing a slight reduction in the amount of cdc37 present in the HSP90 immunoprecipitates. C) Western-blots showing the effects of the HSP90 inhibitor gedunin on BACE-1 expression and APP processing in HEK293 APPsw cells. D) Histogram representing the quantification of BACE-1/Actin chemoluminescent signal showing that the gedunin treatment does not affect BACE-1 expression in HEK293 APPsw cells (ANOVA reveals no significant main effect of gedunin on BACE-1 level (P = 0.572)). E) Representative western-blot showing the level of cdc37 and BACE-1 expression in HEK293 APPsw cells that were knock-down for cdc37 using a shRNA approach for 3 different clones. 1) non silencing scrambled shRNA; 2) cdc37 shRNA clone 51G9; 3) cdc37 shRNA clone 97H1; 4) cdc37 shRNA clone 95C4. F) Histogram representing the quantification of cdc37 and BACE-1 expression for 9 different clones of HEK293 APPsw cells stably transfected with a silencing cdc37 shRNA vector (cdc37 shRNA) and 4 different clones of HEK293 APPsw stably transfected with a non silencing scrambled shRNA vector (control shRNA). Statistically significant inhibition of cdc37 expression (P < 0.001) and no effect on BACE-1 expression (P = 0.947) was observed in HEK293 APPsw cells knock-down for cdc37.

Article Snippet: Western-blots were run with cell lysates from these clones to confirm silencing of the cdc37 gene using a 1:1000 dilution of an anti-cdc37 (V367) antibody (Cell Signaling Technology Inc, MA, USA).

Techniques: Western Blot, Expressing, Knockdown, shRNA, Clone Assay, Stable Transfection, Transfection, Plasmid Preparation, Control, Inhibition

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

Journal: Cancers

Article Title: Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

doi: 10.3390/cancers13040927

Figure Lengend Snippet: Conglobatin A selectively targets K-Ras via the inhibition of Hsp90/Cdc37. ( A , B ) FLIM-FRET images of HEK cells transfected with FRET-pairs of mGFP-/mCherry-K-RasG12V ( A , top) or mGFP-/mCherry-H-RasG12V ( B , top). The labelling indicates donor-only control or treatments. A decrease in the fluorescence lifetime indicates an increase in FRET due to nanoclustering. The transfected cells were treated with 0.1% DMSO vehicle control, 2 μM conglobatin A, or 1.3 μM salinomycin for 24 h. The nanoclustering-FRET analysis by treatment for K-RasG12V ( A , bottom) or H-RasG12V ( B , bottom). The numbers in the bars indicate the number of analyzed cells; n = 3 independent biological repeats. ( C ) Conglobatin A (purple surface) was docked into the crystal structure of human N-Hsp90 (green; PDB ID 3T0Z). The docked complex was overlaid on top of the crystal structure of yeast N-Hsp90 in a complex with human Cdc37 (PDB ID 1US7), of which only Cdc37 is depicted (orange). The magnification shows the details of how conglobatin A (cyan sticks) sterically blocks and disturbs critical interactions, notably between Glu47 of Hsp90 (gray sticks) and Arg167 of Cdc37 (orange sticks) at the interface of the complex. ATP is shown as magenta sticks, and magnesium is shown as a deep purple sphere. The polar interactions between conglobatin A and Hsp90 are shown as dashed lines, with distances in Ångströms. ( D , E ) The K-RasG12V- ( D ) and H-RasG12V- ( E ) nanoclustering-FRET in HEK cells co-transfected with mGFP- and mCherry-tagged RasG12V, together with 50 nM siRNA Hsp90, siRNA Cdc37, or 50 nM scrambled siRNA for compound/vehicle (control) treated samples. The next day, the cells were treated for 24 h with 0.1% DMSO vehicle control, 2 μM conglobatin A, or 2 μM 17-AAG. The numbers in the bars indicate the number of analyzed cells. The number of independent biological repeats n = 3 for K-Ras and n = 1 for H-Ras. **** p < 0.0001.

Article Snippet: For the silencing of human Hsp90, Cdc37, galectin-3 and HIF-1α, we used siRNA Hsp90 (cat. no. sc 35608), siRNA Cdc37 (cat. no. sc 29255) and siRNA galectin-3 (cat. no. sc 155994) from Santa Cruz Biotechnology.

Techniques: Inhibition, Transfection, Control, Fluorescence

Journal: Cancers

Article Title: Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

doi: 10.3390/cancers13040927

Figure Lengend Snippet: Inhibition of the Hsp90/Cdc37 interaction measured in the split Renilla luciferase assays.

Article Snippet: For the silencing of human Hsp90, Cdc37, galectin-3 and HIF-1α, we used siRNA Hsp90 (cat. no. sc 35608), siRNA Cdc37 (cat. no. sc 29255) and siRNA galectin-3 (cat. no. sc 155994) from Santa Cruz Biotechnology.

Techniques: Inhibition, Luciferase

Journal: Cancers

Article Title: Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

doi: 10.3390/cancers13040927

Figure Lengend Snippet: Screening for small molecule Hsp90/Cdc37 interface inhibitors. ( A ) Combined computational and in vitro compound screening workflow to identify novel small molecules with activities below 60 µM in the Hsp90/Cdc37 split Renilla luciferase assay. ( B ) Chemical structures of the selected top hit compounds, arranged by scaffolds. The structure of conglobatin A is shown for comparison at the bottom. ( C ) Competition with Hsp90 N-terminal ATP-binder FITC-geldanamycin decreases the polarization signal, indicating ATP-pocket binding (with 17-AAG, luminespib as positive controls). All of the compounds were tested at 20 µM. ( D , E ) The docked models for ×6506 ( D ) and ×1540 ( E ) in a complex with human N-Hsp90 (green; PDB ID 3T0Z) at the Hsp90/Cdc37 interface. Both compounds are shown as cyan sticks, the interacting residues in Hsp90 are shown as gray sticks, ATP is shown as magenta sticks, and magnesium is shown as a deep purple sphere. The polar interactions between the compounds and the protein are shown as dashed lines with distances in Ångströms. **** p < 0.0001.

Article Snippet: For the silencing of human Hsp90, Cdc37, galectin-3 and HIF-1α, we used siRNA Hsp90 (cat. no. sc 35608), siRNA Cdc37 (cat. no. sc 29255) and siRNA galectin-3 (cat. no. sc 155994) from Santa Cruz Biotechnology.

Techniques: In Vitro, Luciferase, Comparison, Binding Assay

Journal: Cancers

Article Title: Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

doi: 10.3390/cancers13040927

Figure Lengend Snippet: Top compounds inhibit 2D and 3D cancer cell growth and microtumor formation in the CAM model. ( A ) Compounds ×6506 or ×1540 dose-dependently reduced the 2D proliferation of several cancer and non-transformed cell lines after 72 h; n = 3 independent biological repeats. ( B ) Compounds ×6506 or ×1540 dose-dependently reduced the 3D spheroid growth of several cancer cell lines cultured under low attachment and serum-free conditions after 72 h; n = 3 independent biological repeats. ( C ) Microtumor formation of MDA-MB-231 cells on chick embryo CAM. The cells were treated with 0.1% DMSO vehicle control, 20 μM ×6506 or ×1540, or 10 μM 17-AAG for 5 days; n = 2 independent biological repeats. The number of analyzed microtumors per condition is given in brackets in the plot. ( D ) Summary scheme showing, on the left, the compound screening success, leading to six new small molecule Hsp90/Cdc37 protein interface inhibitors that could serve as a starting point for drug development. The major part on the right illustrates how Hsp90 activity is linked to the K-Ras signaling output via HIF-1α and Gal3, which can stabilize the K-Ras nanocluster and thus increase the MAPK-output. According to our nanocluster model, Raf proteins are integral components; hence, we here indicate a potential mechanistic contribution via Raf. However, it is currently unknown which Raf-dimers preferentially stabilize the K-Ras nanocluster. ** p < 0.005, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the silencing of human Hsp90, Cdc37, galectin-3 and HIF-1α, we used siRNA Hsp90 (cat. no. sc 35608), siRNA Cdc37 (cat. no. sc 29255) and siRNA galectin-3 (cat. no. sc 155994) from Santa Cruz Biotechnology.

Techniques: Transformation Assay, Cell Culture, Control, Activity Assay