Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Outline of this study. Comparative analysis of USP7 inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Inhibition, Derivative Assay, Variant Assay, Ubiquitin Proteomics, Cleavage Assay, Incubation, Activity Assay, Fluorescence, Mass Spectrometry

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic of the synthesis of USP7-targeting degraders. Functionalized USP7 ligands (top left, pink triangle) were either coupled directly to VHL-linker conjugates (yellow triangle with grey stick, through steps A and B) or first further functionalized with linkers followed by coupling to the VHL ligand (yellow triangle, through steps A, C and D). See the Supporting Information for detailed chemical procedures. b. Chemical structures of USP7-targeting degrader library. A general structure of the PROTACs is shown on the left, consisting of a USP7 binding ligand (top) and E3 ligase binding ligand (bottom) separated by a linker. Linker attachment points to both ligands are indicated by waved lines (black: USP7, red: VHL). Linkers are shown in the table on the right. c.-d. Assessment of USP7 levels upon treatment with compounds in Panc89 cells (c) and Ma-Mel-47 cells (d). Cells were treated with 5 µM of indicated compounds for 24 h, and cell lysates were analyzed through Western blots with indicated antibodies. See the Supporting Information for uncropped blots. e. Chemical structure of NK225.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Chemical structures of improved VHL-targeting PROTACs NK250, NK266 and non-VHL binding control compound NK245. The additional methyl groups in the VHL ligand in NK250 and NK266 (leading to enhanced VHL binding) as well as the inversed hydroxyproline stereocenter in NK245 (abrogating VHL binding) are highlighted. b. USP7 degradation assay. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with 5 µM of indicated compounds for 24 h and analyzed by Western blot. See the Supporting Information for uncropped blots. c. Assessment of USP7 degradation efficiency. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with increasing concentrations of either NK250 (left) or NK266 (right). d. Determination of degradation kinetics. Panc89 cells (left) or Ma-Mel47 cells (right) were treated with of 1 µM of NK250 (left) and NK266 (right) for indicated times. e. Degradation rescue experiment. Panc89 cells (left) or Ma-Mel47 cells (right) were pretreated for 2 h with either DMSO, NEDDylation inhibitor MLN4924 (500 nM), proteasome inhibitor Carfilzomib (CFZ, 250 nM) or VHL ligand NK249 (10 µM) followed by 1 µM of NK250 or N266 for 20 h. f. Chemical structure of VHL ligand NK249. g. Orthogonal confirmation of USP7 depletion by immune fluorescence. Panc89 cells were treated with 1 µM NK250 for 24 h before staining for USP7 (green), actin (red), and with DAPI (blue). Scale bar: 10 µm.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Control, Degradation Assay, Western Blot, Fluorescence, Staining

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic representation of HiBiT endpoint degradation assay. b. HiBiT-based quantification of USP7 levels. MV4-11 cells stably expressing HiBiT-USP7 generated through lentiviral transduction were treated with PROTACs for either 6 or 24 h. Remaining HiBiT-USP7 could be detected through the luciferase signal using HiBiT Lytic Detection System. Data are shown as mean ± S.D. (N=3), normalized to DMSO treatment. Half-maximal degradation concentrations (DC 50 ) derived from these data are given. c. Schematic representation of a cellular ternary complex formation assay. Halotag-VHL- and NanoLuc-USP7-expressing cells are sequentially treated with Halotag-Fluorophor and PROTAC. Compound-induced proximity is read out by a bioluminescence resonance energy transfer (BRET) signal as shown, demonstrating ternary complex formation. d. Cellular ternary complex formation assay. HEK293T cells overexpressing Halotag-VHL and NanoLuc-USP7 were treated with HaloTag NanoBRET 618 Ligand for 20 h, followed by NK250 or NK266 treatment at indicated concentrations for 2 h prior BRET measurement. The determined half-maximal ternary complex formation concentration (EC 50 ) for NK250 is given. Data are shown as mean ± S.D. (N=4). mBu, milli BRET units.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Degradation Assay, Stable Transfection, Expressing, Generated, Transduction, Luciferase, Derivative Assay, Tube Formation Assay, Bioluminescence Resonance Energy Transfer, Concentration Assay

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: Proteomic analysis of Panc89 cells treated with PROTAC NK250 (a-c) or inhibitor NK192 (d-f) for indicated time points. Volcano blots report proteins identified by data-independent acquisition mass spectrometry in Panc89 cells treated with NK192 (5 µM) or NK250 (1 µM) for 6, 24 or 72 h. Proteins annotated as members of the non-canonical repressive complex 1.6 are highlighted in orange, chromatin bound E3 ligases are highlighted in black, the most upregulated proteins by USP7 inhibition in blue. g.-l. Proteomic analysis of Ma-Mel-47 cells treated with PROTAC NK266 (g-I, 1 µM) or inhibitor NK192 (j-l, 5 µM) for indicated time points.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Data-independent acquisition, Mass Spectrometry, Inhibition

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: a His-tag pulldown-HPLC-MS/MS showing that His-TOP1 interacted USP7 under unperturbed condition. After transfection of 6×His-tagged TOP1 expression plasmid or empty vector control (pTrex), HCT116 cells were subjected to His-tag pulldown using Ni-NTA agarose, followed by HPLC-MS. b In vitro assay showing that USP7 reversed TOP1 ubiquitylation. Recombinant TOP1 was subjected to ubiquitylation with ubiquitin, Ube1 (E1), Ubc5Hα (E2), and RNF4 (E3) for 30 min, followed by termination with EDTA and incubation with increasing concentrations of recombinant USP7 for another 30 min. Samples were Western blotted with α-Ub antibody. c His-tag pulldown assay showing that PARGi enhanced TOP1-USP7 interaction. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. His-tag pulldown was performed with Ni-NTA agarose under native conditions. Western blotting was performed with the indicated antibodies. d PLA assay showing TOP1-USP7 interaction in PARGi-treated cells. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. PLA assays were performed using rabbit α-His-tag antibody and mouse α-FLAG tag antibody. The scale bar represents 10 μm. e Inhibiting USP7 restored TOP1-DPC ubiquitylation in the presence but not in the absence of PARGi. Upper panel : HEK293 cells were treated as indicated: CPT (20 µM, 1 h), CPT + FLAG-USP7 transfection, CPT + USP7i (10 µM, 1 h pre-treatment), CPT + PARGi (10 µM, 1 h pre-treatment), CPT + PARGi + FLAG-USP7 transfection, CPT + PARGi + USP7i. Following treatments, cells were subjected to the modified RADAR assay for detection of TOP1-DPCs and their ubiquitylation using α-TOP1 and α-Ub antibodies. Lower panel : densitometric quantitation of ubiquitylated TOP1-DPC signals generated from triplicate experiments including representative blots shown in ( c ) using ImageJ. n = 3 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). *: p = . NS not significant. f Inhibiting USP7 did not impact the induction of γH2AX upon exposure to CPT. U2OS cells were synchronized in the S phase by double thymidine and treated with CPT (1 µM) in the absence of presence of USP7i (10 µM, 1 h pre-treatment) and collected for IF by iSIM using an anti-γH2AX antibody. The scale bar represents 10 μm.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Tandem Mass Spectroscopy, Transfection, Expressing, Plasmid Preparation, Control, In Vitro, Recombinant, Ubiquitin Proteomics, Incubation, Western Blot, FLAG-tag, Modification, Quantitation Assay, Generated, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: a PARP1 and TOP1 form cellular protein complexes (present study and refs. , ). b TOP1-DPC trapped by CPT is rapidly modified with PAR by PARP1 and with ubiquitin (by RNF4 and potentially other E3 ligases, not shown). The PARylation recruits TDP1, PARG, and USP7 to the TOP1-DPC. c TOP1-DPC PARylation is readily and rapidly reversed by PARG, enabling the 26S proteasome to target the ubiquitylated TOP1-DPC for degradation. d TDP1 hydrolyzes the TOP1 peptide to expose the DNA ends for repair. e In the presence of PARGi, TOP1-DPC dePARylation is blocked and the persistent PAR polymers on TOP1-DPC obstruct the proteasome hence stabilize TOP1-DPC. f The stabilization of TOP1-DPC triggers USP7 to deubiquitylate the DPC to recycle the ubiquitin molecules.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Modification, Ubiquitin Proteomics

Journal: Journal of Cancer

Article Title: Potential Anti-Cancer Drug 6RK73 Suppresses Ovarian Cancer Growth by Inactivating the AKT1/Sp1 Induced c-Myc Signaling Pathway

doi: 10.7150/jca.113511

Figure Lengend Snippet: USP7 binds to c-Myc and regulates its ubiquitination. (A-E). A. Western blot analysis showing c-Myc expression levels following USP7 overexpression; B. Co-IP and Western blot assays confirming the interaction between c-Myc and USP7; C. Schematic representation of c-Myc and USP7 protein docking: c-Myc (purple) and USP7 (blue); D. Western blot analysis assessing the effects of CHX treatment on c-Myc stability in USP7-overexpressing and control cells; E. Co-IP and Western blot assays demonstrating the effect of USP7 overexpression on the ubiquitination level of c-Myc.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA, BioFroxx, Germany) and incubated overnight at 4°C with the following primary antibodies: Sp1 (21962-1-AP, Proteintech, 1:2000), c-Myc(10828-1-AP, Proteintech, 1:2000), AKT1 (10176-2-AP, Proteintech, 1:2000), P-AKT (80462-1-RR, Proteintech, 1:2000), USP7 (66514-1-Ig, Proteintech, 1:5000), GAPDH (AP0063, Bioworld, 1:10000), β-tubulin (AP0064, Bioworld, 1:5000), and Ubiquitin (10201-2-AP, Proteintech, 1:1000).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Over Expression, Co-Immunoprecipitation Assay, Control

Journal: Journal of Cancer

Article Title: Potential Anti-Cancer Drug 6RK73 Suppresses Ovarian Cancer Growth by Inactivating the AKT1/Sp1 Induced c-Myc Signaling Pathway

doi: 10.7150/jca.113511

Figure Lengend Snippet: Sp1 recruits USP7 to deubiquitinate C-MYC. (A-C). A. Co-IP assays confirming the interaction between Sp1 and USP7; B. Western blot analysis detecting the effects of Sp1 overexpression, USP7 knockdown, and their co-transfection on c-Myc expression; C. Compared to Sp1 overexpression alone, co-transfection of the Sp1 overexpression plasmid and si-USP7 increased c-Myc ubiquitination levels.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA, BioFroxx, Germany) and incubated overnight at 4°C with the following primary antibodies: Sp1 (21962-1-AP, Proteintech, 1:2000), c-Myc(10828-1-AP, Proteintech, 1:2000), AKT1 (10176-2-AP, Proteintech, 1:2000), P-AKT (80462-1-RR, Proteintech, 1:2000), USP7 (66514-1-Ig, Proteintech, 1:5000), GAPDH (AP0063, Bioworld, 1:10000), β-tubulin (AP0064, Bioworld, 1:5000), and Ubiquitin (10201-2-AP, Proteintech, 1:1000).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Over Expression, Knockdown, Cotransfection, Expressing, Plasmid Preparation, Ubiquitin Proteomics

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: ( A , B ) 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 ( A ) for ubiquitin with quantification ( B ) of Itgb1 ubiquitination levels. The intensity of the Ub signals was normalized to the intensity of the IP-ed Itgb1 signals. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test comparing the BSA group with USP12 WT , USP12 C48S , UCHL5, or USP7 group in ESCRT-KD condition ( P = 0.0194, 0.2707, 0.9997, and 0.9844, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. BSA served as a negative control. ( C ) Coomassie-blue staining of recombinant proteins used in the in vitro deubiquitination assay. MS was used to determine the identity of the protein bands indicated in the DUB complexes. Band *1, WDR48; band *2, WDR20; band *3, a cleaved WDR20; band *4, USP12. ( D ) 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. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. ( E – G ) Superplots showing the PCC between Ub and EGFP-USP12 ( E ), Ub and Itgb1 ( F ), and Itgb1 and EGFP-USP12 ( G ) 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. The P values in ( E – G ) are 0.0287, 0.0011, and 0.0048, respectively. * P < 0.05; ** P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. ( H ) Representative SIM images of Itgb1 and EEA1 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. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. ( I – K ) Superplots showing the PCC between EEA1 and EGFP-USP12 ( I ), EEA1 and Itgb1 ( J ), and Itgb1 and EGFP-USP12 ( K ) 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. The P values in ( I – K ) are 0.0178, 0.0040, and 0.0208, respectively. * P < 0.05; ** P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. .

Article Snippet: Recombinant USP7 , Novus Biologicals , E-519-025.

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

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant USP7 , Novus Biologicals , E-519-025.

Techniques: Recombinant, Expressing, Sequencing, Real-time Polymerase Chain Reaction, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, BIA-KA, Western Blot, Software, Microscopy