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PubMed2024

Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA.

Ubiquitin-specific protease 7 (USP7) is implicated in many cancers including colorectal cancer in which it regulates cellular pathways such as Wnt signaling and the P53-MDM2 pathway. With the discovery of small-molecule inhibitors, USP7 has also become a promising target for cancer therapy and therefore systematically identifying USP7 deubiquitinase interaction partners and substrates has become an important goal. In this study, we selected a colorectal cancer cell model that is highly dependent on USP7 and in which USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, and cell-cell adhesion. We then used inducible knockdown of USP7 followed by LC-MS/MS to quantify USP7-dependent proteins. We identified the Ajuba LIM domain protein as an interacting partner of USP7 through co-IP, its substantially reduced protein levels in response to USP7 knockdown, and its sensitivity to the specific USP7 inhibitor FT671. The Ajuba protein has been shown to have oncogenic functions in colorectal and other tumors, including regulation of cell-cell adhesion. We show that both knockdown of USP7 or Ajuba results in a substantial reduction of cell-cell adhesion, with concomitant effects on other proteins associated with adherens junctions. Our findings underlie the role of USP7 in colorectal cancer through its protein interaction networks and show that the Ajuba protein is a component of USP7 protein networks present in colorectal cancer.

Ubiquitin-Specific Peptidase 7↗

PubMed2026

Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network.

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro) development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by data-independent acquisition and label free quantitation mass spectrometry. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-ε-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7's activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

Ubiquitin-Specific Peptidase 7↗

PubMed2025

USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

Ubiquitin-Specific Peptidase 7↗

PubMed2026

PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer is characterized by redox adaptation and metabolic plasticity, but the mechanisms linking these processes remain incompletely understood. Integrating bulk, single-cell, and spatial transcriptomic analyses, we identified PRDX1 as a malignant epithelial cell-associated factor linked to adverse outcome. Genetic gain- and loss-of-function studies showed that PRDX1 promoted proliferation, motility, and xenograft growth while limiting reactive oxygen species accumulation and mitochondrial apoptosis. Proteomic and biochemical analyses identified an association between PRDX1 and SCD1. PRDX1 prolonged the SCD1 protein half-life without detectably altering SCD1 transcript abundance and increased USP7-SCD1 co-precipitation. USP7 removed K48-linked polyubiquitin chains from SCD1 and prevented its proteasomal degradation, whereas catalytically inactive USP7 failed to deubiquitinate SCD1. Deletion of PRDX1 residues 157-199 weakened its association with SCD1 and reduced USP7-SCD1 co-precipitation. Depletion of SCD1 or USP7 suppressed PRDX1-dependent growth in vitro and in xenografts. These findings support a model in which PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer↗

PubMed2026

Molecular mechanism of HUWE1-HAPSTR1-USP7-mediated ubiquitin chain amplification on nuclear proteins.

Rapid protein turnover is essential for cellular stress adaptation. HUWE1 (HECT, UBA, and WWE domain containing 1), a large HECT-type E3 ligase, regulates many short-lived stress-responsive proteins, yet the mechanisms underlying its substrate selectivity remain unclear. Here, we reveal that HUWE1 functions as a ubiquitin chain amplifier that captures pre-ubiquitinated substrates and amplifies the degradation signal by assembling long ubiquitin chains containing K11-K48 branch points, a process regulated by its partners HUWE1-associated protein stress response 1 (HAPSTR1) and USP7 (ubiquitin-specific-processing protease 7). Structural and biochemical analyses show that HAPSTR1 engages HUWE1's ubiquitin-binding motifs to drive nuclear import and modulate substrate recruitment. A cryo-EM structure of the HUWE1-USP7 complex reveals a bidirectional regulatory mechanism: HUWE1 activates USP7's catalytic activity, while USP7 modulates HUWE1 conformational states. Global proteomic analyses demonstrate that this axis drives extensive remodeling of the short-lived nuclear proteome. These findings establish the HUWE1-HAPSTR1-USP7 complex as a key ubiquitin code modifier, providing a molecular rationale for HUWE1 dysregulation in neurodevelopmental disorders and cancer.

Ubiquitin-Specific Peptidase 7↗

PubMed2023

RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

Animals↗

PubMed2026

3D epigenomic remodelling mediated by Foxa1 drives gemcitabine resistance in pancreatic cancer.

Gemcitabine remains a cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), yet the emergence of resistance constitutes a major clinical challenge with poorly understood epigenomic mechanisms. Here, we identified the pioneer transcription factor Foxa1 as a master regulator of gemcitabine resistance through multi-omics analysis. Mechanistically, Foxa1 drives widespread super-enhancer (SE) reprogramming and 3D genome remodelling in resistant cells, which coordinately activates the expression of key resistance genes, notably Rrm1 and Cdadc1. This is accompanied by increased chromatin accessibility, elevated H3K27ac enrichment at SEs, and enhanced Foxa1 binding at regulatory elements. Moreover, post-translational stabilization of Foxa1 via USP7-mediated deubiquitination sustains this epigenomic program. Genetic ablation of Foxa1 or specific SE regions near Rrm1 resensitizes resistant cells to gemcitabine. Building upon this mechanism, we demonstrate that bromodomain and extraterminal (BET) inhibitors, which disrupt SE function, potently reverse resistance. Notably, the clinical-stage BET inhibitor AZD5153, in combination with gemcitabine, achieves robust tumor suppression and overcomes resistance in cell-derived xenograft (CDX) models by dismantling the Foxa1-mediated resistant transcriptome and reinvigorating drug sensitivity. Our findings establish Foxa1-orchestrated enhancer reprogramming as a fundamental mechanism of gemcitabine resistance and unveil a promising epigenetic therapy to restore treatment efficacy in PDAC.

Hepatocyte Nuclear Factor 3-alpha↗

PubMed2024

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Substrate Specificity↗

PubMed2025

STUPPIT is a proximity labeling tool for labeling intermediary proteins that bridge two non-interacting proteins.

Decoding the complexities of signaling pathways is fundamental for deciphering the mechanisms underlying tissue development, homeostasis, and disease pathogenesis. Proximity labeling tools have been instrumental in identifying upstream or downstream effectors of specific proteins within signaling pathways. However, currently, there are no tools available to directly label and capture intermediary proteins that bridge two non-interacting proteins. Here, we developed Split-TurboID and PUP-IT based Protein Identification Tool (STUPPIT), a novel method combining split-TurboID and PUP-IT to biotinylate intermediary proteins of two non-interacting proteins through a two-step enzymatic reaction. STUPPIT was validated using three well-characterized protein triads, including YAP1/AMOT/β-actin, YAP1/LATS1/MOB1A, and β-catenin/α-catenin/β-actin using HEK293T human cell lines. Combining STUPPIT and proteomics, we identified novel intermediary proteins including ERC1 and USP7, which interacted both with β-catenin and SMAD4, key components of the Wnt and BMP signaling pathways. In conclusion, STUPPIT represents a powerful tool for labeling and capturing intermediary proteins between non-interacting partners, offering new insights into protein-protein interactions and advancing signal transduction research.

Humans↗