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At least 19 recordsLinked to original sources
PubMed2026

Derivation and characterization of ubiquitin-specific protease 18 inhibitors.

Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity, with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-Lapachone treatments statistically significantly repressed lung cancer xenograft growth. β-Lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. IFN-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 cotransfection antagonized this activity. β-Lapachone treatments increased global ISGylation. RNA-seq of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.

Humans↗
PubMed1992

Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family.

In eukaryotes, both natural and engineered ubiquitin (Ub) fusions to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, we have isolated two other yeast genes for ubiquitin-specific proteases, named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 was active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae.

Amino Acid Sequence↗
PubMed1994

UBP5 encodes a putative yeast ubiquitin-specific protease that is related to the human Tre-2 oncogene product.

A gene from chromosome V of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The deduced amino acid sequence encoded by this gene is similar to several ubiquitin-specific proteases from yeast, especially at the highly conserved domain. It is thus named UBP5. UBP5 is also closely related to the human Tre-2 and the mouse Unp oncogene products. This study adds a new member to the ubiquitin protease family and suggests that alteration of ubiquitin protease activity may result in cancer in mammals. However, disruption of the UBP5 gene in a haploid strain did not result in a noticeable phenotypic alteration.

Amino Acid Sequence↗
PubMed1991

Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae.

In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.

Alleles↗
PubMed2025

USP2 reversed cisplatin resistance through p53-mediated ferroptosis in NSCLC.

BACKGROUND: It has demonstrated the indispensable role of ferroptosis in conferring cisplatin resistance in non-small cell lung cancer (NSCLC), as well as the involvement of ubiquitin-specific protease (USP) in regulating ferroptosis. This paper aspired to the mechanism of USP2 and ferroptosis on NSCLC cisplatin resistance. METHODS: Ubiquitin-specific protease mRNA expression, was detected through RT-qPCR. In vitro functional assays assessed the effects of USP2 overexpression on DDP resistance, cell proliferation capability, and ferroptosis markers in A549/DDP and H1299/DDP cells. Ubiquitination assays evaluated the ubiquitination levels of p53 following USP2 overexpression. Co-immunoprecipitation (Co-IP) assays confirmed the binding relationship between USP2 and p53. In vivo experiments in mice explored the specific role of the USP2-p53 axis in a xenograft tumor model. RESULTS: USP2 expression was suppressed in cisplatin-resistant NSCLC cells. USP2 overexpression inhibited cell viability in cisplatin-resistant cells. Among the ferroptosis markers, the results showed that USP2 overexpression promoted LDH release, Fe2+ level, MDA and Lipid ROS, while inhibited GPX4 activity and GSH levels. The WB results revealed that USP2 overexpression inhibited GPX4, SLC7A11 and cytoplasm p53 protein expression, while promoted the nucleus p53 protein expression. Moreover, USP2 directly bound to p53 and USP2 overexpression stabilized p53 protein by suppressing its ubiquitination. In vivo experiments further suggest that the USP2-p53 pathway plays a crucial role in regulating cisplatin sensitivity in A549/DDP cells. CONCLUSION: USP2 acted on the K305R site of p53, which resulted in its deubiquitination. This cellular process could modulate cisplatin resistance through ferroptosis in NSCLC. This study could provide a potential therapeutic target to NSCLC.

Ferroptosis↗
PubMed2025

Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

The growing interest in deubiquitinases (DUBs) as drug targets for modulating critical molecular pathways in disease is fueled by the discovery of their specific cellular roles. A crucial aspect of this fact is the identification of DUB substrates. While mass spectrometry-based proteomic methods can be used to study global changes in cellular ubiquitination following DUB activity perturbation, these datasets often include indirect and downstream ubiquitination events. To enrich for the direct substrates of DUB enzymes, we have developed a proximal-ubiquitome workflow that combines proximity labeling methodology (ascorbate peroxidase-2 [APEX2]) with subsequent ubiquitination enrichment based on the K-ε-GG motif. We applied this technology to identify altered ubiquitination events in the vicinity of the DUB ubiquitin-specific protease 30 (USP30) upon its inhibition. Our findings reveal ubiquitination events previously associated with USP30 on TOMM20 and FKBP8, as well as the candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner.

Humans↗
PubMed2026

Thrombin concentration shapes endothelial extracellular vesicle profiles with divergent inflammatory functions.

Thrombin, a central enzyme in the coagulation cascade, also regulates diverse cellular processes, including inflammation and vascular barrier function, primarily by activating protease-activated receptor 1. Previous studies demonstrated that thrombin elicits concentration-dependent, opposing effects; low concentrations confer anti-inflammatory and barrier-protective responses, whereas high concentrations promote inflammation and barrier disruption. The underlying mechanisms, however, remain incompletely understood. Here, we showed that thrombin stimulates extracellular vesicle (EV) release from endothelial cells across a broad concentration range and that EVs generated at low vs high thrombin concentrations carry distinct microRNA (miR) cargo. Low-thrombin EVs mediate cytoprotective responses via the transfer of miR-409-5p, which targets ubiquitin-specific protease 7 that promotes inflammation via the NF-kB signaling pathway in recipient cells, whereas high-thrombin EVs disrupt barrier integrity and promote inflammation through delivery of miR-155-5p, a regulator of suppressor of cytokine signaling 1 that acts as a crucial negative regulator of the cytokine signaling pathway. Functional manipulation of these EVs confirmed the causal roles. Incorporation of anti-miR-409-5p abrogated the protective effects of low-thrombin EVs, whereas anti-miR-155-5p suppressed the cytopathic effects of high-thrombin EVs. Moreover, control EVs engineered to carry a miR-409-5p mimic reproduced the anti-inflammatory and barrier-protective phenotype of low-thrombin EVs. Collectively, these findings identified EV-associated miRs as key mediators of the concentration-dependent dual actions of thrombin, which may open the therapeutic potential of EVs engineered to deliver selective miRs or anti-miRs for the treatment of inflammatory vascular diseases.

Thrombin↗
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↗
PubMed1993

Augmentation of protein production by a combination of the T7 RNA polymerase system and ubiquitin fusion: overproduction of the human DNA repair protein, ERCC1, as a ubiquitin fusion protein in Escherichia coli.

This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.

Base Sequence↗
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↗
PubMed1994

Split ubiquitin as a sensor of protein interactions in vivo.

We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub. However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.

Animals↗
PubMed1995

Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene.

Ubiquitin is a highly conserved polypeptide found in all eukaryotes. The major function of ubiquitin is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome. Here, the Drosophila fat facets gene, which is required for the appropriate determination of particular cells in the fly eye, was shown to encode a ubiquitin-specific protease (Ubp), an enzyme that cleaves ubiquitin from ubiquitin-protein conjugates. The Fat facets protein (FAF) acts as a regulatory Ubp that prevents degradation of its substrate by the proteasome. Flies bearing fat facets gene mutations were used to show that a Ubp is cell type--and substrate-specific and a regulator of cell fate decisions in a multicellular organism.

Amino Acid Sequence↗
PubMed1994

Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.

Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology. Despite the availability of several E. coli expression vector systems, adequate levels of expression may not be achieved. Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems. We have modified this technique by the co-expression in E. coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2. This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E. coli. This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu. The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected. The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1. As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.

Amino Acid Sequence↗
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↗
PubMed1996

Using ubiquitin to follow the metabolic fate of a protein.

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.

Amino Acids↗
PubMed1996

DUB-1, a deubiquitinating enzyme with growth-suppressing activity.

Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).

Amino Acid Sequence↗
PubMed1995

Elevated expression of Unph, a proto-oncogene at 3p21.3, in human lung tumors.

The murine Unp proto-oncogene encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. We have cloned cDNAs originating from the human homolog of this gene, designated herein as Unph (Unp, human), and have used these cDNAs to map the gene to 3p21.3, a region frequently rearranged in human tumor cells. Unph mRNA levels are consistently elevated in small cell tumors and adenocarcinomas of the lung, suggesting a possible causative role for the gene in the neoplastic process.

3T3 Cells↗
PubMed1994

The Unp proto-oncogene encodes a nuclear protein.

The mouse Unp gene is related to TRE17, a human oncogene, but is not its mouse homolog. Unp is a ubiquitously expressed gene producing two related mRNAs. The protein product of Unp is localized in the nucleus. Expression of Unp from a highly active promoter results in tumorigenic transformation of NIH3T3 cells injected into athymic mice. Unp therefore encodes a novel nuclear oncoprotein.

3T3 Cells↗