Ubiquitin-specific Protease Research Articles

USP7 cardiomyocyte specific knockout causes disordered mitochondrial biogenesis and dynamics and early neonatal lethality in mice

BackgroundUbiquitination is an enzymatic modification involving ubiquitin chains, that can be reversed by deubiquitination (DUB) enzymes. Ubiquitin-specific protease 7 (USP7), which is also known as herpes virus-associated ubiquitin-specific protease (HAUSP), has been shown to play a vital role in cardiovascular diseases. However, the underlying molecular mechanism by which USP7 regulates cardiomyocyte function has not been reported. MethodsTo understand the physiological function of USP7 in the heart, we constructed cardiomyocyte-specific USP7 conditional knockout mice. ResultsWe found that homozygous knockout mice died approximately three weeks after birth, while heterozygous knockout mice grew normally into adulthood. Severe cardiac dysfunction, hypertrophy, fibrosis, and cell apoptosis were observed in cardiomyocyte-specific USP7 knockout mice, and these effects were accompanied by disordered mitochondrial dynamics and cardiometabolic-related proteins. ConclusionsIn summary, we investigated changes in the growth status and cardiac function of cardiomyocyte-specific USP7 knockout mice, and preliminarily explored the underlying mechanism.

USP31 Activates the Wnt/β-catenin Signaling Pathway and Promotes Gastric Cancer Cell Proliferation, Invasion and Migration.

Gastric cancer (GC) has a poor prognosis because it is highly aggressive, yet there are currently few effective therapies available. Although protein ubiquitination has been shown to play a complex role in the development of gastric cancer, to date, no efficient ubiquitinating enzymes have been identified as treatment targets for GC. The TCGA database was used for bioinformatic investigation of ubiquitin-specific protease 31 (USP31) expression in GC, and experimental techniques, including Western blotting, qRT-PCR, and immunohistochemistry, were used to confirm the findings. We also analyzed the relationship between USP31 expression and clinical prognosis in patients with GC. We further investigated the effects of USP31 on the proliferation, invasion, migration, and glycolysis of GC cells in vitro and in vivo by using colony formation, CCK-8 assays, Transwell chamber assays, cell scratch assays, and cell-derived xenograft. Furthermore, we examined the molecular processes by which USP31 influences the biological development of GC. Patients with high USP31 expression have a poor prognosis because USP31 is abundantly expressed in GC. Therefore, USP31 reduces the level of ubiquitination of the Wnt/β-catenin pathway by binding to β-catenin, thereby activating glycolysis, which ultimately promotes GC proliferation and aggressive metastasis. USP31 inhibits ubiquitination of β-catenin by binding to it, stimulates the Wnt/β-- catenin pathway, activates glycolysis, and accelerates the biology of GCs, which are all demonstrated in this work.

Discovery of potent small molecule ubiquitin-specific protease 10 inhibitors with anti-hepatocellular carcinoma activity through regulating YAP expression

High expression of ubiquitin-specific protease 10 (USP10) promote the proliferation of hepatocellular carcinoma (HCC), thus the development of USP10 inhibitors holds promise as a novel therapeutic approach for HCC treatment. However, the development of selective USP10 inhibitor is still limited. In this study, we developed a novel USP10 inhibitor for investigating the feasibility of targeting USP10 for the treatment of HCC. Due to high USP10 inhibition potency and prominent selectivity, compound D1 bearing quinolin-4(1H)-one scaffold was identified as a lead compound. Subsequent research revealed that D1 significantly inhibits cell proliferation and clone formation in HCC cells. Mechanistic insights indicated that D1 targets the ubiquitin pathway, facilitating the degradation of YAP (Yes-associated protein), thereby triggering the downregulation of p53 and its downstream protein p21. Ultimately, this cascade leads to S-phase arrest in HCC cells, followed by cell apoptosis. Collectively, our findings highlight D1 as a promising starting point for USP10-positive HCC treatment, underscoring its potential as a vital tool for unraveling the functional intricacies of USP10.

USP10 alleviates Nε-carboxymethyl-lysine-induced vascular calcification and atherogenesis in diabetes mellitus by promoting AMPK activation

Vascular calcification (VC) is a characteristic feature in patients with diabetes mellitus (DM) and is closely associated with the osteogenic differentiation of vascular smooth muscle cells (VSMCs). Ubiquitin-Specific Protease 10 (USP10) has been shown to regulate multiple cellular processes; however, its relationship with diabetic VC remains unclear. This study aims to elucidate the role of USP10 in VC development and the underlying regulatory mechanisms. Nε-carboxymethyl lysine (CML) was significantly increased in calcified ateries from diabetic atherosclerosis ApoE−/− mice fed with high-fat diets. CML downregulated USP10 expression in VSMCs and calcified mice coronary arteries, as assessd by Western blotting, RT-qPCR,immunofluorescence and immunohistochemistry. Loss-and gain-of-function experiments were conducted both in vitro and in vivo to verify the biological functions of USP10. Ectopic expression of USP10 mitigated the severity of VC. With regard to the mechanism, the interaction between USP10 and AMPKα was investigated through double-label immunofluorescence and Co-immunoprecipitation. In vitro ubiquitination assay revealed that USP10 was capable of mediating AMPKα ubiquitination and caused increased AMPKα phosphorylation level at Thr172. Moreover, the anticalcification effect of USP10 was reversed by pharmacological inhibition of AMPK signaling pathway. The current fundings suggest an important role of USP10 in diabetic VC progression, at least in part, via mediating the ubiquitination and activation of AMPKα. USP10 may serve as a novel therapeutic target for the treatment of diabetic VC.

Understanding the Interactions of Ubiquitin-Specific Protease 7 with Its Substrates through Molecular Dynamics Simulations: Insights into the Role of Its C-Terminal Domains in Substrate Recognition.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme that plays a critical role in regulating various cellular processes by cleaving ubiquitin molecules from target proteins. The C-terminal loop (CTL) motif is a specific region at the C-terminal end of the USP7 enzyme. Recent experiments suggest that the CTL motif plays a role in USP7's catalytic activity by contributing to the enzyme's structural stability, substrate recognition, and catalytic efficiency. The objective of this work is to elucidate these roles through the utilization of computational methods for molecular simulations. For this, we conducted extensive molecular dynamics (MD) simulations to investigate the conformational dynamics and protein-protein interactions within the USP7 enzyme-substrate complex with the substrate consisting of the ubiquitin tagged with the fluorescent label rhodamine 110-gly (Ub-Rho). Our results demonstrate that the CTL motif plays a crucial role in stabilizing the Ubl domains' conformation and augmenting the stability of active conformations within the enzyme-substrate complex. Conversely, the absence of the CTL motif results in increased flexibility and variability in Ubl domains' motion, leading to a reduced percentage of active conformations. Furthermore, our analysis of protein-protein interactions highlights the significance of the CTL motif in anchoring the Ubl45 domains to the catalytic domain (CD), thereby facilitating stable interactions with the substrate. Overall, our findings provide valuable insights into the conformational dynamics and protein-protein interactions inherent in the USP7 enzyme-substrate complex. These insights shed light on some mechanistic details of USP7 concerning the substrate's recognition before its catalytic action.

The regulatory role and mechanism of USP14 in endothelial cell pyroptosis induced by coronary heart disease.

The present study probes into the role and mechanism of ubiquitin specific peptidase 14 (USP14) in coronary heart disease (CHD)-triggered endothelial cell pyroptosis. An in vitro CHD model was established by inducing human coronary artery endothelial cells (HCAECs) with oxidized low-density lipoprotein (ox-LDL). HCAECs were transfected with si-USP14, followed by evaluation of cell viability by CCK-8 assay, detection of lactate dehydrogenase (LDH) activity by assay kit, detection of USP14, miR-15b-5p, NLRP3, GSDMD-N, and Cleaved-Caspase-1 expressions by qRT-PCR or Western blot, as well as IL-1β and IL-18 concentrations by ELISA. Co-IP confirmed the binding between USP14 and NLRP3. The ubiquitination level of NLRP3 in cells was measured after protease inhibitor MG132 treatment. Dual-luciferase reporter assay verified the targeting relationship between miR-15b-5p and USP14. USP14 and NLRP3 were highly expressed but miR-15b-5p was poorly expressed in ox-LDL-exposed HCAECs. USP14 silencing strengthened the viability of ox-LDL-exposed HCAECs, reduced the intracellular LDH activity, and diminished the NLRP3, GSDMD-N, Cleaved-Caspase-1, IL-1β, and IL-18 expressions. USP14 bound to NLRP3 protein and curbed its ubiquitination. Repression of NLRP3 ubiquitination counteracted the inhibitory effect of USP14 silencing on HCAEC pyroptosis. miR-15b-5p restrained USP14 transcription and protein expression. miR-15b-5p overexpression alleviated HCAEC pyroptosis by suppressing USP14/NLRP3. USP14 stabilizes NLRP3 protein expression through deubiquitination, thereby facilitating endothelial cell pyroptosis in CHD. miR-15b-5p restrains endothelial cell pyroptosis by targeting USP14 expression.

Ruthenium red alleviates post-resuscitation myocardial dysfunction by upregulating mitophagy through inhibition of USP33 in a cardiac arrest rat model

Cardiac arrest (CA) remains a leading cause of death, with suboptimal survival rates despite efforts involving cardiopulmonary resuscitation and advanced life-support technology. Post-resuscitation myocardial dysfunction (PRMD) is an important determinant of patient outcomes. Myocardial ischemia/reperfusion injury underlies this dysfunction. Previous reports have shown that ruthenium red (RR) has a protective effect against cardiac ischemia-reperfusion injury; however, its precise mechanism of action in PRMD remains unclear. This study investigated the effects of RR on PRMD and analyzed its underlying mechanisms. Ventricular fibrillation was induced in rats, which were then subjected to cardiopulmonary resuscitation to establish an experimental CA model. At the onset of return of spontaneous circulation, RR (2.5 mg/kg) was administered intraperitoneally. Our study showed that RR improved myocardial function and reduced the production of oxidative stress markers such as malondialdehyde (MDA), glutathione peroxidase (GSSG), and reactive oxygen species (ROS) production. RR also helped maintain mitochondrial structure and increased ATP and GTP levels. Additionally, RR effectively attenuated myocardial apoptosis. Furthermore, we observed downregulation of proteins closely related to mitophagy, including ubiquitin-specific protease 33 (USP33) and P62, whereas LC3B (microtubule-associated protein light chain 3B) was upregulated. The upregulation of mitophagy may play a critical role in reducing myocardial injury. These results demonstrate that RR may attenuate PRMD by promoting mitophagy through the inhibition of USP33. These effects are likely mediated through diverse mechanisms, including antioxidant activity, apoptosis suppression, and preservation of mitochondrial integrity and energy metabolism. Consequently, RR has emerged as a promising therapeutic approach for addressing post-resuscitation myocardial dysfunction.

Abstract PO2-24-04: Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer

Abstract Dysregulated estrogen receptor (ER) function is a key feature of 70% of breast cancers. In this setting ER primarily functions as a growth-controlling transcription factor driving pro-tumorigenic properties, thus there is a need to identify novel modulators of ER transcriptional activity as alternative therapeutic options. Previously, we showed a significant association between high ubiquitin specific protease 11 (USP11) expression and poor survival in ER+ breast cancer patients, but not in ER- patients, indicating a role for USP11 in ER+ subtypes. However, the precise role of USP11 in controlling ER function remains undescribed. To interrogate USP11 function in ER+ breast cancer we generated a CRISPR USP11 knockout MCF7 breast cancer cell line. Our results showed for the first time that USP11 knockout significantly reduces proliferation, induces G2 cell cycle arrest and apoptosis, abrogates ERα transcriptional activity, significantly impairs cell migration and invasion, whilst also impairing MCF7 cancer stem-cell like properties and malignant transformation in vitro. In addition, we also observed significant dysregulation of epithelial-mesenchymal transition-related markers including E-cadherin, N-cadherin, Snail and Slug. Using RNA-seq of MCF7 cells +/- estradiol, +/- stable USP11 knockdown we identified dysregulated pathways in protein binding, cell adhesion molecule binding and cell cycle regulation following USP11 modulation. Of note, gene expression analysis indicated NCOA3 as the most differentially expressed coding gene. Further validation confirmed a significant reduction in NCOA3- encoded Steroid receptor coactivator- 3 (SRC-3) protein expression following USP11 modulation with immunocytochemical analysis also indicating co-localisation within the nucleus. Frequently upregulated in breast cancer and associated with poor outcome, SRC-3 promotes ER transcriptional activity and co-activates a wide range of additional pro-tumorigenic transcription factors. Thus, SRC-3 modulation offers an attractive therapeutic target in breast cancer. This finding may present a novel role for USP11 in controlling SRC-3 and therefore associated ER transcriptional activity. We then aimed to explore the therapeutic potential of USP11. Although rare in primary breast cancer, activating mutations in the ER ligand-binding domain have recently been linked to recurrent, anti-endocrine resistant disease and metastasis. As such cancers remain dependent on ER-signalling for proliferation, but are resistant to ER targeted therapies, we identified USP11 as a potential novel therapeutic target. Using CRISPR knock-in ER mutant MCF7 cell lines expressing the four most common ER activating mutations, we demonstrated that USP11 knockdown continues to significantly reduce proliferation and abrogates mutant ERα transcriptional activity. We then explored amphipathic cell penetrating peptide RALA encapsulation as a delivery method for siRNA targeting USP11. Optimisation of nanoparticle delivery in the Y537S ER-mutant MCF7 breast cancer cell line achieved significant knockdown of USP11 and ER- related target genes including TFF1, PgR, GREB1 and PKIB in 2D with minimal associated cellular toxicity. To overcome the siRNA-related issues of rapid clearance upon systemic delivery, we then explored the use of a freeze-dried collagen-based scaffold model as an in vitro 3D RALA-encapsulated siRNA delivery system. Here we observed successful delivery of nanoparticle to MCF7 breast cancer cells, with a significant reduction in USP11 mRNA expression. Bio-compatible scaffold- facilitated delivery may therefore offer a more feasible translation of this siRNA-based therapeutic. Overall, this study highlights a novel role for USP11 in various oncogenic pathways. USP11 may therefore offer a novel therapeutic target to be exploited for the management of ER-positive breast cancer, including ER mutant breast cancer. Citation Format: Rachel Moore, Anna Blümel, Elspeth Ward, Elizabeth Sainsbury, Emer Conroy, Fergal O’Brien, Caroline Curtin, Simak Ali, Helen McCarthy, William Gallagher, Darran O'Connor. Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-04.

Ubiquitin-specific protease-7 promotes expression of airway mucin MUC5AC via the NF-κB signaling pathway

Chronic obstructive pulmonary disease (COPD) and other respiratory diseases frequently present with airway mucus hypersecretion, which not only affects the patient's quality of life but also poses a constant threat to their life expectancy. Ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme, affects cell differentiation, tissue growth, and disease development. However, its role in airway mucus hypersecretion induced by COPD remains elusive. In this study, USP7 expression was significantly upregulated in airway epithelial samples from patients with COPD, and USP7 was also overexpressed in mouse lung and human airway epithelial cells in models of airway mucus hypersecretion. Inhibition of USP7 reduced the expression of nuclear factor kappa B (NF-κB), phosphorylated-NF-κB (p-NF-κB), and phosphonated inhibitor of nuclear factor kappa B (p-IκBα), and alleviated the airway mucus hypersecretion in vivo and in vitro. Further research revealed that USP7 stimulated airway mucus hypersecretion through the activation of NF-κB nuclear translocation. In addition, the expression of mucin 5AC (MUC5AC) was suppressed by the NF-κB inhibitor erdosteine. These findings suggest that USP7 stimulates the NF-κB signaling pathway, which promotes airway mucus hypersecretion. This study identifies one of the mechanisms regulating airway mucus secretion and provides a new potential target for its prevention and treatment.

A long-term prognosis study of human USP8-mutated ACTH-secreting pituitary neuroendocrine tumours.

Somatic variants in the ubiquitin-specific protease 8 (USP8) gene are the most common genetic cause of Cushing disease. We aimed to explore the relationship between clinical outcomes and USP8 status in a single centre. We investigated the USP8 status in 48 patients with pituitary corticotroph tumours. A median of 62 months of follow-up was conducted after surgery from November 2013 to January 2015. The clinical, biochemical and imaging features were collected and analysed. Seven USP8 variants (p.Ser718Pro, p.Ser719del, p.Pro720Arg, p.Pro720Gln, p.Ser718del, p.Ser718Phe, p.Lys713Arg) were identified in 24 patients (50%). USP8 variants showed a female predominance (100% vs. 75% in wild type [WT], p = .022). Patients with p.Ser719del showed an older age at surgery compared to patients with the p.Pro720Arg variant (47- vs. 24-year-olds, p = .033). Patients with p.Pro720Arg showed a higher rate of macroadenoma compared to patients harbouring the p.Ser718Pro variant (60% vs. 0%, p = .037). No significant differences were observed in serum and urinary cortisol andadrenocorticotropin hormone (ACTH) levels. Immediate surgical remission (79% vs. 75%) and long-term hormone remission (79% vs. 67%) were not significantly different between the two groups. The recurrence rate was 21% (4/19) in patients harbouring USP8 variants and 13% (2/16) in WT patients. Recurrence-free survival presented a tendency to be shorter in USP8-mutated individuals (76.7 vs. 109.2 months, p = .068). Somatic USP8 variants accounted for 50% of the genetic causes in this cohort with a significant female frequency. A long-term follow-up revealed a tendency toward shorter recurrence-free survival in USP8-mutant patients.

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

USP3 inhibition is Active Against Chemo-resistant Hepatocellular Carcinoma Anchorage-independent Growth via Suppressing Wnt/β-catenin.

USPs are a family of enzymes that regulate protein degradation, and their dysregulation has been implicated in the development and progression of cancer. This study aimed to determine whether ubiquitin-specific proteases 3 (USP3) could be a potential target for therapy in hepatocellular carcinoma (HCC), particularly in resistant HCC. This study systematically investigated the role of USP3 in HCC, with a focus on chemo-resistant HCC cells. The level of USP3 from clinical samples was measured using an ELISA assay. Cell proliferation, apoptosis, migration, and anchorage-independent colony formation assays were performed. Transfection was performed to knock down USP3 expression and measure β-catenin activity, and real-time PCR was used to measure levels of MYC and CYCLIN D1 genes. USP3 protein was upregulated in HCC tissues, but its upregulation was not associated with clinicopathology. USP3 knockdown had a similar inhibitory effect on growth in both sensitive and resistant HCC cells, did not affect migration, and induced apoptosis in sensitive but not resistant HCC cells. Furthermore, USP3 knockdown was more effective in suppressing anchorage-independent colony formation in chemoresistant HCC cells compared to their chemo-sensitive counterparts. Pearson correlation coefficient analysis revealed a strong positive correlation between USP3 and CTNNB1, and consistently, USP3 knockdown reduced the levels and activities of β-catenin in HCC cells. Using a Wnt activator (lithium) in rescue studies significantly reversed the inhibitory effects of USP3 knockdown. The findings suggest that inhibiting USP3 is an effective strategy against cancer stem cells and chemo-resistant HCC cells.

Stress-induced epinephrine promotes hepatocellular carcinoma progression via the USP10-PLAGL2 signaling loop

Hepatocellular carcinoma (HCC) is associated with a poor prognosis. Our previous study demonstrated that Pleomorphic adenoma gene like-2 (PLAGL2) was a potential therapeutic target in HCC. However, the mechanisms that lead to the upregulation of PLAGL2 in HCC remain unclear. The present study revealed that stress-induced epinephrine increased the expression of PLAGL2, thereby promoting the progression of HCC. Furthermore, PLAGL2 knockdown inhibited epinephrine-induced HCC development. Mechanistically, epinephrine upregulated ubiquitin-specific protease 10 (USP10) to stabilize PLAGL2 via the adrenergic β-receptor-2-c-Myc (ADRB2-c-Myc) axis. Furthermore, PLAGL2 acted as a transcriptional regulator of USP10, forming a signaling loop. Taken together, these results reveal that stress-induced epinephrine activates the PLAGL2-USP10 signaling loop to enhance HCC progression. Furthermore, PLAGL2 plays a crucial role in psychological stress-mediated promotion of HCC progression.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

Preclinical testing of CT1113, a novel USP28 inhibitor, for the treatment of T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

Mogrol-mediated enhancement of radiotherapy sensitivity in non-small cell lung cancer: a mechanistic study.

This study investigated mogrol's impact on non-small cell lung cancer (NSCLC) radiosensitivity and underlying mechanisms, using various methods including assays, bioinformatics, and xenograft models. CCK-8, clonogenic, flow cytometry, TUNEL, and Western blot assays evaluated mogrol and radiation effects on NSCLC viability and apoptosis. Ubiquitin-specific protease 22 (USP22) expression in NSCLC patient tissues was determined by RT-qPCR and Western blot. A xenograft model validated mogrol's effects on tumor growth. Bioinformatics identified four ubiquitin-specific proteases, including USP22, in NSCLC. Kaplan-Meier analysis confirmed USP22's value in lung cancer survival. Human Protein Atlas (HPA) database analysis indicated higher USP22 expression in lung cancer tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis implicated ERK1/2 in NSCLC progression, and molecular docking showed stability between mogrol and ERK1/2. Further in vivo and in vitro experiments have demonstrated that mogrol enhances the inhibitory effect of radiation on NSCLC cell viability and clonogenic capacity. Cell viability and clonogenic capacity are reduced by >50%, and an increase in cellular apoptosis is observed, with apoptotic levels reaching 10%. USP22 expression was significantly elevated in NSCLC tissues, particularly in radiotherapy-resistant patients. Mogrol downregulated USP22 expression by inhibiting the ERK/CREB pathway, lowering COX2 expression. Mogrol also enhanced radiation's inhibition of tumor growth in mice. Mogrol enhances NSCLC radiosensitivity by downregulating USP22 via the ERK/CREB pathway, leading to reduced COX2 expression.NEW & NOTEWORTHY Mogrol enhances non-small cell lung cancer (NSCLC) cell sensitivity to radiotherapy by downregulating USP22 through the ERK/CREB pathway, reducing COX2 expression. These findings highlight mogrol's potential as an adjunct to improve NSCLC radiotherapy and open avenues for further research and clinical applications.

Fusion of Platelet Derived Growth Factor Receptor Alpha (PDGFRA) With Ubiquitin Specific Peptidase 8 (USP8) in a Calcified Chondroid Mesenchymal Neoplasm Harboring t(4;15)(q12;q21) as a Sole Aberration.

The term "calcified chondroid mesenchymal neoplasm" was introduced in 2021 to describe a group of tumors characterized by various morphological features, including the formation of cartilage or chondroid matrix. These tumors frequently carry chimeric genes where the 5'-end partner gene is fibronectin 1 and the 3'-end partner gene codes for receptor tyrosine kinase. Our study explores fusion of the genes platelet-derived growth factor receptor alpha (PDGFRA) and ubiquitin-specific peptidase 8 (USP8) in calcified chondroid mesenchymal neoplasm. Genetic investigations were conducted on a tumor located in the leg of a 71-year-old woman. G-banding analysis of short-term cultured tumor cells revealed the karyotype 46,XX,t(4;15)(q12;q21)[6]/46,XX[4]. RNA sequencing detected in-frame PDGFRA::USP8 and USP8::PDGFRA chimeric transcripts, which were validated by RT-PCR/Sanger sequencing. The PDGFRA::USP8 chimeric protein is predicted to have cell membrane location and functions as a chimeric ubiquitinyl hydrolase. The USP8::PDGFRA protein was predicted to be nuclear and function as a positive regulator of cellular metabolic process. We report, for the first time, a calcified chondroid mesenchymal neoplasm carrying a balanced t(4;15)(q12;q21) chromosomal translocation, resulting in the generation of both PDGFRA::USP8 and USP8::PDGFRA chimeras. The PDGFRA::USP8 protein is located on the cell membrane and functions as a chimeric ubiquitinyl hydrolase, activated by PDGFs. Conversely, USP8::PDGFRA is a nuclear protein regulating metabolic processes.

20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

Bladder cancer is one of the most common malignancies of the urinary tract and a prevalent cancer worldwide, still requiring efficient therapeutic agents and approaches. 20-Hydroxyecdysone (20-HE), a steroid hormone, can be found in insects and few plants and mediate numerous biological events to control the progression of varying diseases; however, its impacts on bladder cancer remain unclear. In the study, we found that 20-HE treatments effectively inhibited the viability and proliferation of bladder cancer cells and induced apoptosis by activating Caspase-3. The migratory and invasive potential of bladder cancer cells was markedly repressed by 20-HE in a dose-dependent manner. The inhibitory effects of 20-HE on bladder cancer were confirmed in an established xenograft mouse model, as indicated by the markedly reduced tumor growth rates and limited lung and lymph node metastasis. High-throughput RNA sequencing was performed to explore dysregulated genes in bladder cancer cells after 20-HE treatment. We identified ubiquitin-specific protease 21 (USP21) as a key deubiquitinating enzyme for bladder cancer progression and a positive correlation between USP21 and nuclear factor-κB (NF-κB)/p65 in patients. Furthermore, 20-HE treatments markedly reduced USP21 expression, NF-κB/p65 mRNA, stability and phosphorylated NF-κB/p65 expression levels in bladder cancer cells, which were validated in animal tumor tissues. Mechanistic studies showed that USP21 directly interacted with and stabilized p65 by deubiquitinating its K48-linked polyubiquitination in bladder cancer cells, which could be abolished by 20-HE treatment, contributing to p65 degradation. Finally, we found that USP21 overexpression could not only facilitate the proliferation, migration, and invasion of bladder cancer cells, but also significantly eliminated the suppressive effects of 20-HE on bladder cancer. Notably, 20-HE could still perform its anti-tumor role in bladder cancer when USP21 was knocked down with decreased NF-κB/p65 expression and activation, revealing that USP21 suppression might not be the only way for 20-HE during bladder cancer treatment. Collectively, all our results clearly demonstrated that 20-HE may function as a promising therapeutic strategy for bladder cancer treatment mainly through reducing USP21/p65 signaling expression.

USP3: Key deubiquitylation enzyme in human diseases.

Ubiquitination and deubiquitylation are pivotal posttranslational modifications essential for regulating cellular protein homeostasis and are implicated in the development of human diseases. Ubiquitin-specific protease 3 (USP3), a member of the ubiquitin-specific protease family, serves as a key deubiquitylation enzyme, playing a critical role in diverse cellular processes including the DNA damage response, cell cycle regulation, carcinogenesis, tumor cell proliferation, migration, and invasion. Despite notable research efforts, our current understanding of the intricate and context-dependent regulatory networks governing USP3 remains incomplete. This review aims to comprehensively synthesize existing published works on USP3, elucidating its multifaceted roles, functions, and regulatory mechanisms, while offering insights for future investigations. By delving into the complexities of USP3, this review strives to provide a foundation for a more nuanced understanding of its specific roles in various cellular processes. Furthermore, the exploration of USP3's regulatory networks may uncover novel therapeutic strategies targeting this enzyme in diverse human diseases, thereby holding promising clinical implications. Overall, an in-depth comprehension of USP3's functions and regulatory pathways is crucial for advancing our knowledge and developing targeted therapeutic approaches for human diseases.

Recurrent USP6 rearrangement in a subset of atypical myofibroblastic tumours of the soft tissues: low-grade myofibroblastic sarcoma or atypical/malignant nodular fasciitis?

Low-grade myofibroblastic sarcoma (LGMS) is a rarely metastasizing myofibroblastic tumour mostly affecting extremities and the head and neck of adults. Histologically, it shows long infiltrative fascicles of spindle cells with moderate nuclear atypia. By immunohistochemistry, it stains positive for smooth muscle actin (SMA) and sometimes for desmin. To date, no recurrent genetic abnormalities have been described. Ubiquitin-specific peptidase 6 (USP6) gene rearrangement is typically found in some benign bone and soft-tissue tumours including nodular fasciitis (NF), among others. Nevertheless, rare cases of USP6-rearranged tumours resembling NF with atypical features have been reported. One index case of LGMS of the deltoid in a 56-year-old man presented the THBS2::USP6 translocation by RNA sequencing (Archer FusionPlex Sarcoma v2 panel). Further screening of 11 cases of LGMS using fluorescent in situ hybridization (FISH) analysis with a USP6 break-apart probe identified two additional cases. These cases were investigated with RNA-sequencing, and a RRBP1::USP6 translocation was detected in one. The other case was not assessable because of low-quality RNA. Noteworthy, rearranged LGMSs presented distinctive features including variable multinodular/plexiform architecture, prominent vasculature with occasional wall thickening, scattered osteoclast-like multinucleated giant cells, and peripheral lymphoid aggregates. Our findings support the notion that among soft-tissue neoplasms with fibroblastic/myofibroblastic phenotype, USP6 rearrangement is not limited to benign tumours, and warrants further investigation of genetic changes in myofibroblastic sarcomas.