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Methyl‐CpG binding protein 2 (MeCP2) is an important X‐linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss‐of‐function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia–reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin‐6 (IL‐6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48‐linked ubiquitination from MeCP2 prevented its proteasome‐dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin‐specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia‐induced MeCP2 degradation by cleaving lysine 48‐linked ubiquitin chains from MeCP2, primarily targeting its C‐terminal domain. Consistently, USP15 inhibited hypoxia‐induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL‐6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL‐6 signaling. [ABSTRACT FROM AUTHOR]

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML. [ABSTRACT FROM AUTHOR]

Programmed cell death ligand-1 (PD-L1) is a T cell inhibitory immune checkpoint molecule that interacts with programmed cell death-1 (PD-1) to promote immune escape of tumor cells. Compared with antibody therapies, small molecule drugs show better prospects due to their advantages such as higher bioavailability, better tissue penetration, and reduced risk of immunogenicity. Here, we found that the small molecule demethylzeylasteral (Dem) can significantly downregulate the expression of PD-L1 in colorectal cancer cells and enhance the killing effect of T cells on tumor cells. Mechanistically, Dem binds to the deubiquitinating enzyme USP22 and promotes its degradation, resulting in increased ubiquitination and degradation of PD-L1 through the proteasome pathway. In addition, Dem increased the activity of cytotoxic T cells and reduced the number of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in tumor-infiltrating lymphocytes (TILs), thereby activating the tumor immune microenvironment and inhibiting the growth of subcutaneous MC38 tumors in C57BL/6 mice. Moreover, we also found that the combination of Dem and CTLA4 antibodies can further improve the efficacy of antitumor therapy. Our study reveals the mechanism by which Dem promotes PD-L1 degradation and suggests that the combination of Dem and CTLA4 antibodies may improve the efficacy of immunotherapy. Proposed model underlying the role of Dem in promoting PD-L1 degradation in colorectal cancer. Dem specifically binds to and promotes USP22 degradation, which increases the ubiquitination of PD-L1 and leads to its degradation through the proteasome, thereby enhancing T cell activity and activating the tumor immune microenvironment. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Long-term accumulation of misfolded proteins leads to endoplasmic reticulum (ER) stress in colorectal cancer (CRC). However, the precise pathways controlling the decision between survival and apoptosis in CRC are unclear. Therefore, in this study, we investigated the function and molecular mechanism of glucosidase I (GCS1) in regulating ER stress in CRC. Methods: A public database was used to confirm the expression level of GCS1 in CRC and normal tissues. Clinical samples from our center were used to confirm the mRNA and protein expression levels of GCS1. Cell proliferation, migration, invasion, and apoptosis assays revealed the biological role of GCS1. Immunohistochemical techniques were used to evaluate the expression of key proteins in subcutaneous implanted tumors in nude mice, which provided further evidence for the biological function of GCS1 in promoting cancer in vivo. The results of coimmunoprecipitation-mass spectrometry analysis and immunofluorescence colocalization analysis the interaction between GCS1 and GRP78. In addition, the mechanism of action of USP10, GRP78, and GCS1 at the post- translational level was investigated. Finally, a tissue microarray was used to examine the connection between GCS1 and GRP78 expression and intracellular localization of these proteins using immunohistochemistry and immunofluorescence. Results: The experimental results revealed that GCS1 was substantially expressed in CRC, with higher expression indicating a worse prognosis. Thus, GCS1 can enhance the proliferation and metastasis while inhibiting the apoptosis of CRC cells both in vivo and in vitro. Mechanistically, GCS1 binds to GRP78, recruits USP10 for deubiquitination of GRP78 to promote its degradation, and decreases ER stress-mediated apoptosis, increasing CRC cell proliferation and metastasis. Conclusions: In summary, GCS1 stimulates CRC growth and migration and reduces ER stress-mediated apoptosis via USP10-mediated deubiquitination of GRP78. Our findings identify a possible therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles. Synopsis: Hypoxia-inducible transcription Factors (HIFs) are central to the metazoan response to oxygen. This work reveals that USP43, a deubiquitinating enzyme (DUB), is required for activation of the HIF-1 response by facilitating HIF-1 nuclear accumulation. CRISPR/Cas9 mutagenesis screens uncover DUBs involved in HIF regulation and identify USP43 as a DUB required for activation of a HIF-1 response. USP43 associates with HIF-1α through the hypoxia-sensitive recruitment of 14-3-3 proteins and increases the nuclear accumulation of HIF-1α within minutes of hypoxia. USP43 is a catalytically active DUB, but its enzymatic activity is not essential for accumulation of HIF-1 on chromatin. The association of USP43 with 14-3-3 proteins and HIF-1 is dependent on hypoxia-induced serine phosphorylation of USP43. USP43 promotes HIF-1 nuclear accumulation by forming a complex with HIF-1alpha and 14-3-3 proteins in low oxygen. [ABSTRACT FROM AUTHOR]

Background: Lung adenocarcinoma (LUAD) is a highly aggressive and rapidly fatal malignancy worldwide. Collagen XVII (COL17A1) has been implicated in various protumorigenic processes. However, the functions and mechanisms of COL17A1 in LUAD progression still remain elusive. Methods: COL17A1 and ubiquitin‐specific protease 22 (USP22) mRNA analysis was performed by quantitative PCR, and their protein levels were detected by immunoblotting and immunohistochemistry. The functional influence was evaluated by determining cell viability, proliferation, apoptosis, invasion, migration, and ferroptosis in vitro, as well as xenograft growth in vivo. Co‐immunoprecipitation (Co‐IP) and IP experiments were used to examine the USP22/COL17A1 interaction and COL17A1 deubiquitination. Cycloheximide treatment was used to analyze COL17A1 protein stability. Results: COL17A1 and USP22 were upregulated in human LUAD tissues and cell lines. Functionally, COL17A1 knockdown acted for the suppression of LUAD cell growth, invasion, and migration as well as promotion of cell apoptosis and ferroptosis in vitro. COL17A1 knockdown could diminish the tumorigenicity of LUAD cells in vivo. Mechanistically, USP22 stabilized and upregulated COL17A1 by enhancing the deubiquitination of COL17A1. Additionally, reexpression of COL17A1 could reverse USP22 silencing‐induced phenotype changes of LUAD cells in vitro. Conclusion: Our findings demonstrate that USP22‐stabilized COL17A1 possesses oncogenic activity in LUAD. We propose that USP22 and COL17A1 would be potential targets for the establishment of therapeutic approaches against LUAD. [ABSTRACT FROM AUTHOR]

Ubiquitin‐specific peptidase 15 (USP15), a critical deubiquitinating enzyme, has been demonstrated to improve substrate stabilization by hydrolyzing the bond between the substrate and ubiquitin, and is implicated in multiple carcinogenic processes. Prompted by the information cited from The Cancer Genome Atlas (TCGA) database and the Cancer Proteogenomic Data Analysis Site (cProSite), USP15 is selectively overexpressed in clear cell renal cell carcinoma (ccRCC) samples. We aimed to investigate the function of USP15 on ccRCC malignant features, which was emphasized in its deubiquitination of SHC adaptor protein 1 (SHC1). The overexpression of USP15 promoted the capacity of proliferation, migration, and invasion in ccRCC CAKI1 and 769‐P cells, and these malignant biological properties were diminished by USP15 deletion in 786‐O cells. USP15 accelerated tumor growth and lung metastasis in vivo. In addition, deubiquitinase USP15 was further identified as a new protector for SHC1 from degradation by the ubiquitination pathway, the post‐translational modification. In sequence, transcription factor activating enhancer binding protein 4 (TFAP4) was shown to be partly responsible for USP15 expression at the level of transcription, as manifested by the chromatin immunoprecipitation and pull‐down assay. Based on the in vitro and in vivo data, we postulate that USP15 regulated by TFAP4 transcriptionally deteriorates ccRCC malignant biological properties via stabilizing SHC1 by deubiquitination. [ABSTRACT FROM AUTHOR]

Programmed cell death ligand-1 (PD-L1) is a T cell inhibitory immune checkpoint molecule that interacts with programmed cell death-1 (PD-1) to promote immune escape of tumor cells. Compared with antibody therapies, small molecule drugs show better prospects due to their advantages such as higher bioavailability, better tissue penetration, and reduced risk of immunogenicity. Here, we found that the small molecule demethylzeylasteral (Dem) can significantly downregulate the expression of PD-L1 in colorectal cancer cells and enhance the killing effect of T cells on tumor cells. Mechanistically, Dem binds to the deubiquitinating enzyme USP22 and promotes its degradation, resulting in increased ubiquitination and degradation of PD-L1 through the proteasome pathway. In addition, Dem increased the activity of cytotoxic T cells and reduced the number of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in tumor-infiltrating lymphocytes (TILs), thereby activating the tumor immune microenvironment and inhibiting the growth of subcutaneous MC38 tumors in C57BL/6 mice. Moreover, we also found that the combination of Dem and CTLA4 antibodies can further improve the efficacy of antitumor therapy. Our study reveals the mechanism by which Dem promotes PD-L1 degradation and suggests that the combination of Dem and CTLA4 antibodies may improve the efficacy of immunotherapy.

Abstract Background Hydrogen sulfide (H2S), the third gasotransmitter discovered, regulates a variety of physiological functions. Whether H2S alleviates skeletal muscle ageing by regulating autophagy has not been reported. Methods Mice were administered 150 mg/kg/day of D‐galactose (D‐gal), and C2C12 myotubes were cultured in 20 g/L D‐gal to induce ageing. Sodium hydrosulfide (NaHS) was employed as an exogenous donor in the treatment group. The intracellular concentration of H2S was quantified by the 7‐azido‐4‐methylcoumarin fluorescence probe. The proteins involved in the ubiquitin‐mediated degradation of AMPKα1 were detected by liquid chromatography tandem mass spectrometry (LC–MS/MS) and co‐immunoprecipitation (Co‐IP). S‐sulfhydration of USP5 was tested by a biotin‐switch assay. Associated proteins were analysed by western blot. Results NaHS was found to effectively restore the H2S content in both ageing gastrocnemius (+91.89%, P

Abstract Background Long-term accumulation of misfolded proteins leads to endoplasmic reticulum (ER) stress in colorectal cancer (CRC). However, the precise pathways controlling the decision between survival and apoptosis in CRC are unclear. Therefore, in this study, we investigated the function and molecular mechanism of glucosidase I (GCS1) in regulating ER stress in CRC. Methods A public database was used to confirm the expression level of GCS1 in CRC and normal tissues. Clinical samples from our center were used to confirm the mRNA and protein expression levels of GCS1. Cell proliferation, migration, invasion, and apoptosis assays revealed the biological role of GCS1. Immunohistochemical techniques were used to evaluate the expression of key proteins in subcutaneous implanted tumors in nude mice, which provided further evidence for the biological function of GCS1 in promoting cancer in vivo. The results of coimmunoprecipitation-mass spectrometry analysis and immunofluorescence colocalization analysis the interaction between GCS1 and GRP78. In addition, the mechanism of action of USP10, GRP78, and GCS1 at the post- translational level was investigated. Finally, a tissue microarray was used to examine the connection between GCS1 and GRP78 expression and intracellular localization of these proteins using immunohistochemistry and immunofluorescence. Results The experimental results revealed that GCS1 was substantially expressed in CRC, with higher expression indicating a worse prognosis. Thus, GCS1 can enhance the proliferation and metastasis while inhibiting the apoptosis of CRC cells both in vivo and in vitro. Mechanistically, GCS1 binds to GRP78, recruits USP10 for deubiquitination of GRP78 to promote its degradation, and decreases ER stress-mediated apoptosis, increasing CRC cell proliferation and metastasis. Conclusions In summary, GCS1 stimulates CRC growth and migration and reduces ER stress-mediated apoptosis via USP10-mediated deubiquitination of GRP78. Our findings identify a possible therapeutic target for CRC.

Abstract Background MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. Methods The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. Results Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. Conclusion USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells.

Abstract Breast cancer is the most common female malignancy worldwide. Ubiquitin-specific peptidase 53 (USP53) has been shown to exert cancer-suppressing functions in several solid tumors, but its role and the underlying mechanism in breast cancer has not been clearly elucidated. Therefore, we have carried out a series of detailed studies on this matter at the levels of bioinformatics, clinical tissue, cell function and animal model. We found that USP53 expression was downregulated in breast cancer specimens and was negatively correlated with the clinical stages. Gain- and loss-of-function experiments demonstrated USP53 inhibited proliferation, clonogenesis, cell cycle and xenograft growth, as well as induced apoptosis and mitochondrial damage of breast cancer cells. Co-immunoprecipitation data suggested that USP53 interacted with zinc finger MYND-type containing 11 (ZMYND11), and catalyzed its deubiquitination and stabilization. The 33–50 amino acid Cys-box domain was key for USP53 enzyme activity, but not essential for its binding with ZMYND11. The rescue experiments revealed that the anti-tumor role of USP53 in breast cancer cells was at least partially mediated by ZMYND11. Both USP53 and ZMYND11 were prognostic protective factors for breast cancer. USP53-ZMYND11 axis may be a good potential biomarker or therapeutic target for breast cancer, which can provide novel insights into the diagnosis, treatment and prognosis.

Abstract Background Hepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood. Methods The binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models. Results UCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells. Conclusion Our findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC.

Abstract Background Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported. Methods Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2. Results Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS. Conclusion Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels.

Background: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. Methods: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. Results: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. Conclusion: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common female malignancy worldwide. Ubiquitin-specific peptidase 53 (USP53) has been shown to exert cancer-suppressing functions in several solid tumors, but its role and the underlying mechanism in breast cancer has not been clearly elucidated. Therefore, we have carried out a series of detailed studies on this matter at the levels of bioinformatics, clinical tissue, cell function and animal model. We found that USP53 expression was downregulated in breast cancer specimens and was negatively correlated with the clinical stages. Gain- and loss-of-function experiments demonstrated USP53 inhibited proliferation, clonogenesis, cell cycle and xenograft growth, as well as induced apoptosis and mitochondrial damage of breast cancer cells. Co-immunoprecipitation data suggested that USP53 interacted with zinc finger MYND-type containing 11 (ZMYND11), and catalyzed its deubiquitination and stabilization. The 33–50 amino acid Cys-box domain was key for USP53 enzyme activity, but not essential for its binding with ZMYND11. The rescue experiments revealed that the anti-tumor role of USP53 in breast cancer cells was at least partially mediated by ZMYND11. Both USP53 and ZMYND11 were prognostic protective factors for breast cancer. USP53-ZMYND11 axis may be a good potential biomarker or therapeutic target for breast cancer, which can provide novel insights into the diagnosis, treatment and prognosis. Keypoints: 1. USP53 has been shown to play an anticancer role in a variety of solid malignancies, but its role in breast cancer and its mechanism remain unclear. 2. Our results suggested that USP53 was low expressed in breast cancer and negatively correlated with TNM stage; USP53 suppressed proliferation and triggered apoptosis of breast cancer cells; USP53 inhibited breast cancer in vitro and in vivo by deubiquitinating ZMYND11. 3. USP53-ZMYND11 axis may be a potential breast cancer marker and therapeutic target. [ABSTRACT FROM AUTHOR]

Myricetin, a natural flavonoid found in various foods, was investigated for its antiviral effect against transmissible gastroenteritis virus (TGEV). This a-coronavirus causes significant economic losses in the global swine industry. The study focused on the papain-like protease (PLpro), which plays a crucial role in coronavirus immune evasion by mediating deubiquitination. Targeting PLpro could potentially disrupt viral replication and enhance antiviral responses. The results demonstrated that myricetin effectively inhibited TGEV-induced cytopathic effects in a dose-dependent manner, with an EC50 value of 31.19 mM. Myricetin significantly reduced TGEV viral load within 48 h after an 8-h co-incubation period. Further investigations revealed that myricetin at a concentration of 100 mM directly inactivated TGEV and suppressed its intracellular replication stage. Moreover, pretreatment with 100 mM myricetin conferred a protective effect on PK-15 cells against TGEV infection. Myricetin competitively inhibited PLpro with an IC50 value of 6.563 mM. Molecular docking experiments show that myricetin binds to the Cys102 residue of PLpro through conventional hydrogen bonds, Pi-sulfur, and Pi-alkyl interactions. This binding was confirmed through site-directed mutagenesis experiments, indicating myricetin as a potential candidate for preventing and treating TGEV infection. [ABSTRACT FROM AUTHOR]

Ubiquitination is a highly conserved and dynamic post-translational modification in which protein substrates are modified by ubiquitin to influence their activity, localization, or stability. Deubiquitination enzymes (DUBs) counter ubiquitin signaling by removing ubiquitin from the substrates. Ubiquitin-specific proteases (UBPs), the largest subfamily of DUBs, are conserved in plants, serving diverse functions across various cellular processes, although members within the same group often exhibit functional redundancy. Here, we briefly review recent advances in understanding the biological roles of UBPs, particularly the molecular mechanism by which UBPs regulate plant development and growth, morphogenesis, and stress response, which sheds light on the mechanistic roles of deubiquitination in plants. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood. Methods: The binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models. Results: UCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells. Conclusion: Our findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non–small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. Methods: The detection of mRNA and protein of MRPL35, ubiquitin‐specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT‐PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α‐ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis. Results: MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. Conclusion: MRPL35 expression was stabilized by USP39‐induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC. [ABSTRACT FROM AUTHOR]

Background: Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported. Methods: Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2. Results: Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS. Conclusion: Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels. [ABSTRACT FROM AUTHOR]

Hepatic ischemia/reperfusion (I/R) injury is an important cause of liver function impairment following liver surgery. The ubiquitin-proteasome system (UPS) plays a crucial role in protein quality control and has substantial impact on the hepatic I/R process. Although OTU deubiquitinase 1 (OTUD1) is involved in diverse biological processes, its specific functional implications in hepatic I/R are not yet fully understood. This study demonstrates that OTUD1 alleviates oxidative stress, apoptosis, and inflammation induced by hepatic I/R injury. Mechanistically, OTUD1 deubiquitinates and activates nuclear factor erythroid 2-related factor 2 (NRF2) through its catalytic site cysteine 320 residue and ETGE motif, thereby attenuating hepatic I/R injury. Additionally, administration of a short peptide containing the ETGE motif significantly mitigates hepatic I/R injury in mice. Overall, our study elucidates the mechanism and role of OTUD1 in ameliorating hepatic I/R injury, providing a theoretical basis for potential treatment using ETGE-peptide.

ABSTRACT Background Lung adenocarcinoma (LUAD) is a highly aggressive and rapidly fatal malignancy worldwide. Collagen XVII (COL17A1) has been implicated in various protumorigenic processes. However, the functions and mechanisms of COL17A1 in LUAD progression still remain elusive. Methods COL17A1 and ubiquitin‐specific protease 22 (USP22) mRNA analysis was performed by quantitative PCR, and their protein levels were detected by immunoblotting and immunohistochemistry. The functional influence was evaluated by determining cell viability, proliferation, apoptosis, invasion, migration, and ferroptosis in vitro, as well as xenograft growth in vivo. Co‐immunoprecipitation (Co‐IP) and IP experiments were used to examine the USP22/COL17A1 interaction and COL17A1 deubiquitination. Cycloheximide treatment was used to analyze COL17A1 protein stability. Results COL17A1 and USP22 were upregulated in human LUAD tissues and cell lines. Functionally, COL17A1 knockdown acted for the suppression of LUAD cell growth, invasion, and migration as well as promotion of cell apoptosis and ferroptosis in vitro. COL17A1 knockdown could diminish the tumorigenicity of LUAD cells in vivo. Mechanistically, USP22 stabilized and upregulated COL17A1 by enhancing the deubiquitination of COL17A1. Additionally, reexpression of COL17A1 could reverse USP22 silencing‐induced phenotype changes of LUAD cells in vitro. Conclusion Our findings demonstrate that USP22‐stabilized COL17A1 possesses oncogenic activity in LUAD. We propose that USP22 and COL17A1 would be potential targets for the establishment of therapeutic approaches against LUAD.

Abstract Aberrant upregulation of the ubiquitin-specific protease 14 (USP14) has been found in some malignant tumors, including oral squamous cell carcinoma (OSCC). In this study, we further demonstrated that aberrantly overexpressed USP14 was also closely related to adverse clinicopathological features and poor prognosis in patients with OSCC, so we hypothesized that USP14 might act as a tumor-promoting factor during the progression of OSCC. Notably, we originally proved that USP14 is a deubiquitinating enzyme for phosphofructokinase-1 liver type (PFKL), a key rate-limiting enzyme involved in the glycolytic pathway. USP14 interacts with PFKL and enhances its stability through deubiquitination in OSCC cells, which in turn enhances PFKL-mediated glycolytic metabolism and ultimately promote cellular proliferation, migration, and tumorigenesis. In this work, we have also demonstrated for the first time that USP14 is a critical regulator of glycolysis in OSCC and verified a novel mechanism whereby it is involved in tumor metastasis and growth. Collectively, our findings provide novel insights into the tumor-promoting role of USP14 and establish mechanistic foundations for USP14-targeting therapies.

ABSTRACT: The development and maturation of follicles are intricately linked to egg production and reproductive performance of chickens. Granulosa cells death directly affects the development and maturation of follicles, thereby impacting the reproductive performance of hens. Ferroptosis is a new type of cell death, it is unknown how it affects the growth and development of chicken follicles. In this study, RNA-seq analysis revealed significant differences in the expression of ferroptosis-related genes between normal follicles and atretic follicles, suggesting a potential role for ferroptosis in follicle growth and development. In addition, we found that ubiquitin-specific protease 13 (USP13) was significantly upregulated in atrophic follicles. Overexpression of USP13 results in depletion of glutathione (GSH), peroxidation of lipids, accumulation of iron, and activation of ferroptosis in chicken granulosa cells. In contrast, USP13 knockdown significantly inhibited ferroptosis events. Mechanistically, USP13 prevents the degradation of autophagy related 7 (ATG7) by deubiquitinating it, thereby enhancing the stability of ATG7 protein and ultimately promoting ferroptosis. In conclusion, this study elucidates the crucial role of the USP13-ATG7 axis in regulating ferroptosis in chicken follicle granulosa cells, thereby presenting a novel avenue for molecular breeding research in chickens.

Ovarian cancer is a common cancer for females, and the incidence and mortality rates are on the rise. Many treatment strategies have been developed for ovarian cancer, including chemotherapy and immunotherapy, but they are often ineffective and prone to drug resistance. Protein ubiquitination is an important class of post-translation modifications that have been found to be associated with various human diseases and cancer development. Recent studies have revealed that protein ubiquitination is involved in the progression of ovarian cancer and plays an important role in the tumor immune process. Moreover, the combination of ubiquitinase/deubiquitinase inhibitors and cancer immunotherapy approaches can effectively reduce treatment resistance and improve treatment efficacy, which provides new ideas for cancer treatment. Herein, we review the role of protein ubiquitination in relation to ovarian cancer immunotherapy and recent advances in the use of ubiquitinase/deubiquitinase inhibitors in combination with cancer immunotherapy.

Objective: To elucidate the relationship between USP19 and O(6)‐methylguanine‐DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. Methods: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. Results: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. Conclusion: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response. [ABSTRACT FROM AUTHOR]

Fms‐like tyrosine kinase‐3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal‐tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3‐ITD and FLT3‐TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3‐ITD or ‐TKD differently. We found that BRCA1/BRCA2‐containing complex subunit 36 (BRCC36), a specific K63‐linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3‐ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3‐ITD, which may shed light on developing a novel therapeutic approach for AML. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) are multifactorial chronic diseases and have the highest rates of morbidity and mortality worldwide. The ubiquitin-proteasome system (UPS) plays a crucial role in posttranslational modification and quality control of proteins, maintaining intracellular homeostasis via degradation of misfolded, short-lived, or nonfunctional regulatory proteins. Noncoding RNAs (ncRNAs, such as microRNAs, long noncoding RNAs, circular RNAs and small interfering RNAs) serve as epigenetic factors and directly or indirectly participate in various physiological and pathological processes. NcRNAs that regulate ubiquitination or are regulated by the UPS are involved in the execution of target protein stability. The cross-linked relationship between the UPS, ncRNAs and CVDs has drawn researchers' attention. Herein, we provide an update on recent developments and perspectives on how the crosstalk of the UPS and ncRNAs affects the pathological mechanisms of CVDs, particularly myocardial ischemia/reperfusion injury, myocardial infarction, cardiomyopathy, heart failure, atherosclerosis, hypertension, and ischemic stroke. In addition, we further envision that RNA interference or ncRNA mimics or inhibitors targeting the UPS can potentially be used as therapeutic tools and strategies. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific proteases (USPs), as one of the deubiquitinating enzymes (DUBs) families, regulate the fate of proteins and signaling pathway transduction by removing ubiquitin chains from the target proteins. USPs are essential for the modulation of a variety of physiological processes, such as DNA repair, cell metabolism and differentiation, epigenetic modulations as well as protein stability. Recently, extensive research has demonstrated that USPs exert a significant impact on innate and adaptive immune reactions, metabolic syndromes, inflammatory disorders, and infection via post-translational modification processes. This review summarizes the important roles of the USPs in the onset and progression of inflammatory diseases, including periodontitis, pneumonia, atherosclerosis, inflammatory bowel disease, sepsis, hepatitis, diabetes, and obesity. Moreover, we highlight a comprehensive overview of the pathogenesis of USPs in these inflammatory diseases as well as post-translational modifications in the inflammatory responses and pave the way for future prospect of targeted therapies in these inflammatory diseases. [ABSTRACT FROM AUTHOR]

N6 methyladenosine (m6A) is the most prevalent RNA epigenetic modification, regulated by methyltransferases and demethyltransferases and recognized by methylation-related reading proteins to impact mRNA splicing, translocation, stability, and translation efficiency. It significantly affects a variety of activities, including stem cell maintenance and differentiation, tumor formation, immune regulation, and metabolic disorders. Ubiquitination refers to the specific modification of target proteins by ubiquitin molecule in response to a series of enzymes. E3 ligases connect ubiquitin to target proteins and usually lead to protein degradation. On the contrary, deubiquitination induced by deubiquitinating enzymes (DUBs) can separate ubiquitin and regulate the stability of protein. Recent studies have emphasized the potential importance of ubiquitination and deubiquitination in controlling the function of m6A modification. In this review, we discuss the impact of ubiquitination and deubiquitination on m6A functional molecules in diseases, such as metabolism, cellular stress, and tumor growth. [ABSTRACT FROM AUTHOR]

Programmed death ligand 1 (PD-L1) plays a pivotal role in cancer immune evasion and is a critical target for cancer immunotherapy. This review focuses on the regulation of PD-L1 through the dynamic processes of ubiquitination and deubiquitination, which are crucial for its stability and function. Here, we explored the intricate mechanisms involving various E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) that modulate PD-L1 expression in cancer cells. Specific ligases are discussed in detail, highlighting their roles in tagging PD-L1 for degradation. Furthermore, we discuss the actions of DUBs that stabilize PD-L1 by removing ubiquitin chains. The interplay of these enzymes not only dictates PD-L1 levels but also influences cancer progression and patient response to immunotherapies. Furthermore, we discuss the therapeutic implications of targeting these regulatory pathways and propose novel strategies to enhance the efficacy of PD-L1/PD-1-based therapies. Our review underscores the complexity of PD-L1 regulation and its significant impact on the tumor microenvironment and immunotherapy outcomes. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense. [ABSTRACT FROM AUTHOR]

Aberrant upregulation of the ubiquitin-specific protease 14 (USP14) has been found in some malignant tumors, including oral squamous cell carcinoma (OSCC). In this study, we further demonstrated that aberrantly overexpressed USP14 was also closely related to adverse clinicopathological features and poor prognosis in patients with OSCC, so we hypothesized that USP14 might act as a tumor-promoting factor during the progression of OSCC. Notably, we originally proved that USP14 is a deubiquitinating enzyme for phosphofructokinase-1 liver type (PFKL), a key rate-limiting enzyme involved in the glycolytic pathway. USP14 interacts with PFKL and enhances its stability through deubiquitination in OSCC cells, which in turn enhances PFKL-mediated glycolytic metabolism and ultimately promote cellular proliferation, migration, and tumorigenesis. In this work, we have also demonstrated for the first time that USP14 is a critical regulator of glycolysis in OSCC and verified a novel mechanism whereby it is involved in tumor metastasis and growth. Collectively, our findings provide novel insights into the tumor-promoting role of USP14 and establish mechanistic foundations for USP14-targeting therapies. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non–small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. Methods The detection of mRNA and protein of MRPL35, ubiquitin‐specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT‐PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α‐ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis. Results MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. Conclusion MRPL35 expression was stabilized by USP39‐induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the thyroid. Fibroblast growth factor receptor 1 (FGFR1) is highly expressed in PTC and works as an oncogenic protein in this disease. In this report, we wanted to uncover a new mechanism that drives overexpression of FGFR1 in PTC. Analysis of FGFR1 expression in clinical specimens and PTC cells revealed that FGFR1 expression was enhanced in PTC. Using siRNA/shRNA silencing experiments, we found that FGFR1 downregulation impeded PTC cell growth, invasion, and migration and promoted apoptosis in vitro, as well as suppressed tumor growth in vivo. Bioinformatic analyses predicted the potential USP7-FGFR1 interplay and the potential binding between YY1 and the FGFR1 promoter. The mechanism study found that USP7 stabilized FGFR1 protein via deubiquitination, and YY1 could promote the transcription of FGFR1. Our rescue experiments showed that FGFR1 re-expression had a counteracting effect on USP7 downregulation-imposed in vitro alterations of cell functions and in vivo suppression of xenograft growth. In conclusion, our study identifies the deubiquitinating enzyme USP7 and the oncogenic transcription factor YY1 as potent inducers of FGFR1 overexpression. Designing inhibitors targeting FGFR1 or its upstream inducers USP7 and YY1 may be foreseen as a promising strategy to control PTC development.

Abstract Background Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. It is an aggressive tumor characterized by rapid proliferation, diffuse tumor morphology, and poor prognosis. Unfortunately, current treatments, such as surgery, radiotherapy, and chemotherapy, are unable to achieve good outcomes. Therefore, there is an urgent need to explore new treatment targets. A detailed mechanistic exploration of the role of the nuclear pore transporter KPNB1 in GBM is lacking. This study demonstrated that KPNB1 regulated GBM progression through a transcription factor YBX1 to promote the expression of post-protrusion membrane protein NLGN3. This regulation was mediated by the deubiquitinating enzyme USP7. Methods A tissue microarray was used to measure the expression of KPNB1 and USP7 in glioma tissues. The effects of KPNB1 knockdown on the tumorigenic properties of glioma cells were characterized by colony formation assays, Transwell migration assay, EdU proliferation assays, CCK-8 viability assays, and apoptosis analysis using flow cytometry. Transcriptome sequencing identified NLGN3 as a downstream molecule that is regulated by KPNB1. Mass spectrometry and immunoprecipitation were performed to analyze the potential interaction between KPNB1 and YBX1. Moreover, the nuclear translocation of YBX1 was determined with nuclear-cytoplasmic fractionation and immunofluorescence staining, and chromatin immunoprecipitation assays were conducted to study DNA binding with YBX1. Ubiquitination assays were performed to determine the effects of USP7 on KPNB1 stability. The intracranial orthotopic tumor model was used to detect the efficacy in vivo. Results In this study, we found that the nuclear receptor KPNB1 was highly expressed in GBM and could mediate the nuclear translocation of macromolecules to promote GBM progression. Knockdown of KPNB1 inhibited the progression of GBM, both in vitro and in vivo. In addition, we found that KPNB1 could regulate the downstream expression of Neuroligin-3 (NLGN3) by mediating the nuclear import of transcription factor YBX1, which could bind to the NLGN3 promoter. NLGN3 was necessary and sufficient to promote glioma cell growth. Furthermore, we found that deubiquitinase USP7 played a critical role in stabilizing KPNB1 through deubiquitination. Knockdown of USP7 expression or inhibition of its activity could effectively impair GBM progression. In vivo experiments also demonstrated the promoting effects of USP7, KPNB1, and NLGN3 on GBM progression. Overall, our results suggested that KPNB1 stability was enhanced by USP7-mediated deubiquitination, and the overexpression of KPNB1 could promote GBM progression via the nuclear translocation of YBX1 and the subsequent increase in NLGN3 expression. Conclusion This study identified a novel and targetable USP7/KPNB1/YBX1/NLGN3 signaling axis in GBM cells.

Abstract Background Bladder cancer is the second most common genitourinary malignancy worldwide. The death rate of bladder cancer has increased every year. However, the molecular mechanism of bladder cancer is not sufficiently studied. Deubiquitinating enzymes (DUBs) play an important role in carcinogenesis. Several studies have demonstrated that USP5 associated with malignancy and pathological progression in hepatocellular carcinoma, colorectal and non-small cell lung cancer. However, the role of USP5 in bladder cancer need to be explored. Methods The USP5 expression was analysed using the web server GEPIA. To explore USP5 function in bladder cancer, we constructed USP5-knockout cell lines in T24 cells. A FLAG-USP5 (WT USP5) plasmid and a plasmid FLAG-USP5 C335A (catalytic-inactive mutant) used to overexpress USP5 in EJ cells. CCK8, colony formation, transwell and scratch assays were used to assess cell viability, proliferation and migration. RNA sequencing (RNA-seq) and dual-luciferase reporter assays were performed to screen the pathway. Coimmunoprecipitation and immunofluorescence were used to explore the interaction between USP5 and c-Jun. Cycloheximide (CHX) chase assays were performed to establish the effect of USP5 on c-Jun stability. Xenograft mouse model was used to study the role of USP5 in bladder cancer. Results USP5 expression is increased in bladder cancer patients. Genetic ablation of USP5 markedly inhibited bladder cancer cell proliferation, viability, and migration both in vitro and in vivo. RNA-seq and luciferase pathway screening showed that USP5 activated JNK signalling, and we identified the interaction between USP5 and c-Jun. USP5 was found to activate c-Jun by inhibiting its ubiquitination. Conclusions Our results show that high USP5 expression promotes bladder cancer progression by stabilizing c-Jun and that USP5 is a potential therapeutic target in bladder cancer.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

Background: Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. Methods: Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbβ3 in washed platelets was analyzed using flow cytometry. Results: USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbβ3 in mice. Conclusion: USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbβ3.

Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitinproteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3KAKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. It is an aggressive tumor characterized by rapid proliferation, diffuse tumor morphology, and poor prognosis. Unfortunately, current treatments, such as surgery, radiotherapy, and chemotherapy, are unable to achieve good outcomes. Therefore, there is an urgent need to explore new treatment targets. A detailed mechanistic exploration of the role of the nuclear pore transporter KPNB1 in GBM is lacking. This study demonstrated that KPNB1 regulated GBM progression through a transcription factor YBX1 to promote the expression of post-protrusion membrane protein NLGN3. This regulation was mediated by the deubiquitinating enzyme USP7. Methods: A tissue microarray was used to measure the expression of KPNB1 and USP7 in glioma tissues. The effects of KPNB1 knockdown on the tumorigenic properties of glioma cells were characterized by colony formation assays, Transwell migration assay, EdU proliferation assays, CCK-8 viability assays, and apoptosis analysis using flow cytometry. Transcriptome sequencing identified NLGN3 as a downstream molecule that is regulated by KPNB1. Mass spectrometry and immunoprecipitation were performed to analyze the potential interaction between KPNB1 and YBX1. Moreover, the nuclear translocation of YBX1 was determined with nuclear-cytoplasmic fractionation and immunofluorescence staining, and chromatin immunoprecipitation assays were conducted to study DNA binding with YBX1. Ubiquitination assays were performed to determine the effects of USP7 on KPNB1 stability. The intracranial orthotopic tumor model was used to detect the efficacy in vivo. Results: In this study, we found that the nuclear receptor KPNB1 was highly expressed in GBM and could mediate the nuclear translocation of macromolecules to promote GBM progression. Knockdown of KPNB1 inhibited the progression of GBM, both in vitro and in vivo. In addition, we found that KPNB1 could regulate the downstream expression of Neuroligin-3 (NLGN3) by mediating the nuclear import of transcription factor YBX1, which could bind to the NLGN3 promoter. NLGN3 was necessary and sufficient to promote glioma cell growth. Furthermore, we found that deubiquitinase USP7 played a critical role in stabilizing KPNB1 through deubiquitination. Knockdown of USP7 expression or inhibition of its activity could effectively impair GBM progression. In vivo experiments also demonstrated the promoting effects of USP7, KPNB1, and NLGN3 on GBM progression. Overall, our results suggested that KPNB1 stability was enhanced by USP7-mediated deubiquitination, and the overexpression of KPNB1 could promote GBM progression via the nuclear translocation of YBX1 and the subsequent increase in NLGN3 expression. Conclusion: This study identified a novel and targetable USP7/KPNB1/YBX1/NLGN3 signaling axis in GBM cells. [ABSTRACT FROM AUTHOR]

Background: Bladder cancer is the second most common genitourinary malignancy worldwide. The death rate of bladder cancer has increased every year. However, the molecular mechanism of bladder cancer is not sufficiently studied. Deubiquitinating enzymes (DUBs) play an important role in carcinogenesis. Several studies have demonstrated that USP5 associated with malignancy and pathological progression in hepatocellular carcinoma, colorectal and non-small cell lung cancer. However, the role of USP5 in bladder cancer need to be explored. Methods: The USP5 expression was analysed using the web server GEPIA. To explore USP5 function in bladder cancer, we constructed USP5-knockout cell lines in T24 cells. A FLAG-USP5 (WT USP5) plasmid and a plasmid FLAG-USP5 C335A (catalytic-inactive mutant) used to overexpress USP5 in EJ cells. CCK8, colony formation, transwell and scratch assays were used to assess cell viability, proliferation and migration. RNA sequencing (RNA-seq) and dual-luciferase reporter assays were performed to screen the pathway. Coimmunoprecipitation and immunofluorescence were used to explore the interaction between USP5 and c-Jun. Cycloheximide (CHX) chase assays were performed to establish the effect of USP5 on c-Jun stability. Xenograft mouse model was used to study the role of USP5 in bladder cancer. Results: USP5 expression is increased in bladder cancer patients. Genetic ablation of USP5 markedly inhibited bladder cancer cell proliferation, viability, and migration both in vitro and in vivo. RNA-seq and luciferase pathway screening showed that USP5 activated JNK signalling, and we identified the interaction between USP5 and c-Jun. USP5 was found to activate c-Jun by inhibiting its ubiquitination. Conclusions: Our results show that high USP5 expression promotes bladder cancer progression by stabilizing c-Jun and that USP5 is a potential therapeutic target in bladder cancer. [ABSTRACT FROM AUTHOR]

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections. IMPORTANCE The ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections. [ABSTRACT FROM AUTHOR]

Radiotherapy, a treatment method employing radiation to eradicate tumor cells and subsequently reduce or eliminate tumor masses, is widely applied in the management of numerous patients with tumors. However, its therapeutic effectiveness is somewhat constrained by various drug-resistant factors. Recent studies have highlighted the ubiquitination/deubiquitination system, a reversible molecular modification pathway, for its dual role in influencing tumor behaviors. It can either promote or inhibit tumor progression, impacting tumor proliferation, migration, invasion, and associated therapeutic resistance. Consequently, delving into the potential mechanisms through which ubiquitination and deubiquitination systems modulate the response to radiotherapy in malignant tumors holds paramount significance in augmenting its efficacy. In this paper, we comprehensively examine the strides made in research and the pertinent mechanisms of ubiquitination and deubiquitination systems in governing radiotherapy resistance in tumors. This underscores the potential for developing diverse radiosensitizers targeting distinct mechanisms, with the aim of enhancing the effectiveness of radiotherapy. [ABSTRACT FROM AUTHOR]

The interaction of viruses with hosts is complex, especially so with the antiviral immune systems of hosts, and the underlying mechanisms remain perplexing. Infection with SARS-CoV-2 may result in cytokine syndrome in the later stages, reflecting the activation of the antiviral immune response. However, viruses also encode molecules to negatively regulate the antiviral immune systems of hosts to achieve immune evasion and benefit viral replication during the early stage of infection. It has been observed that the papain-like protease (PLP) encoded by coronavirus could negatively regulate the host's IFNβ innate immunity. In this study, we first found that eight inflammasome-related genes were downregulated in CD14+ monocytes from COVID-19 patients. Subsequently, we observed that SARS-CoV-2 PLP negatively regulated the NLRP3 inflammasome pathway, inhibited the secretion of IL-1β, and decreased the caspase-1-mediated pyroptosis of human monocytes. The mechanisms for this may arise because PLP coimmunoprecipitates with ASC, reduces ASC ubiquitination, and inhibits ASC oligomerization and the formation of ASC specks. These findings suggest that PLP may inhibit strong immune defenses and provide the maximum advantage for viral replication. This research may allow us to better understand the flex function of CoV-encoding proteases and provide a new perspective on the innate immune responses against SARS-CoV-2 and other viruses. [ABSTRACT FROM AUTHOR]

Deubiquitination, a post-translational modification regulated by deubiquitinases, is essential for cancer initiation and progression. Ubiquitin-specific proteases (USPs) are essential elements of the deubiquitinase family, and are overexpressed in gastric cancer (GC). Through the regulation of several signaling pathways, such as Wnt/β-Catenin and nuclear factor-κB signaling, and the promotion of the expression of deubiquitination- and stabilization-associated proteins, USPs promote the proliferation, metastasis, invasion, and epithelial-mesenchymal transition of GC. In addition, the expression of USPs is closely related to clinicopathological features, patient prognosis, and chemotherapy resistance. USPs therefore could be used as prognostic biomarkers. USP targeting small molecule inhibitors have demonstrated strong anticancer activity. However, they have not yet been tested in the clinic. This article provides an overview of the latest fundamental research on USPs in GC, aiming to enhance the understanding of how USPs contribute to GC progression, and identifying possible targets for GC treatment to improve patient survival.

Abstract Ubiquitination is one of the most significant post-translational modifications that regulate almost all physiological processes like cell proliferation, autophagy, apoptosis, and cell cycle progression. Contrary to ubiquitination, deubiquitination removes ubiquitin from targeted protein to maintain its stability and thus regulate cellular homeostasis. Ubiquitin-Specific Protease 12 (USP12) belongs to the biggest family of deubiquitinases named ubiquitin-specific proteases and has been reported to be correlated with various pathophysiological processes. In this review, we initially introduce the structure and biological functions of USP12 briefly and summarize multiple substrates of USP12 as well as the underlying mechanisms. Moreover, we discuss the influence of USP12 on tumorigenesis, tumor immune microenvironment (TME), disease, and related signaling pathways. This study also provides updated information on the roles and functions of USP12 in different types of cancers and other diseases, including prostate cancer, breast cancer, lung cancer, liver cancer, cardiac hypertrophy, multiple myeloma, and Huntington's disease. Generally, this review sums up the research advances of USP12 and discusses its potential clinical application value which deserves more exploration in the future.