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1.
Proc Natl Acad Sci U S A ; 120(4): e2217840120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36656861

RESUMO

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Mesotelioma Maligno , Mesotelioma , Ubiquitina Tiolesterase , Humanos , Heterozigoto , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma Maligno/genética , Mesotelioma Maligno/complicações , Mutação , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo
2.
Cell Death Dis ; 14(1): 56, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36693850

RESUMO

MAPK/JNK signaling is pivotal in carcinogenesis. However, ubiquitin-mediated homeostasis of JNK remains to be verified. Here, with results from RNA sequencing (RNA-seq) and luciferase reporter pathway identification, we show that USP14 orchestrates MAPK/JNK signaling and identify USP14 as a deubiquitinase that interacts and stabilizes JNK. USP14 is elevated in colorectal cancer patients and is positively associated with JNK protein and downstream gene expression. USP14 ablation reduces cancer cell proliferation in vitro and colorectal tumorigenesis in vivo by downregulating MAPK/JNK pathway activation. Moreover, USP14 expression is induced by TNF-α, forming a feedback loop with JNK and leading to tumor amplification. Our study suggests that elevated expression of USP14 promotes MAPK/JNK signaling by stabilizing JNK, which in turn augments colorectal carcinogenesis, indicating a potential therapeutic target for colorectal cancer patients with increased USP14 expression.


Assuntos
Neoplasias Colorretais , Ubiquitina Tiolesterase , Humanos , Linhagem Celular Tumoral , Ubiquitina Tiolesterase/metabolismo , Proliferação de Células/genética , Transformação Celular Neoplásica , Carcinogênese/genética , Neoplasias Colorretais/genética
3.
Nat Commun ; 14(1): 251, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36646704

RESUMO

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.


Assuntos
Interferon Tipo I , Neoplasias , Camundongos , Animais , Humanos , Piroptose , Pool Gênico , Transdução de Sinais , NF-kappa B/metabolismo , Interferon Tipo I/genética , Ubiquitina Tiolesterase/metabolismo , Neoplasias/genética
4.
Carbohydr Polym ; 303: 120453, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36657860

RESUMO

The water-soluble Morchella esculenta polysaccharide 2 (MEP2) was purified and isolated from an aqueous extract of the Morchella esculenta fruiting bodies. MEP2, having a molecular weight of 959 kDa, has a →4)-α-D-Glcp-(1→ glucan backbone, and this branch was substituted at the H-6 position by an α-D-Glcp-(1 â†’ 4)-α-D-Glcp-(1→ residue and an α-D-Glcp-(1→ residue. The hepatoprotective activity and potential mechanism of action of MEP2 were also investigated. MEP2 ameliorated severe liver damage and regulated the liver function indicators and the alcohol-related enzyme levels in chronic alcohol-induced mice. Combined with biochemical detection, the gut microbiota, metabolites, and proteomics results revealed that MEP2 regulates the levels of hepatic cytokines related to inflammatory response and oxidative stress, as well as those of intestinal Bacteroides, Oscillospira, Parabacteroides, Alistipes, and Prevotella, through the ubiquitin-specific peptidase 10 (Usp10)/nuclear factor κB (NF-κB)/nuclear factor erythroid-2 related factor 2 (Nrf2) signaling pathway in the liver of mice induced by long-term alcohol intake. These data provide experimental evidence for the application of MEP2 in chronic alcohol-induced liver injury.


Assuntos
Fator 2 Relacionado a NF-E2 , NF-kappa B , Camundongos , Animais , NF-kappa B/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Fígado/metabolismo , Estresse Oxidativo , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Polissacarídeos/metabolismo , Ubiquitina Tiolesterase/metabolismo
5.
Cell Death Dis ; 14(1): 40, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36653359

RESUMO

Liver fibrosis is a complex fibrotic process that develops early in the course of cirrhosis and is caused by chronic liver damage. The activation of hepatic stellate cells is primarily responsible for the fibrosis process. Studies show that NRP1 influences HSC motility and migration. However, whether NRP1 regulates HSC activation remains unknown. C57BL/6 male mice (6-8 weeks old) were intraperitoneally injected with 10% CCl4 in olive oil (5 µl/g body weight) every three days for four weeks to create an animal model of liver fibrosis. Control mice received olive oil (5 µl/g body weight). Different assays such as immunohistochemistry, immunostaining, Western blotting, qRT-PCR, immunoprecipitation, immunoprecipitation, and GST pull-down assays, and in vivo and in vitro ubiquitination assays were conducted. We found that NRP1 expression was significantly elevated both in mouse and human fibrotic livers, mainly in activated HSCs at the fibrotic foci. NRP1 promoted HSC activation via the cytokine TGF-ß1, VEGFA, and PDGF-BB. Moreover, USP9X was found to be a critical deubiquitinating enzyme for the stability and high activity of NRP1 and NRP1 deubiquitination mediated by USP9X enhanced HSC activation and liver fibrosis. NRP1 deubiquitination mediated by USP9X enhances HSC activation, implying that targeting NRP1 or USP9X potentiates novel options in the treatment of liver fibrosis.


Assuntos
Células Estreladas do Fígado , Fígado , Masculino , Humanos , Camundongos , Animais , Fígado/metabolismo , Células Estreladas do Fígado/metabolismo , Azeite de Oliva/metabolismo , Proliferação de Células , Camundongos Endogâmicos C57BL , Cirrose Hepática/patologia , Fibrose , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
6.
Biochem Biophys Res Commun ; 641: 132-138, 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36527747

RESUMO

Cervical cancer is one of the most lethal gynaecological malignancies in females. The deubiquitylase UCHL3 has been studied as an oncogenic factor in multiple cancers. However, the expression pattern and function profile of UCHL3 in cervical cancer hasn't been fully characterized. Here, we revealed that UCHL3 was highly expressed in cervical cancer and overexpressed UCHL3 predicted a poor survival probability in cervical cancer patients. Our findings showed that knockdown of UCHL3 inhibited cell growth, migration and invasion in cervical cancer cells while UCHL3 knockdown inhibited cervical cancer development and metastasis in vivo in mouse models. Mechanistically, co-immunoprecipitation assay showed that UCHL3 directly interacted with NRF2. Knockdown of UCHL3 decreased NRF2 expression while overexpression of UCHL3 stabilized NRF2 via deubiquitination. In addition, overexpression of UCHL3 with C92A mutation didn't affect NRF2 stability. Moreover, we revealed that overexpression of NRF2 could antagonize the function of UCHL3 knockdown in cervical cancer cells. Collectively, our findings suggest that UCHL3 promotes cervical cancer development and metastasis by stabilizing NRF2 via deubiquitination. Thus, UCHL3/NRF2 axis could be utilized to develop efficient treatments for cervical cancer patients.


Assuntos
Neoplasias do Colo do Útero , Humanos , Feminino , Animais , Camundongos , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Colo do Útero/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
7.
Gen Physiol Biophys ; 41(6): 569-577, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36454117

RESUMO

As COVID-19 spreads over the world, the treatment of acute lung injury (ALI) has attracted much attention. Considering ubiquitin-specific protease (USP) 25 has been relevant to inflammation, this article focused on its role in ALI and its regulatory mechanism. Lipopolysaccharide (LPS) was applied to separately stimulate mice and human lung epithelial cells to establish in vivo and in vitro ALI models. To discover the effects of USP25 overexpression on mouse, lung pathology, inflammatory factor levels, edema, number of inflammatory cells, and downstream protein levels were evaluated. USP25 overexpression in mice could alleviate LPS-induced lung tissue lesions and edema, and reduce inflammatory factors and inflammatory cells. It also inhibited the levels of downstream TRAF6, MAPK pathway-related proteins, and Fos Proto-Oncogene (FOS) in vivo. Furthermore, BEAS-2B cells were transfected with TNF receptor-associated factor 6 (TRAF6) plasmids to study the role of TRAF6 in the regulatory mechanism of USP25. TRAF6 overexpression was found to reverse the functions of USP25 overexpression on cells. In conclusion, USP25 reduced ALI and inhibited inflammation in lung epithelial cells via regulating TRAF6/MAPK/FOS signaling.


Assuntos
Lesão Pulmonar Aguda , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina , Animais , Humanos , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Células Epiteliais , Inflamação , Lipopolissacarídeos , Pulmão , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
8.
Biomed Res Int ; 2022: 1525216, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36567903

RESUMO

Background: Ubiquitination is an important regulator in physiological and pathological conditions. Ubiquitin-specific protease 2 (USP2), as a member of the USP family, exhibits oncogenic effects in multiple malignancies. However, the exact role of USP2 has not been well clarified in lung cancer pathogenesis and progression. Therefore, we aimed to further investigate the regulatory roles of USP2 in lung cancer in this study. Methods: Firstly, immunoprecipitation-Mass Spectrometry (IP-MS), Co-immunoprecipitation (Co-IP), combined with immunofluorescent colocalization method, was conducted for USP2 protein interaction analysis in lung cancer cell lines. qRT-PCR, Western blot, and immunohistochemistry assays explored the USP2 expression pattern and USP2/ARID2- (AT-rich interactive domain 2-) specific shRNAs and overexpression vectors. Co-IP assays were designed to validate USP2-ARID2 protein interaction. Further functional studies including CHX chase assay, transwell assay, and wound healing assay were subsequently applied to evaluate the impact of USP2 modulation on lung cancer cells. Results: USP2 suppression was characteristic in lung cancer cell line models and lung cancer samples. USP2 and ARID2 demonstrated protein-protein interaction and overlapping localization in cancer cell models. Functional experiments suggested USP2 inhibited lung cancer cell invasion and migration by reducing ARID2 protein degradation. Subsequent ubiquitination assays indicated ARID2 protein degradation via the ubiquitination was significantly reduced by USP2 interaction. Conclusions: Our study provided novel insight that USP2 might suppress lung cancer by reducing ARID2 protein degradation via ubiquitination.


Assuntos
Neoplasias Pulmonares , Proteólise , Ubiquitinação , Humanos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
9.
Cell Mol Biol Lett ; 27(1): 111, 2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36528617

RESUMO

BACKGROUND: Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments. METHODS: The yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed. RESULTS: We identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi. CONCLUSION: Our findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi.


Assuntos
Complexo de Endopeptidases do Proteassoma , Interferência de RNA , Humanos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Imunoprecipitação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interferência de RNA/fisiologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
10.
Exp Mol Med ; 54(11): 1814-1821, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36385557

RESUMO

PTEN is among the most commonly lost or mutated tumor suppressor genes in human cancer. PTEN, a bona fide lipid phosphatase that antagonizes the highly oncogenic PI3K-AKT-mTOR pathway, is considered a major dose-dependent tumor suppressor. Although PTEN function can be compromised by genetic mutations in inherited syndromes and cancers, posttranslational modifications of PTEN may also play key roles in the dynamic regulation of its function. Notably, deregulated ubiquitination and deubiquitination lead to detrimental impacts on PTEN levels and subcellular partitioning, promoting tumorigenesis. While PTEN can be targeted by HECT-type E3 ubiquitin ligases for nuclear import and proteasomal degradation, studies have shown that several deubiquitinating enzymes, including HAUSP/USP7, USP10, USP11, USP13, OTUD3 and Ataxin-3, can remove ubiquitin from ubiquitinated PTEN in cancer-specific contexts and thus reverse ubiquitination-mediated PTEN regulation. Researchers continue to reveal the precise molecular mechanisms by which cancer-specific deubiquitinases of PTEN regulate its roles in the pathobiology of cancer, and new methods of pharmacologically for modulating PTEN deubiquitinases are critical areas of investigation for cancer treatment and prevention. Here, we assess the mechanisms and functions of deubiquitination as a recently appreciated mode of PTEN regulation and review the link between deubiquitinases and PTEN reactivation and its implications for therapeutic strategies.


Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Carcinogênese/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Enzimas Desubiquitinantes , Tioléster Hidrolases/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
11.
Cells ; 11(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36359803

RESUMO

The role of hypoxia-regulated long non-coding RNA (lncRNA) in the development of head and neck squamous cell carcinoma (HNSCC) remains to be elucidated. In the current study, we initially screened hypoxia-regulated lncRNA in HNSCC cells by RNA-seq, before focusing on the rarely annotated lncRNA USP2 antisense RNA 1 (USP2-AS1). We determined that USP2-AS1 is a direct target of HIF1α and is remarkably elevated in HNSCC compared with matched normal tissues. Patients with a higher level of USP2-AS1 suffered a poor prognosis. Next, loss- and gain-of-function assays revealed that USP2-AS1 promoted cell proliferation and invasion in vitro and in vivo. Mechanically, RNA pulldown and LC-MS/MS demonstrated that the E3 ligase DDB1- and CUL4-associated factor 13 (DCAF13) is one of the binding partners to USP2-AS1 in HNSCC cells. In addition, we assumed that USP2-AS1 regulates the activity of DCAF13 by targeting its substrate ATR. Moreover, the knockdown of DCAF13 restored the elevated cell proliferation and growth levels achieved by USP2-AS1 overexpression. Altogether, we found that lncRNA USP2-AS1 functions as a HIF1α-regulated oncogenic lncRNA and promotes HNSCC cell proliferation and growth by interacting and modulating the activity of DCAF13.


Assuntos
Neoplasias de Cabeça e Pescoço , RNA Longo não Codificante , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Cromatografia Líquida , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Hipóxia/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Espectrometria de Massas em Tandem , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , RNA Antissenso
12.
Exp Cell Res ; 421(2): 113402, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36328194

RESUMO

Aberrant ubiquitin-proteasome system (UPS) contributes to tumorigeneisis or drug resistance of Pancreatic Adenocarcinoma (PAAD). Previous studies have implicated the deubiquitinase UCHL5 was abnormally expressed in multiple malignancies. However, little was reported about the specific roles of UCHL5 in PAAD. We aimed to identify the biological roles of UCHL5 in PAAD and demonstrate its prognostic significance. Differential analysis revealed that UCHL5 expressed highly in tumors versus normal tissues, like TCGA-PAAD, GSE28735, GSE15471 and collected samples. Patients with high UCHL5 expressions had worse survival outcomes relative to those with low UCHL5 levels. Experimental assays showed that UCHL5 overexpression could significantly enhance cell proliferation, colony formation and self-renewal capacities. UCHL5 could also promote PAAD migration in vitro and in vivo. Mechanistically, UCHL5 could directly deubiquitinate and stabilize ELK3 proteins. UCHL5 relied on accumulated ELK3 proteins to drive cell growth, stem-like properties and migration abilities. In addition, enrichment analysis based on RNA-seq data implicated that ELK3 mainly correlated with Notch1 signaling and ELK3 could notably elevate ELK3 mRNA levels. UCHL5 could thus promote self-renewal abilities of PAAD and targeting ELK3 could inhibit the stemness features. In contrast, UCHL5 deficiency could suppress PAAD stemness features, and ectopic expression of ELK3 could rescue this effect. Last of all, we utilized the UCHL5 inhibitor, b-AP15, to treat PAAD cells and found that b-AP15 could inhibit the growth of PAAD cells in a dose-dependent manner. Collectively, our study uncovered the underlying mechanisms of UCHL5/ELK3/Notch1 axis in PAAD progression and stemness maintaince, shedding light on individualized treatment and risk stratification for PAAD patients.


Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Adenocarcinoma/patologia , Proliferação de Células/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteínas Proto-Oncogênicas c-ets
13.
Int J Mol Sci ; 23(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36361894

RESUMO

The Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family, involved in inducing apoptosis at the mitochondrial level. Regulating the protein levels of Bax is essential to enhancing apoptosis. In the current study, we ascertained the presence of deubiquitinating enzymes (DUBs) associated with Bax by performing the yeast two-hybrid screening (Y2H). We determined that ubiquitin-specific protease 12 (USP12), one of the DUBs, is associated with Bax. The binding of USP12 to Bax shows the interaction as a DUB, which regulates ubiquitination on Bax. Taken together, we believe that USP12 regulates Bax by detaching ubiquitin on K63-linked chains, indicating that USP12 affects the cellular functions of Bax, but it is not related with proteasomal degradation. The half-life of the Bax protein was determined by performing the site-directed mutagenesis of putative ubiquitination sites on Bax (K128R, K189R, and K190R). Of these, Bax (K128R and K190R) showed less ubiquitination; therefore, we compared the half-life of Bax (WT) and Bax K mutant forms in vitro. Interestingly, Bax (K189R) showed a higher ubiquitination level and shorter half-life than Bax (WT), and the (K128R and K190R) mutant form has a longer half-life than Bax (WT).


Assuntos
Proteínas Reguladoras de Apoptose , Ubiquitina Tiolesterase , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Ubiquitinação , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Apoptose
14.
Int J Mol Sci ; 23(19)2022 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-36233276

RESUMO

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells' survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.


Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Antagonistas do Ácido Fólico , Neoplasias Hepáticas , Alanina/farmacologia , Aminoácidos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores/metabolismo , Calpaína/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Ácido Fólico/farmacologia , Glucose/farmacologia , Humanos , L-Iditol 2-Desidrogenase/metabolismo , Neoplasias Hepáticas/metabolismo , Redes e Vias Metabólicas , Nucleotídeos/metabolismo , Fosfatidilcolinas/farmacologia , Prolina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Proteômica , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Ácido Succínico/farmacologia , Superóxido Dismutase/metabolismo , Tirosina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Difosfato de Uridina/metabolismo
15.
Nat Commun ; 13(1): 5950, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216817

RESUMO

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.


Assuntos
Ubiquitina Tiolesterase , Ubiquitina , Animais , Humanos , Camundongos , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
16.
J Med Chem ; 65(20): 13645-13659, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36221183

RESUMO

Deubiquitinating enzymes (DUBs) are key regulatory components of the ubiquitination system. Many DUBs have been revealed to play key roles in normal physiology and diseases. However, only very limited DUB members have well-characterized inhibitors. OTUB1 and USP8 are two DUBs reported to promote both immune evasion and tumorigenesis in tumor models, yet their targeted inhibitors are in the early stages of development. Here, we describe the lead identification and optimization of an OTUB1/USP8 dual inhibitor, 61, which exhibits highly potent and selective inhibition of both targets with subnanomolar IC50s in vitro. By inhibiting both DUBs, 61 phenocopies the double knockdown of OTUB1/USP8 and exerts pronounced antiproliferative effects in H1975 and other non-small-cell lung cancer (NSCLC) cell lines. Moreover, 61 efficaciously mitigates tumor growth in vivo. Collectively, our results provide a useful tool for pharmacological perturbation of OTUB1/USP8 and introduce a promising therapeutic strategy of dual DUB inhibition for treating NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteostase , Neoplasias Pulmonares/tratamento farmacológico , Ubiquitinação , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo
17.
Biomolecules ; 12(10)2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36291686

RESUMO

The diagnosis of brain gliomas is mainly based on imaging methods. The gold standard in this area is MRI. Recommendations for the prevention, diagnosis, and treatment of gliomas are periodically modified and updated. One of the diagnostic techniques used when a brain glioma is suspected is liquid biopsy. However, this technique requires further development to confirm its effectiveness. This paper presents a proposal of three potential biomarkers of brain gliomas-extracellular matrix metalloproteinase-1 (MMP-1), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and the 20S proteasome-which were quantified in blood plasma using SPRi biosensors. A statistical analysis of the results indicated no significant changes in the concentrations between the control group (K) and grades G1 and G2, and similarly between grades G3 and G4. However, the differences in the concentrations between the groups K/G1/G2 and G3/G4 were statistically significant. A positive average correlation was found between the concentrations of the proteins and the patient's age. The individual tested proteins were also highly correlated with each other. Our work proposes a new diagnostic technique that may aid in the diagnosis of brain gliomas.


Assuntos
Glioma , Ubiquitina Tiolesterase , Humanos , Ubiquitina Tiolesterase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Glioma/diagnóstico , Glioma/patologia , Biomarcadores , Encéfalo/metabolismo
18.
Genes Dev ; 36(17-18): 1016-1030, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36302555

RESUMO

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.


Assuntos
Proteômica , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteoma , Ubiquitina/metabolismo , Ubiquitinação
19.
Oncogene ; 41(47): 5061-5075, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36243803

RESUMO

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.


Assuntos
Glioma , Proteína Supressora de Tumor p53 , Humanos , Peptidase 7 Específica de Ubiquitina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Apoptose , Glioma/genética , Linhagem Celular Tumoral , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
20.
Mil Med Res ; 9(1): 54, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36163081

RESUMO

BACKGROUND: Melatonin, a natural hormone secreted by the pineal gland, has been reported to exhibit antitumor properties through diverse mechanisms of action. However, the oncostatic function of melatonin on esophageal squamous cell carcinoma (ESCC) remains elusive. This study was conducted to investigate the potential effect and underlying molecular mechanism of melatonin as single anticancer agent against ESCC cells. METHODS: ESCC cell lines treated with or without melatonin were used in this study. In vitro colony formation and EdU incorporation assays, and nude mice tumor xenograft model were used to confirm the proliferative capacities of ESCC cells. RNA-seq, qPCR, Western blotting, recombinant lentivirus-mediated target gene overexpression or knockdown, plasmids transfection and co-IP were applied to investigate the underlying molecular mechanism by which melatonin inhibited ESCC cell growth. IHC staining on ESCC tissue microarray and further survival analyses were performed to explore the relationship between target genes' expression and prognosis of ESCC. RESULTS: Melatonin treatment dose-dependently inhibited the proliferative ability and the expression of histone deacetylase 7 (HDAC7), c-Myc and ubiquitin-specific peptidase 10 (USP10) in ESCC cells (P < 0.05). The expressions of HDAC7, c-Myc and USP10 in tumors were detected significantly higher than the paired normal tissues from 148 ESCC patients (P < 0.001). Then, the Kaplan-Meier survival analyses suggested that ESCC patients with high HDAC7, c-Myc or USP10 levels predicted worse overall survival (Log-rank P < 0.001). Co-IP and Western blotting analyses further revealed that HDAC7 physically deacetylated and activated ß-catenin thus promoting downstream target c-Myc gene transcription. Notably, our mechanistic study validated that HDAC7/ß-catenin/c-Myc could form the positive feedback loop to enhance ESCC cell growth, and USP10 could deubiquitinate and stabilize HDAC7 protein in the ESCC cells. Additionally, we verified that inhibition of the HDAC7/ß-catenin/c-Myc axis and USP10/HDAC7 pathway mediated the anti-proliferative action of melatonin on ESCC cells. CONCLUSIONS: Our findings elucidate that melatonin mitigates the HDAC7/ß-catenin/c-Myc positive feedback loop and inhibits the USP10-maintained HDAC7 protein stability thus suppressing ESCC cell growth, and provides the reference for identifying biomarkers and therapeutic targets for ESCC.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Melatonina , Animais , Cateninas/metabolismo , Proliferação de Células , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Retroalimentação , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Melatonina/farmacologia , Melatonina/uso terapêutico , Camundongos , Camundongos Nus , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
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