RESUMO
BACKGROUND: The vast majority of pancreatic cancers have been shown to be insensitive to single-agent immunotherapy. Exploring the mechanisms of immune resistance and implementing combination therapeutic strategies are crucial for PDAC patients to derive benefits from immunotherapy. Deletion of BAP1 occurs in approximately 27% of PDAC patients and is significantly correlated with poor prognosis, but the mechanism how BAP1-deletion compromises survival of patients with PDAC remain a puzzle. METHODS: Bap1 knock-out KPC (KrasG12D/+; LSLTrp53R172H/+; Pdx-1-Cre) mice and control KPC mice, syngeneic xenograft models were applied to analysis the correlation between BAP1 and immune therapy response in PDAC. Immunoprecipitation, RT-qPCR, luciferase and transcriptome analysis were combined to revealing potential mechanisms. Syngeneic xenograft models and flow cytometry were constructed to examine the efficacy of the inhibitor of SIRT1 and its synergistic effect with anti-PD-1 therapy. RESULT: The deletion of BAP1 contributes to the resistance to immunotherapy in PDAC, which is attributable to BAP1's suppression of the transcriptional activity of HSF1. Specifically, BAP1 competes with SIRT1 for binding to the K80 acetylated HSF1. The BAP1-HSF1 interaction preserves the acetylation of HSF1-K80 and promotes HSF1-HSP70 interaction, facilitating HSF1 oligomerization and detachment from the chromatin. Furthermore, we demonstrate that the targeted inhibition of SIRT1 reverses the immune insensitivity in BAP1 deficient PDAC mouse model. CONCLUSION: Our study elucidates an unrevealed mechanism by which BAP1 regulates immune therapy response in PDAC via HSF1 inhibition, and providing promising therapeutic strategies to address immune insensitivity in BAP1-deficient PDAC.
Assuntos
Neoplasias Pancreáticas , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Animais , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Humanos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Linhagem Celular Tumoral , Camundongos Knockout , Imunoterapia/métodosRESUMO
Uveal melanoma (UM) is a neuroectodermal tumor that results from malignant transformation of melanocytes in the eye uvea, including the iris, the ciliary body, and the choroid. UM accounts for 5% of all melanoma cases and is extremely aggressive with half of the UM patients developing metastases within the first 1-2 years after tumor development. Molecular mechanisms of UM carcinogenesis are poorly understood, but are known to differ from those of skin melanoma. Activating mutations of the GNAQ and GNA11 genes, which code for the large G protein subunits Gq and G11, respectively, are found in 90% of UM patients. The Gaq/PKC/MAPK signaling pathway is a main signaling cascade that leads to the transformation of melanocytes of the uveal tract, and major regulators of the cascade provide targets for the development of drugs. Metastatic UM (MUM) is most often associated with mutations of BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. A combination of a commercial expression test panel of 15 genes and a mutation panel of 7 genes, supplemented with data on the size of the primary tumor, is highly efficient in predicting the risk of metastasis. The risk of metastasis determines the choice of therapy and the patient follow-up regimen. However, no systemic therapy for MUM has been developed to date. New drugs undergoing clinical trials are mostly targeted drugs designed to inhibit the protein products of mutant genes or immunotherapeutic agents designed to stimulate the immune response against specific antigens. In addition to these approaches, potential therapeutic targets of epigenetic regulation of UM development are considered in the review.
Assuntos
Melanoma , Mutação , Neoplasias Uveais , Humanos , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Neoplasias Uveais/metabolismo , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/terapia , Melanoma/genética , Melanoma/patologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/terapia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Premature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear. METHODS: We measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose. RESULTS: USP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo. CONCLUSIONS: Upregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.
Assuntos
Senescência Celular , Dano ao DNA , Reparo do DNA , Células da Granulosa , Insuficiência Ovariana Primária , Ubiquitina Tiolesterase , Feminino , Insuficiência Ovariana Primária/patologia , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/genética , Células da Granulosa/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/patologia , Senescência Celular/efeitos dos fármacos , Animais , Humanos , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Reparo do DNA/efeitos dos fármacos , Camundongos , Adulto , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
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.
Assuntos
Neoplasias Colorretais , Progressão da Doença , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico , Ubiquitina Tiolesterase , Ubiquitinação , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Chaperona BiP do Retículo Endoplasmático/metabolismo , Animais , Camundongos , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Camundongos Nus , Proliferação de Células , Masculino , Linhagem Celular Tumoral , Apoptose , Feminino , Movimento CelularRESUMO
Lenvatinib is a targeted drug used for first-line treatment of hepatocellular carcinoma (HCC). A deeper insight into the resistance mechanism of HCC against lenvatinib is urgently needed. In this study, we aimed to dissect the underlying mechanism of lenvatinib resistance (LR) and provide effective treatment strategies. We established an HCC model of acquired LR. Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were used to detect the stemness of HCC cells. Molecular and biochemical strategies such as RNA-sequencing, immunoprecipitation, mass spectrometry and ubiquitination assays were used to explore the underlying mechanisms. Patient-derived HCC models and HCC samples from patients were used to demonstrate clinical significance. We identified that increased cancer stemness driven by the hypoxia-inducible factor-1α (HIF-1α) pathway activation is responsible for acquired LR in HCC. Phosphorylated non-muscle myosin heavy chain 9 (MYH9) at Ser1943, p-MYH9 (Ser1943), could recruit ubiquitin-specific protease 22 (USP22) to deubiquitinate and stabilize HIF-1α in lenvatinib-resistant HCC. Clinically, p-MYH9 (Ser1943) expression was upregulated in HCC samples, which predicted poor prognosis and LR. A casein kinase-2 (CK2) inhibitor and a USP22 inhibitor effectively reversed LR in vivo and in vitro. Therefore, the p-MYH9 (Ser1943)/USP22/HIF-1α axis is critical for LR and cancer stemness. For the diagnosis and treatment of LR in HCC, p-MYH9 (Ser1943), USP22, and HIF-1α might be valuable as novel biomarkers and targets.
Assuntos
Carcinoma Hepatocelular , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Hepáticas , Células-Tronco Neoplásicas , Compostos de Fenilureia , Quinolinas , Ubiquitina Tiolesterase , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Humanos , Quinolinas/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Compostos de Fenilureia/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Camundongos , Linhagem Celular Tumoral , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , MasculinoRESUMO
Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.
Assuntos
Glucose , Mitocôndrias , Fosfatos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucose/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosfatos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Glicólise , Trealose/metabolismo , EndopeptidasesRESUMO
BACKGROUND: Compared with conventional chemotherapy and targeted therapy, immunotherapy has improved the treatment outlook for a variety of solid tumors, including lung cancer, colorectal cancer (CRC), and melanoma. However, it is effective only in certain patients, necessitating the search for alternative strategies to targeted immunotherapy. The deubiquitinating enzyme USP18 is known to play an important role in various aspects of the immune response, but its role in tumor immunity in CRC remains unclear. METHODS: In this study, multiple online datasets were used to systematically analyze the expression, prognosis, and immunomodulatory role of USP18 in CRC. The effect of USP18 on CRC was assessed via shRNA-mediated knockdown of USP18 expression in combination with CCK-8 and colony formation assays. Finally, molecular docking analysis of USP18/ISG15 and programmed death-ligand 1 (PD-L1) was performed via HDOCK, and an ELISA was used to verify the potential of USP18 to regulate PD-L1. RESULTS: Our study revealed that USP18 expression was significantly elevated in CRC patients and closely related to clinicopathological characteristics. The experimental data indicated that silencing USP18 significantly promoted the proliferation and population-dependent growth of CRC cells. In addition, high USP18 expression was positively correlated with the CRC survival rate and closely associated with tumor-infiltrating CD8+ T cells and natural killer (NK) cells. Interestingly, USP18 was correlated with the expression of various chemokines and immune checkpoint genes. The results of molecular docking simulations suggest that USP18 may act as a novel regulator of PD-L1 and that its deficiency may potentiate the antitumor immune response to PD-L1 blockade immunotherapy in CRC. CONCLUSIONS: In summary, USP18 shows great promise for research and clinical application as a potential target for CRC immunotherapy.
Assuntos
Antígeno B7-H1 , Neoplasias Colorretais , Ubiquitina Tiolesterase , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Prognóstico , Proliferação de Células , Linhagem Celular Tumoral , Feminino , Masculino , Regulação Neoplásica da Expressão Gênica , Simulação de Acoplamento MolecularRESUMO
The nucleotide binding oligomerization domain containing 2 (NOD2) protein and its ligand N-acetyl muramyl dipeptide (MDP) are crucially involved in Crohn's disease (CD). However, the mechanism by which NOD2 signaling is regulated in CD patients remains unclear. Ubiquitin specific protease (USP14) is a deubiquitylase that plays an important role in immunity. This study aimed to investigate the mechanism by which UPS14 regulates NOD2 induced inflammatory response in CD and inflammatory bowel diseases (IBD). Our results showed that USP14 protein and mRNA levels in intestinal tissues of CD patients were significantly higher than those in healthy controls. In addition, USP14 was upregulated in IBD mouse model. While treatment with MDP, TNF-α or the Toll-like receptor 1/2 agonist Pam3CSK4 all led to significantly higher mRNA levels of TNF-α, IL-8 and IL-1ß in THP-1 cells, pretreatment with USP14 inhibitor IU1 could stimulate further upregulation of TNF-α, IL-8 and IL-1ß. In particular, MDP promoted the activation of JNK, ERK1/2 and p38 as well as NF-kB in THP-1 cells, and IU1 significantly enhanced the MDP-induced activation of these proteins without effects on USP14 protein level. Furthermore, the JNK inhibitor sp600125, ERK1/2 inhibitor U0126 or P38 MAPK inhibitor PD169316 significantly decreased the mRNA levels of TNF-α, IL-8 and IL-1ß in THP-1 cells stimulated by both IU1 and MDP. In conclusion, our findings suggest that USP14 could inhibit MDP-induced activation of MAPK signaling and the inflammation response involved in IBD, and that USP14 is a potential therapeutic target for IBD.
Assuntos
Doença de Crohn , Proteína Adaptadora de Sinalização NOD2 , Ubiquitina Tiolesterase , Regulação para Cima , Doença de Crohn/metabolismo , Humanos , Proteína Adaptadora de Sinalização NOD2/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Ubiquitina Tiolesterase/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Masculino , Inflamação/metabolismo , Feminino , Adulto , Camundongos Endogâmicos C57BL , Células THP-1RESUMO
BACKGROUND: Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. METHODS: Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. RESULTS: In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. CONCLUSION: Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.
Assuntos
Movimento Celular , Proliferação de Células , Proteína Semelhante a ELAV 1 , Subunidade alfa do Fator 1 Induzível por Hipóxia , Ubiquitina Tiolesterase , Humanos , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Células Cultivadas , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Vasos Retinianos/metabolismo , AngiogêneseRESUMO
Background: Concussion and the damage resulting from this event related to brain function have been widely studied; however, little is known about subconcussive impacts, especially in Mixed Martial Arts (MMA) fighters, which is a combat and full contact sport in which most blows are aimed at the head. Objective: This study aims to evaluate the biomarker levels associated with subconcussive hits to the head in MMA fighters. Methods: This is an exploratory study in which 30 male subjects (10 MMA fighters, 10 healthy individuals who practice muscle training, and 10 healthy sedentary individuals) aged between 18 and 32 years (25.4 ± 3.8) were evaluated. These individuals underwent blood collection to assess their Ubiquitin C-terminal hydrolase (UCH-L1), Glial Fibrillary Acidic Protein (GFAP) and Brain Derived Neurotrophic Factor (BDNF) levels before, immediately after and 72 hours after the sparring session (for the fighters) and were compared between groups. Results: Significant differences were found at baseline between active and healthy fighters in BDNF levels (p = 0.03). A significant reduction of BDNF levels were also observed between the post-immediate and 72h after the sparring session (p = 0.03). No differences were observed in the number or severity of symptoms reported by the fighters. Conclusion: Despite the exploratory approach, the findings of this study may help to understand the influence of repeated subconcussive hits to the head in MMA fighters, as well as to propose preventive interventions which can minimize the effects of the impact of hits, preserving fighters' neuronal integrity and function.
Assuntos
Biomarcadores , Concussão Encefálica , Fator Neurotrófico Derivado do Encéfalo , Artes Marciais , Humanos , Masculino , Artes Marciais/lesões , Concussão Encefálica/sangue , Concussão Encefálica/fisiopatologia , Biomarcadores/sangue , Fator Neurotrófico Derivado do Encéfalo/sangue , Adulto , Adulto Jovem , Proteína Glial Fibrilar Ácida/sangue , Adolescente , Ubiquitina Tiolesterase/sangue , Ubiquitina Tiolesterase/metabolismoRESUMO
Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.
Assuntos
Diferenciação Celular , Condrogênese , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fatores de Transcrição SOX9 , Ubiquitina Tiolesterase , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Diferenciação Celular/genética , Condrogênese/genética , Animais , Humanos , Cartilagem/metabolismo , Condrócitos/metabolismo , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Sistemas CRISPR-Cas , CamundongosRESUMO
Prostate cancer (PCa) is one of the most common male genitourinary system malignancies. Despite the significant benefits of anti-PD-L1 immune checkpoint inhibitor therapy in other cancers, the reasons for its poor therapeutic efficacy in prostate cancer (PCa) remain unclear.NDR1 plays an important role in innate immunity, but its role in tumor immunity and immunotherapy has not been investigated. The role of NDR1 in the immune microenvironment of PCa and the related mechanisms are unknown. Here, we found a positive correlation between NDR1 and PD-L1 expression in PCa. NDR1 significantly inhibits CD8 + T cell infiltration and function, thereby promoting immune escape in prostate cancer.More importantly, NDR1 inhibition significantly enhanced CD8 + T cell activation, which enhanced the therapeutic effect of anti-PD-L1. Mechanistic studies revealed that NDR1 inhibits ubiquitination-mediated PD-L1 degradation via the deubiquitinase USP10, upregulates PD-L1, and promotes PCa immune escape. Thus, our study suggests a unique PD-L1 regulatory mechanism underlying PCa immunotherapy failure. The significance of NDR1 in PCa immune escape and its mechanism of action were clarified, and combined NDR1/PD-L1 inhibition was suggested as an approach to boost PCa immunotherapy effectiveness.
Assuntos
Antígeno B7-H1 , Neoplasias da Próstata , Evasão Tumoral , Ubiquitina Tiolesterase , Ubiquitinação , Masculino , Humanos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Animais , Camundongos , Linfócitos T CD8-Positivos/imunologia , Microambiente Tumoral/imunologiaRESUMO
Lung cancer is one of the most common malignant tumors. Despite decades of research, the treatment of lung cancer remains challenging. Non-small cell lung cancer (NSCLC) is the primary type of lung cancer and is a significant focus of research in lung cancer treatment. The deubiquitinase ubiquitin-specific protease 28 (USP28) plays a role in the progression of various tumors and serves as a potential therapeutic target. This study aims to determine the role of USP28 in the progression of NSCLC. We examined the impact of the USP28 inhibitor AZ1 on the cell cycle, apoptosis, DNA damage response, and cellular immunogenicity in non-small cell lung cancer. We observed that AZ1 and siUSP28 induce DNA damage, leading to the activation of Noxa-mediated mitochondrial apoptosis. The dsDNA and mtDNA released from DNA damage and mitochondrial apoptosis activate tumor cell immunogenicity through the cGAS-STING signaling pathway. Simultaneously, targeting USP28 promotes the degradation of c-MYC, resulting in cell cycle arrest and inhibition of DNA repair. This further promotes DNA damage-induced cell apoptosis mediated by the Noxa protein, thereby enhancing tumor cell immunogenicity mediated by dsDNA and mtDNA. Moreover, we found that the combination of AZ1 and cisplatin (DDP) can enhance therapeutic efficacy, thereby providing a new strategy to overcome cisplatin resistance in NSCLC. These findings suggest that targeting USP28 and combining it with cisplatin are feasible strategies for treating NSCLC.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas , Cisplatino , Dano ao DNA , Neoplasias Pulmonares , Ubiquitina Tiolesterase , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Animais , Camundongos , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , PiperidonasRESUMO
Atherosclerotic cardiovascular disease is closely correlated with elevated low density lipoprotein-cholesterol. In feeding state, glucose and insulin activate mammalian target of rapamycin 1 that phosphorylates the deubiquitylase ubiquitin-specific peptidase 20 (USP20). USP20 then stabilizes HMG-CoA reductase, thereby increasing lipid biosynthesis. In this study, we applied clinically approved lipid nanoparticles to encapsulate the siRNA targeting Usp20. We demonstrated that silencing of hepatic Usp20 by siRNA decreased body weight, improved insulin sensitivity, and increased energy expenditure through elevating UCP1. In Ldlr-/- mice, silencing Usp20 by siRNA decreased lipid levels and prevented atherosclerosis. This study suggests that the RNAi-based therapy targeting hepatic Usp20 has a translational potential to treat metabolic disease.
Assuntos
Síndrome Metabólica , Nanopartículas , RNA Interferente Pequeno , Ubiquitina Tiolesterase , Animais , Camundongos , Nanopartículas/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , RNA Interferente Pequeno/metabolismo , Síndrome Metabólica/metabolismo , Síndrome Metabólica/tratamento farmacológico , Masculino , Receptores de LDL/metabolismo , Receptores de LDL/genética , Camundongos Knockout , Lipídeos/sangue , Lipídeos/química , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Fígado/efeitos dos fármacos , Resistência à Insulina , Aterosclerose/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/prevenção & controle , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteína Desacopladora 1RESUMO
BACKGROUND: Despite improvements in colorectal cancer (CRC) treatment, the prognosis for advanced CRC patients remains poor. Disruption of protein stability is one of the important factors in cancer development and progression. In this study, we aim to identify and analyze novel dysregulated proteins in CRC, assessing their significance and the mechanisms. METHODS: Using quantitative proteomics, expression pattern analysis, and gain-of-function/loss-of-function experiments, we identify novel functional protein dysregulated by ubiquitin-proteasome axis in CRC. Prognostic significance was evaluated in a training cohort of 546 patients and externally validated in 794 patients. Mechanistic insights are gained through molecular biology experiments, deubiquitinating enzymes (DUBs) expression library screening, and RNA sequencing. RESULTS: MAFF protein emerged as the top novel candidate substrate regulated by ubiquitin-proteasome in CRC. MAFF protein was preferentially downregulated in CRC compared to adjacent normal tissues. More importantly, multicenter cohort study identified reduced MAFF protein expression as an independent predictor of overall and disease-free survival in CRC patients. The in vitro and vivo assays showed that MAFF overexpression inhibited CRC growth, while its knockdown had the opposite effect. Intriguingly, we found the abnormal expression of MAFF protein was predominantly regulated via ubiquitination of MAFF, with K48-ubiquitin being dominant. BAP1 as a nuclear deubiquitinating enzyme (DUB), bound to and deubiquitinated MAFF, thereby stabilizing it. Such stabilization upregulated DUSP5 expression, resulting in the inhibition of ERK phosphorylation. CONCLUSIONS: This study describes a novel BAP1-MAFF signaling axis which is crucial for CRC growth, potentially serving as a therapeutic target and a promising prognostic biomarker for CRC.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Ubiquitinação , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão , Proliferação de Células , Progressão da Doença , Neoplasias Pulmonares , Ubiquitina Tiolesterase , Humanos , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Camundongos , Proliferação de Células/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Apoptose/genética , Masculino , Feminino , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , UbiquitinaçãoRESUMO
Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitina/metabolismo , Dano ao DNA , Ligação Proteica , Proteases Específicas de UbiquitinaRESUMO
Induction of resistin-like molecule ß (Relm-ß) and mitofusin 2 (MFN2) mediated aberrant mitochondrial fission have been found to be involved in the pathogenesis of pulmonary arterial hypertension (PAH). However, the molecular mechanisms underlying Relm-ß regulation of MFN2 therefore mitochondrial fission remain unclear. This study aims to address these issues. Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. The results showed that Relm-ß promoted cells proliferation in PASMCs, this was accompanied with the upregulation of USP18, Twist1 and miR-214, and downregulation of MFN2. We found that Relm-ß increased USP18 expression which in turn raised Twist1 by suppressing its proteasome degradation. Elevation of Twist1 increased miR-214 expression and then reduced MFN2 expression and mitochondrial fragmentation leading to PASMCs proliferation. In vivo study, we confirmed that Relm-ß was elevated in MCT-induced PAH rat model, and USP18/Twist1/miR-214/MFN2 axis was altered similar as in vitro. Targeting this cascade by Relm-ß receptor inhibitor Calhex231, proteasome inhibitor MG-132, Twist1 inhibitor Harmine or miR-214 antagomiR prevented the development of pulmonary vascular remodeling and therefore PAH in MCT-treated rats. In conclusion, we demonstrate that Relm-ß promotes PASMCs proliferation and vascular remodeling by activating USP18/Twist1/miR-214 dependent MFN2 reduction and mitochondrial fission, suggesting that this signaling pathway might be a promising target for management of PAH.
Assuntos
Proliferação de Células , GTP Fosfo-Hidrolases , MicroRNAs , Mitocôndrias , Ratos Sprague-Dawley , Transdução de Sinais , Proteína 1 Relacionada a Twist , Ubiquitina Tiolesterase , Animais , Masculino , Ratos , Proliferação de Células/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais , Monocrotalina/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/induzido quimicamente , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
The placenta, a pivotal organ in mammalian reproduction, allows nutrient exchange and hormonal signaling between the mother and the developing fetus. Understanding its molecular intricacies is essential for deciphering normal embryonic development and pathological conditions such as tumorigenesis. Here, we explore the multifaceted role of the tumor suppressor BRCA1-associated protein 1 (BAP1) in cancer and placentation. Initially recognized for its tumor-suppressive properties, BAP1 has emerged as a key regulator at the intersection of tumorigenesis and placental development. BAP1 influences crucial cellular processes such as cell death, proliferation, metabolism, and response to hypoxic conditions. By integrating insights from tumor and developmental biology, we illuminate the complex molecular pathways orchestrated by BAP1. This perspective highlights BAP1's significant impact on both cancer and placental development, and suggests novel therapeutic strategies that could improve outcomes for pregnancy disorders and cancer.