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1.
Nat Commun ; 12(1): 1826, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758188

RESUMO

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.


Assuntos
Envelhecimento/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo , Idoso , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Técnicas de Introdução de Genes , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
2.
Nat Commun ; 12(1): 1865, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767158

RESUMO

Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of this global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Feminino , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitina Tiolesterase/genética
3.
Bull Cancer ; 108(4): 385-398, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33685627

RESUMO

Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histonas/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Ubiquitinadas/fisiologia , Carcinoma/etiologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/terapia , Dano ao DNA , Reparo do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Progressão da Doença , Humanos , Proteínas de Neoplasias/genética , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisão , Elongação da Transcrição Genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668938

RESUMO

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteases/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/metabolismo
5.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530560

RESUMO

Ubiquitin specific protease (USP) 2 is a multifunctional deubiquitinating enzyme. USP2 modulates cell cycle progression, and therefore carcinogenesis, via the deubiquitination of cyclins and Aurora-A. Other tumorigenic molecules, including epidermal growth factor and fatty acid synthase, are also targets for USP2. USP2 additionally prevents p53 signaling. On the other hand, USP2 functions as a key component of the CLOCK/BMAL1 complex and participates in rhythmic gene expression in the suprachiasmatic nucleus and liver. USP2 variants influence energy metabolism by controlling hepatic gluconeogenesis, hepatic cholesterol uptake, adipose tissue inflammation, and subsequent systemic insulin sensitivity. USP2 also has the potential to promote surface expression of ion channels in renal and intestinal epithelial cells. In addition to modifying the production of cytokines in immune cells, USP2 also modulates the signaling molecules that are involved in cytokine signaling in the target cells. Usp2 knockout mice exhibit changes in locomotion and male fertility, which suggest roles for USP2 in the central nervous system and male genital tract, respectively. In this review, we summarize the cellular events with USP2 contributions and list the signaling molecules that are upstream or downstream of USP2. Additionally, we describe phenotypic differences found in the in vitro and in vivo experimental models.


Assuntos
Suscetibilidade a Doenças , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Biomarcadores , Ciclo Celular/genética , Relógios Circadianos/genética , Metabolismo Energético , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Ubiquitina Tiolesterase/genética
6.
Nat Commun ; 12(1): 51, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397955

RESUMO

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin-Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fusão bcr-abl , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-Box/metabolismo , Proteínas ras/metabolismo
7.
Virchows Arch ; 478(1): 59-72, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33475835

RESUMO

The 2015 WHO classification of pleural mesotheliomas includes three major histologic subtypes-epithelioid, sarcomatoid, and biphasic. Recent genomic data has supported the need for a more granular and clinically valid classification beyond the three current subtypes. Because of tumor rarity and overlapping histologic features with other tumor types, diagnostic immunohistochemical work up is essential component in establishing the final diagnosis of mesothelioma. The use of BAP1 and CDKN2A/MTAP improves the diagnostic sensitivity of effusion specimens and are valuable in establishing the diagnosis of epithelioid mesothelioma. The major change in the forthcoming WHO classification is the inclusion of mesothelioma in situ as a diagnostic category. In this review, we discuss recently proposed changes in the histologic classification of pleural mesothelioma, differential diagnosis, and importance of ancillary diagnostic studies.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Pleurais/diagnóstico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , /patologia , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo
8.
Phytomedicine ; 80: 153383, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33091855

RESUMO

BACKGROUND: Caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) is a natural polyphenolic ester isolated as a minor component from a water extract of the Chinese medicine Zhongjiefeng [Sarcandra glabra (Thunb.) Nakai (Chloranthaceae)] and has previously shown to have activity against solid tumors through the modulation of multiple targets or signal pathways. However, the activity and potential mechanism of CADPE against leukemia cells have not yet been characterized. PURPOSE: To investigate whether and how CADPE kills leukemia cells. METHOD: (1) The activity of CADPE inhibiting the growth of different leukemia cell lines was evaluated by MTT assay; (2) Cell cycle arrest and apoptosis induced by CADPE were determined by flow cytometry with FlowJo software for quantification; (3) The protein levels were analyzed by Western blot and ubiquitin-binding c-Myc was acquired by co-immunoprecipitation. RESULTS: CADPE exerted potent activity against different leukemia cell lines with low toxicity in normal cells. In terms of mechanism of action, CADPE promoted ubiquitin-proteasome-dependent degradation of c-Myc through activating glycogen synthase kinase-3ß (GSK3ß) and downregulating deubiquitinating enzyme USP28 to trigger the interaction of c-Myc with ubiquitin ligase Fbw7, resulting in the downregulation of cell cycle regulators and anti-apoptotic proteins and consequently, cell cycle arrest and cell apoptosis. CONCLUSION: CADPE is a novel c-Myc inhibitor with high activity and a unique mechanism for killing leukemia cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ácidos Cafeicos/farmacologia , Leucemia/tratamento farmacológico , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas F-Box/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo
9.
Int Heart J ; 61(5): 1034-1040, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32999190

RESUMO

Low-density lipoprotein (LDL) particles are known to be atherogenic agents in coronary artery diseases. They adjust to other electronegative forms and can be the subject for the enhancement of inflammatory events in vessel subendothelial spaces. The LDL uptake is related to the membrane scavenger receptors, including LDL receptor (LDLR). The LDLR expression is closely associated with LDL uptake and occurrence of diseases, such as atherosclerotic cardiovascular diseases. Our findings identified USP16 as a novel regulator of LDLR due to its ability to prevent ubiquitylation-dependent LDLR degradation, further promoting the uptake of LDL. The enhancement of USP16-mediated deubiquitination andthe suppressive degradation of the LDLR cause the presentation of a potential strategy to increase LDL cholesterol clearance.


Assuntos
Receptores de LDL/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Células HeLa , Humanos , Lipoproteínas LDL/metabolismo , Processamento de Proteína Pós-Traducional
10.
Mol Carcinog ; 59(10): 1199-1208, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32808708

RESUMO

Long noncoding RNAs (lncRNAs) can act as oncogene and tumor suppressor genes in many types of cancers including breast cancer (BC). Our previous study has indicated microRNA (miR)-125a-5p was downregulated and function as a tumor suppressor in BC. However, its upstream regulation mechanism is still unclear. In this study, we used bioinformatics algorithms, RNA pulldown assay, and dual-luciferase reports assay to predict and confirm lncRNA CERS6-AS1 interacted with miR-125a-5p. Then we found CERS6-AS1 was upregulated in BC tissues. Experimental results of tumor growth in nude mice show that CERS6-AS1 promotes tumor growth. Furthermore, CERS6-AS1 regulated BC susceptibility gene 1-associated protein 1 (BAP1) expression via sponging miR-125a-5p via Western blot analysis and quantitative polymerase chain reaction arrays. Finally, we showed that miR-125a-5p had opposing effects to those of CERS6-AS1 on BC cells, demonstrating that CERS6-AS1 may promote cell proliferation and inhibit cell apoptosis via sponging miR-125a-5p. Our results indicated CERS6-AS1 promote BC cell proliferation and inhibit cell apoptosis via sponging miR-125a-5p to upregulate BAP1 expression.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Proteínas de Membrana/antagonistas & inibidores , MicroRNAs/genética , Oligonucleotídeos Antissenso/genética , RNA Longo não Codificante/genética , Esfingosina N-Aciltransferase/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Esfingosina N-Aciltransferase/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Am J Physiol Lung Cell Mol Physiol ; 319(4): L652-L660, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726133

RESUMO

Mesothelial cells are arranged as a monolayer on covering membranes that invest surfaces of body cavities like the pleura and peritoneum. Primary human mesothelial cell (HMC) cultures are needed for studying mesothelial cell homeostasis and developing disease models, such as wound healing or cancers. Remarkably, there is a paucity of useable HMC lines that are currently available that faithfully recapitulate normal in vivo phenotypic characteristics. Here, we present a strategy to recover HMC from human pleural tissue and to immortalize them for extended in vitro culturing. Human pleural membrane was harvested by minimally invasive surgical techniques. HMC were isolated using a two-step process combining explant cellular outgrowth from biopsy tissue and flow cytometry based on cell surface expression of cadherin-1 and CD71. Cell cultures were generated after lentiviral transfection with human telomerase. The new HMC cultures retain the same phenotypic traits and physiologic features as their in vivo counterparts, yet they can be adapted for short-term or long-term culture in large-scale in vitro experimentation. In particular, we generated a new HMC line harboring a germline mutation in breast cancer type-1-associated protein-1 (BAP1), a causal tumor suppressor gene, that could be instrumental to malignant mesothelioma research. Patient-specific, normal HMC may serve as novel discovery tools allowing more powerful research models of both normal physiology and disease processes. Our surgically driven approach leads to a limitless resource of novel mesothelial cell cultures.


Assuntos
Epitélio/patologia , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Pleura/patologia , Neoplasias Pleurais/patologia , Biópsia/métodos , Humanos , Neoplasias Pleurais/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo
12.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32631902

RESUMO

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Citoplasma/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Proteínas Ubiquitinadas/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(30): 17510-17512, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32665439

RESUMO

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.


Assuntos
Endossomos/metabolismo , Interferon Tipo I/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Endossomos/ultraestrutura , Humanos , Transporte Proteico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
14.
Cancer Sci ; 111(9): 3174-3183, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32539182

RESUMO

Programmed cell death-ligand 1 (PD-L1) expressed on cancer cells can cause immune escape of non-small-cell lung cancer (NSCLC). Elucidation of the regulatory mechanisms of the PD-L1 expression is a prerequisite for establishing new tumor immunotherapy strategies. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a regulator of cellular signaling transduction that is aberrantly expressed in NSCLC. However, it is not known whether UCHL1 regulates the expression of PD-L1 in NSCLC cells. In the present study, we found that UCHL1 promotes the expression of PD-L1 in NSCLC cell lines. In addition, UCHL1 expressed in NSCLC cells inhibited activation of Jurkat cells through upregulation of PD-L1 expression in in vitro experiments. Moreover, UCHL1 upregulates PD-L1 expression through facilitating activation of the AKT-P65 signaling pathway. In conclusion, these results indicated that UCHL1 promoted PD-L1 expression in NSCLC cells. This finding implied that inhibition of UCHL1 might suppress immune escape of NSCLC through downregulation of PD-L1 expression in NSCLC cells.


Assuntos
Antígeno B7-H1/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Imunomodulação , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo
15.
Nat Commun ; 11(1): 3018, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541668

RESUMO

Chronic pancreatitis represents a risk factor for the development of pancreatic cancer. We find that heterozygous loss of histone H2A lysine 119 deubiquitinase BAP1 (BRCA1 Associated Protein-1) associates with a history of chronic pancreatitis and occurs in 25% of pancreatic ductal adenocarcinomas and 40% of acinar cell carcinomas. Deletion or heterozygous loss of Bap1 in murine pancreata causes genomic instability, tissue damage, and pancreatitis with full penetrance. Concomitant expression of KrasG12D leads to predominantly intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, while pancreatic intraepithelial neoplasias are rarely detected. These lesions progress to metastatic pancreatic cancer with high frequency. Lesions with histological features mimicking Acinar Cell Carcinomas are also observed in some tumors. Heterozygous mice also develop pancreatic cancer suggesting a haploinsufficient tumor suppressor role for BAP1. Mechanistically, BAP1 regulates genomic stability, in a catalytic independent manner, and its loss confers sensitivity to irradiation and platinum-based chemotherapy in pancreatic cancer.


Assuntos
Neoplasias Pancreáticas/metabolismo , Pancreatite Crônica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Haploinsuficiência , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
16.
Nature ; 582(7812): 416-420, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499641

RESUMO

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.


Assuntos
Sistemas CRISPR-Cas , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/biossíntese , Edição de Genes , Regulação da Expressão Gênica , Humanos , Imunoterapia , Masculino , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/prevenção & controle , Estabilidade Proteica , Reprodutibilidade dos Testes , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo
17.
Gene ; 753: 144798, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32445925

RESUMO

Gastric carcinoma (GC) ranks fifth in terms of cancer morbidity and third in cancer-related death worldwide and imposes enormous health and economic burdens. The molecular mechanisms underlying GC formation and progression remain unclear. Our aim was to identify the involvement of circular RNA circFOXO3 in GC, and to determine the underlying mechanisms. In this study, we revealed a stimulatory role of circular RNA circFOXO3 in tumor growth in vivo. CircFOXO3 enhanced GC cell proliferation and migration in vitro and promoted tumor growth of GC cells in vivo. Bioinformatic analysis revealed that circFOXO3 might regulate USP44 expression by specifically binding to microRNA (miR)-143-3p. Existence of circFOXO3-miR-143-3p-USP44 axis in GC cells was confirmed by RNA-binding protein immunoprecipitation, luciferase reporter assay, and an RNA pull-down experiments. All the data indicate that circFOXO3 promotes GC cell proliferation and migration by upregulating USP44 expression via targeting of miR-143-3p.


Assuntos
Ácidos Nucleicos Livres/metabolismo , Proteína Forkhead Box O3/genética , MicroRNAs/metabolismo , Neoplasias Gástricas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ácidos Nucleicos Livres/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Proteína Forkhead Box O3/metabolismo , Células HEK293 , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ativação Transcricional , Ubiquitina Tiolesterase/genética , Regulação para Cima
18.
Sci Rep ; 10(1): 6515, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296077

RESUMO

DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m2) and 24 obese women (BMI: 43.3 ± 5.7 kg/m2) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip (cutoff of p ≤ 0.05 and fold change ≥2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.


Assuntos
Adiposidade/genética , Cirurgia Bariátrica , Metilação de DNA , Obesidade/genética , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Aminopeptidases/genética , Aminopeptidases/metabolismo , Fator 1 de Resposta a Butirato/genética , Fator 1 de Resposta a Butirato/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Obesidade/cirurgia , Período Pós-Operatório , Período Pré-Operatório , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
19.
BMC Cancer ; 20(1): 214, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164618

RESUMO

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer. Ubiquitin-specific protease (USP)44 has been reported to be involved in various cancers. We investigated the function, role and molecular mechanism of USP44 in ccRCC. METHODS: Data obtained from the Cancer Genome Atlas Data Portal and Gene Expression Omnibus database were analyzed to uncover the clinical relevance of USP44 expression and tumor development. USP44 function in the proliferation and migration of tumor cells was assessed by cellular and molecular analyses using ccRCC lines (786-O cells and Caki-1 cells). RESULTS: USP44 showed low expression in ccRCC cancer tissues compared with that in normal tissue. USP44 expression was negatively correlated with tumor stage, tumor grade, and patient survival. USP44 overexpression inhibited the proliferation and migration of 786-O cells and Caki-1 cells significantly. USP44 overexpression also prohibited cell proliferation by upregulating expression of P21, downregulating cyclin-D1 expression, and inhibiting cell migration by downregulating expression of matrix metalloproteinase (MMP)2 and MMP9. USP44 knockdown enhanced the proliferation and migration of 786-O cells and Caki-1 cells. USP44 function in inhibiting the proliferation and migration of 786-O cells and Caki-1 cells was associated with phosphorylation of Jun N-terminal kinase (JNK). CONCLUSION: USP44 may be a marker in predicting ccRCC progression. Inhibition by USP44 of the proliferation and migration of 786-O cells and Caki-1 cells is dependent upon the JNK pathway.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/patologia , Regulação para Baixo , Neoplasias Renais/patologia , Ubiquitina Tiolesterase/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Gradação de Tumores , Análise de Sobrevida , Ubiquitina Tiolesterase/metabolismo
20.
Sci Adv ; 6(11): eaay1109, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32195343

RESUMO

Protein modification with ISG15 (ISGylation) represents a major type I IFN-induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies.


Assuntos
Infecções por Coxsackievirus/metabolismo , Citocinas/metabolismo , Enterovirus Humano B/metabolismo , Fígado/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções por Coxsackievirus/genética , Citocinas/genética , Feminino , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
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