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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203850

RESUMO

Steroid receptor coactivator-1 (SRC-1) is a transcription coactivator playing a pivotal role in mediating a wide range of signaling pathways by interacting with related transcription factors and nuclear receptors. Aberrantly elevated SRC-1 activity is associated with cancer metastasis and progression, and therefore, suppression of SRC-1 is emerging as a promising therapeutic strategy. In this study, we developed a novel SRC-1 degrader for targeted degradation of cellular SRC-1. This molecule consists of a selective ligand for SRC-1 and a bulky hydrophobic group. Since the hydrophobic moiety on the protein surface could mimic a partially denatured hydrophobic region of a protein, SRC-1 could be recognized as an unfolded protein and experience the chaperone-mediated degradation in the cells through the ubiquitin-proteasome system (UPS). Our results demonstrate that a hydrophobic-tagged chimeric molecule is shown to significantly reduce cellular levels of SRC-1 and suppress cancer cell migration and invasion. Together, these results highlight that our SRC-1 degrader represents a novel class of therapeutic candidates for targeting cancer metastasis. Moreover, we believe that the hydrophobic tagging strategy would be widely applicable to develop peptide-based protein degraders with enhanced cellular activity.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Coativador 1 de Receptor Nuclear/metabolismo , Proteólise , Transativadores/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Nat Commun ; 12(1): 4147, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230470

RESUMO

The TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate cancer. However, its role in disease progression is still unclear. In this study, we uncover an important mechanism promoting ERG oncogenic activity. We show that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 methylation modifies intra-domain interactions, favors DNA binding and enhances ERG transcriptional activity. In a genetically engineered mouse model of ERG fusion-positive prostate cancer (Pb-Cre4 Pten flox/flox Rosa26-ERG, ERG/PTEN), ERG K362 methylation is associated with PTEN loss and progression to invasive adenocarcinomas. In both ERG positive VCaP cells and ERG/PTEN mice, PTEN loss results in AKT activation and EZH2 phosphorylation at serine 21 that favors ERG methylation. We find that ERG and EZH2 interact and co-occupy several sites in the genome forming trans-activating complexes. Consistently, ERG/EZH2 co-regulated target genes are deregulated preferentially in tumors with concomitant ERG gain and PTEN loss and in castration-resistant prostate cancers. Collectively, these findings identify ERG methylation as a post-translational modification sustaining disease progression in ERG-positive prostate cancers.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Lisina/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/metabolismo , Regulador Transcricional ERG/metabolismo , Adenocarcinoma/genética , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Serina Endopeptidases/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Regulador Transcricional ERG/genética
3.
Nat Commun ; 12(1): 4164, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230493

RESUMO

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismo , Transativadores/metabolismo , beta Catenina/metabolismo , Animais , Transplante de Medula Óssea , Carcinogênese/genética , Modelos Animais de Doenças , Feminino , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Fator 1 de Transcrição de Linfócitos T/genética , Linfócitos T/metabolismo , Transativadores/genética , Transcriptoma , beta Catenina/genética
4.
Nat Commun ; 12(1): 3965, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172724

RESUMO

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.


Assuntos
Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Fosforilação , RNA Helicases/genética , RNA Helicases/metabolismo , Telomerase/metabolismo , Transativadores/genética , Transativadores/metabolismo
5.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
6.
Int J Nanomedicine ; 16: 3833-3859, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34113102

RESUMO

Background: Hematopoietic stem cells' commitment to myelopoiesis builds immunity to prevent infection. This process is controlled through transcription factor, especially Purine rich box 1 (PU.1) protein, which plays a central role in regulating myelopoiesis. The ß3/ß4 region of PU.1 accommodates a coactivator transcription factor, c-Jun, to activate myelopoiesis. However, an erythroid transcription factor, GATA-1, competes with c-Jun for the ß3/ß4 region, abolishing myelopoiesis and promoting erythropoiesis. This competitive regulation decides the hematopoietic stem cells' commitment towards either erythroid or myeloid lineage. Methods: Therefore, this study investigated the in vitro and in vivo effect of novel synthetic PU.1 ß3/ß4 mimic peptide analogs and peptide-loaded hydrophilic poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles. Results: The designed peptides significantly increase the expression of corresponding myeloid markers, specifically CD33 in vitro. However, the in vivo delivery of peptide-loaded PLGA nanoparticles, which have sustained release effect of peptides, increases 10.8% of granulocytes as compared to control. Conclusion: The observations showed that the fabricated nanoparticles protected the loaded peptides from the harsh intracellular environment for a longer duration without causing any toxicity. These findings highlight the possibility to use these peptides and peptide-loaded nanoparticles to increase hematopoietic stem cell commitment to myeloid cells in case of opportunistic infection.


Assuntos
Eritropoese , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Mielopoese , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Diferenciação Celular , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Proto-Oncogênicas/genética , Transativadores/genética
7.
Aging (Albany NY) ; 13(11): 15523-15537, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099591

RESUMO

S100 calcium-binding protein A8 (S100A8) and S100A9 are small molecular weight calcium-binding regulatory proteins that have been involved in multiple chronic inflammatory diseases. However, the role of S100A8 and S100A9 in keratinocytes in wounded skin and how they are regulated during this process are still unclear. Here, we found that S100A8 and S100A9 were both upregulated in burn-wounded skins in vivo and thermal-stimulated epidermal keratinocytes in vitro, accompanied by increased levels of epithelial-mesenchymal transition (EMT). Then, we demonstrated that upregulation of S100A8 and S100A9 alone or together enhanced characteristics of EMT in normal keratinocytes, manifested by excessive proliferation rate, abnormal ability of cell invasion, and high expression levels of EMT marker proteins. The transcription factor PU box-binding protein (PU.1) bound to the promoter regions and transcriptionally promoted the expression of S100A8 and S100A9 both in the human and mice, and it had strong positive correlations with both S100A8 and S100A9 protein levels in burned skin in vivo. Moreover, PU.1 positively regulated expression of S100A8 and S100A9 in a dose-dependent manner, and enhanced EMT of keratinocytes in vitro. Finally, through the burn mouse model, we found that PU.1-/- mice displayed a lower ability of scar formation, manifested by smaller scar volume, thickness, and collagen content, which could be enhanced by S100A8 and S100A9. In conclusion, PU.1 transcriptionally promotes expression of S100A8 and S100A9, thus positively regulating epithelial-mesenchymal transformation (EMT) and invasive growth of dermal keratinocytes during scar formation post burn.


Assuntos
Queimaduras/patologia , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Movimento Celular , Cicatriz/patologia , Transição Epitelial-Mesenquimal , Queratinócitos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Adulto , Animais , Proliferação de Células , Modelos Animais de Doenças , Feminino , Temperatura Alta , Humanos , Masculino , Camundongos Endogâmicos BALB C , Regulação para Cima/genética
8.
Nat Commun ; 12(1): 3388, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099676

RESUMO

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos Vestíveis
9.
Nat Commun ; 12(1): 3385, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099679

RESUMO

Obesity is the key driver of peripheral insulin resistance, one of the key features of type 2 diabetes (T2D). In insulin-resistant individuals, the expansion of beta-cell mass is able to delay or even prevent the onset of overt T2D. Here, we report that beta-arrestin-1 (barr1), an intracellular protein known to regulate signaling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice maintained on an obesogenic diet. Specifically, insulin-resistant beta-cell-specific barr1 knockout mice display marked reductions in beta-cell mass and the rate of beta-cell proliferation, associated with pronounced impairments in glucose homeostasis. Mechanistic studies suggest that the observed metabolic deficits are due to reduced Pdx1 expression levels caused by beta-cell barr1 deficiency. These findings indicate that strategies aimed at enhancing barr1 activity and/or expression in beta-cells may prove useful to restore proper glucose homeostasis in T2D.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/patologia , Obesidade/metabolismo , beta-Arrestina 1/metabolismo , Animais , Glicemia/metabolismo , Proliferação de Células , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Proteínas de Homeodomínio/metabolismo , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologia , Transativadores/metabolismo , beta-Arrestina 1/genética
10.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065474

RESUMO

Obesity-induced adipose tissue dysfunction and disorders of glycolipid metabolism have become a worldwide research priority. Zfp217 plays a crucial role in adipogenesis of 3T3-L1 preadipocytes, but about its functions in animal models are not yet clear. To explore the role of Zfp217 in high-fat diet (HFD)-induced obese mice, global Zfp217 heterozygous knockout (Zfp217+/-) mice were constructed. Zfp217+/- mice and Zfp217+/+ mice fed a normal chow diet (NC) did not differ significantly in weight gain, percent body fat mass, glucose tolerance, or insulin sensitivity. When challenged with HFD, Zfp217+/- mice had less weight gain than Zfp217+/+ mice. Histological observations revealed that Zfp217+/- mice fed a high-fat diet had much smaller white adipocytes in inguinal white adipose tissue (iWAT). Zfp217+/- mice had improved metabolic profiles, including improved glucose tolerance, enhanced insulin sensitivity, and increased energy expenditure compared to the Zfp217+/+ mice under HFD. We found that adipogenesis-related genes were increased and metabolic thermogenesis-related genes were decreased in the iWAT of HFD-fed Zfp217+/+ mice compared to Zfp217+/- mice. In addition, adipogenesis was markedly reduced in mouse embryonic fibroblasts (MEFs) from Zfp217-deleted mice. Together, these data indicate that Zfp217 is a regulator of energy metabolism and it is likely to provide novel insight into treatment for obesity.


Assuntos
Metabolismo Energético/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Transativadores/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos Brancos/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/fisiopatologia , Animais , Dieta Hiperlipídica , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Termogênese/fisiologia , Ganho de Peso/fisiologia
11.
Mol Cell ; 81(11): 2332-2348.e9, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974912

RESUMO

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.


Assuntos
Carcinogênese/genética , DNA Helicases/genética , Proteínas Intrinsicamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Análise de Sobrevida , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
12.
Angew Chem Int Ed Engl ; 60(9): 4666-4672, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33935588

RESUMO

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.


Assuntos
Proteínas de Escherichia coli/metabolismo , Etionina/metabolismo , Transativadores/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/química , Etionina/análogos & derivados , Etionina/química , Radicais Livres/química , Radicais Livres/metabolismo , Modelos Moleculares , Estrutura Molecular , Transativadores/química
13.
Zhonghua Gan Zang Bing Za Zhi ; 29(4): 350-355, 2021 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-33979962

RESUMO

Objective: To construct a recombinant HBV replication-type plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter in order to elucidate the role of HBx-enhanced HBV replication. Methods: Site-directed mutagenesis technology was used to construct a recombinant plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter on the basis of wild-type HBV replicating plasmid and HBV replicating plasmid lacking HBx expression. Subsequently, plasmid transfection was carried out in HBV liver cancer cell replication model and mouse replication model, and HBV replication intermediates of cells and mouse liver tissue were extracted for detection. Results: Based on the HBV replicating plasmid, the HBV replicating plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter was successfully constructed. HBx-enhanced HBV replication were detected in both the HBV liver cancer replication model and the mouse replication model. After mutating liver-enriched transcription factor binding site mutation at proximal of HBV C promoter, the effect of HBx on the enhancement of HBV replication was not significantly affected. Conclusion: HBx may not enhance HBV replication through liver-enriched transcription factor binding site mutation at proximal of HBV C promoter. The role of other liver-enriched transcription factor binding sites in HBx-enhanced HBV replication needs further study.


Assuntos
Vírus da Hepatite B , Hepatite B , Animais , Sítios de Ligação , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Camundongos , Mutação , Regiões Promotoras Genéticas , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral
14.
Theranostics ; 11(12): 5794-5812, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897882

RESUMO

Rationale: Resistance to androgen-deprivation therapy (ADT) associated with metastatic progression remains a challenging clinical task in prostate cancer (PCa) treatment. Current targeted therapies for castration-resistant prostate cancer (CRPC) are not durable. The exact molecular mechanisms mediating resistance to castration therapy that lead to CRPC progression remain obscure. Methods: The expression of MYB proto-oncogene like 2 (MYBL2) was evaluated in PCa samples. The effect of MYBL2 on the response to ADT was determined by in vitro and in vivo experiments. The survival of patients with PCa was analyzed using clinical specimens (n = 132) and data from The Cancer Genome Atlas (n = 450). The mechanistic model of MYBL2 in regulating gene expression was further detected by subcellular fractionation, western blotting, quantitative real-time PCR, chromatin immunoprecipitation, and luciferase reporter assays. Results: MYBL2 expression was significantly upregulated in CRPC tissues and cell lines. Overexpression of MYBL2 could facilitate castration-resistant growth and metastatic capacity in androgen-dependent PCa cells by promoting YAP1 transcriptional activity via modulating the activity of the Rho GTPases RhoA and LATS1 kinase. Importantly, targeting MYBL2, or treatment with either the YAP/TAZ inhibitor Verteporfin or the RhoA inhibitor Simvastatin, reversed the resistance to ADT and blocked bone metastasis in CRPC cells. Finally, high MYBL2 levels were positively associated with TNM stage, total PSA level, and Gleason score and predicted a higher risk of metastatic relapse and poor prognosis in patients with PCa. Conclusions: Our results reveal a novel molecular mechanism conferring resistance to ADT and provide a strong rationale for potential therapeutic strategies against CRPC.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Antagonistas de Androgênios/farmacologia , Castração/métodos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células PC-3 , Prognóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
15.
Theranostics ; 11(12): 5847-5862, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897885

RESUMO

Metastasis and chemoresistance are major causes of poor prognosis in patients with esophageal squamous cell carcinoma (ESCC), manipulated by multiple factors including deubiquitinating enzyme (DUB). DUB PSMD14 is reported to be a promising therapeutic target in various cancers. Here, we explored the antitumor activity of Thiolutin (THL), the PSMD14 inhibitor, as a new therapy strategy in ESCC. Methods: Through 4-NQO-induced murine ESCC model, we investigated the expression of PSMD14 in esophageal tumorigenesis. Ubiquitin-AMC assay was performed to evaluate DUB activity of PSMD14 with THL treatment. The effect of THL on epithelial-to-mesenchymal transition (EMT), invasion, stemness and chemosensitivity was detected by using in vitro and in vivo experiments. Immunoprecipitation and in vivo ubiquitination assay were conducted to examine whether THL could impair the deubiquitination and stability of SNAIL regulated by PSMD14. Results: Compared with normal esophageal epithelium, PSMD14 was upregulated in 4-NQO-induced murine esophageal epithelium dysplasia and ESCC tissues. THL could significantly weaken DUB activity of PSMD14. Furthermore, the results of in vitro and in vivo assays showed that THL efficiently suppressed motility and stemness and increased sensitivity to cisplatin in ESCC. Mechanically, THL impaired the interaction between PSMD14 and SNAIL, then promoted the ubiquitination and degradation of SNAIL to inhibit EMT which plays a crucial role in ESCC metastasis, stemness and chemosensitivity. TCGA database analysis revealed that high concomitant PSMD14/SNAIL expression predicted shorter overall survival in esophageal cancer. Conclusion: Our findings demonstrate for the first time that suppression of PSMD14/SNAIL axis by THL could be a novel and promising therapeutic approach for ESCC clinical therapy.


Assuntos
Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/patologia , Pirrolidinonas/farmacologia , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
16.
Oxid Med Cell Longev ; 2021: 5521503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815654

RESUMO

Background: Bu Shen Yi Sui capsule (BSYS) is a traditional Chinese medicine prescription that has shown antineuroinflammatory and neuroprotective effects in treating multiple sclerosis (MS) and its animal model of experimental autoimmune encephalomyelitis (EAE). Microglia play an important role in neuroinflammation. The M1 phenotype of microglia is involved in the proinflammatory process of the disease, while the M2 phenotype plays an anti-inflammatory role. Promoting the polarization of microglia to M2 in MS/EAE is a promising therapeutic strategy. This study is aimed at exploring the effects of BSYS on microglial polarization in mice with EAE. Methods: The EAE model was established by the intraperitoneal injection of pertussis toxin and subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG)35-55 in C57BL/6J mice. The mice were treated with BSYS (3.02 g/kg), FTY720 (0.3 mg/kg), or distilled water by intragastric administration. H&E and LFB staining, transmission electron microscopy, qRT-PCR, immunofluorescence, ELISA, fluorescence in situ hybridization, and western blotting were used to detect the histological changes in myelin, microglial M1/M2 polarization markers, and the expression of key genes involved in EAE. Results and Conclusions. BSYS treatment of EAE mice increased the body weight, decreased the clinical score, and reduced demyelination induced by inflammatory infiltration. BSYS also inhibited the mRNA expression of M1 microglial markers while increasing the mRNA level of M2 markers. Additionally, BSYS led to a marked decrease in the ratio of M1 microglia (iNOS+/Iba1+) and an obvious increase in the number of M2 microglia (Arg1+/Iba1+). In the EAE mouse model, miR-124 expression was decreased, and miR-155 expression was increased, while BSYS treatment significantly reversed this effect and modulated the levels of C/EBP α, PU.1, and SOCS1 (target genes of miR-124 and miR-155). Therefore, the neuroprotective effect of BSYS against MS/EAE was related to promoting microglia toward M2 polarization, which may be correlated with changes in miR-124 and miR-155 in vivo.


Assuntos
Encéfalo/patologia , Doenças Desmielinizantes/genética , Medicamentos de Ervas Chinesas/farmacologia , Encefalomielite Autoimune Experimental/genética , Inflamação/patologia , MicroRNAs/metabolismo , Microglia/patologia , Animais , Peso Corporal/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cápsulas , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/patologia , Exossomos/metabolismo , Feminino , Inflamação/genética , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , MicroRNAs/genética , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Medula Espinal/patologia , Transativadores/metabolismo , Regulação para Cima/genética
17.
Biochem Biophys Res Commun ; 555: 128-133, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33813271

RESUMO

mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.


Assuntos
Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Animais , Sistemas CRISPR-Cas , Feminino , Coração/embriologia , Cardiopatias Congênitas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos
18.
FEBS Lett ; 595(11): 1569-1586, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33835503

RESUMO

Among breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive with the worst prognosis and the highest rates of metastatic disease. To identify TNBC gene signatures, we applied the network-based methodology implemented by the SWIM software to gene expression data of TNBC patients in The Cancer Genome Atlas (TCGA) database. SWIM enables to predict key (switch) genes within the co-expression network, whose perturbations in expression pattern and abundance may contribute to the (patho)biological phenotype. Here, SWIM analysis revealed an interesting interplay between the genes encoding the transcription factors HMGA1, FOXM1, and MYBL2, suggesting a potential cooperation among these three switch genes in TNBC development. The correlative nature of this interplay in TNBC was assessed by in vitro experiments, demonstrating how they may actually modulate the expression of each other.


Assuntos
Carcinoma Ductal de Mama/genética , Proteínas de Ciclo Celular/genética , Proteína Forkhead Box M1/genética , Redes Reguladoras de Genes , Proteína HMGA1a/genética , Transativadores/genética , Neoplasias de Mama Triplo Negativas/genética , Atlas como Assunto , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Mineração de Dados , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Feminino , Proteína Forkhead Box M1/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteína HMGA1a/metabolismo , Humanos , Família Multigênica , Ligação Proteica , Transdução de Sinais , Transativadores/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
19.
FASEB J ; 35(5): e21529, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33813778

RESUMO

To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.


Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição MafG/metabolismo , Miosina não Muscular Tipo IIA/química , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fator de Transcrição MafG/antagonistas & inibidores , Fator de Transcrição MafG/genética , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Oligonucleotídeos Antissenso/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias/genética
20.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33830911

RESUMO

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa.Methodology. Computer-based virtual screening under consideration of the 'eNTRy' rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus.Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.


Assuntos
Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Transativadores/antagonistas & inibidores , Animais , Drosophila , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/genética , Taxa de Sobrevida , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Virulência/efeitos dos fármacos , Virulência/genética
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