RESUMO
Ferroptosis, an iron-triggered modality of cellular death, has been reported to closely relate to human aging progression and aging-related diseases. However, the involvement of ferroptosis in the development and maintenance of senescent cells still remains elusive. Here, we established a doxorubicin-induced senescent HSkM cell model and found that both iron accumulation and lipid peroxidation increase in senescent cells. Moreover, such iron overload in senescent cells has changed the expression panel of the ferroptosis-response proteins. Interestingly, the iron accumulation and lipid peroxidation does not trigger ferroptosis-induced cell death. Oppositely, senescent cells manifest resistance to the ferroptosis inducers, compared to the proliferating cells. To further investigate the mechanism of ferroptosis-resistance for senescent cells, we traced the iron flux in cell and found iron arrested in lysosome. Moreover, disruption of lysosome functions by chloroquine and LLOMe dramatically triggered the senescent cell death. Besides, the ferroitinophagy-related proteins FTH1/FTL and NCOA4 knockdown also increases the senescent cell death. Thus, we speculated that iron retardation in lysosome of senescent cells is the key mechanism for ferroptosis resistance. And the lysosome is a promising target for senolytic drugs to selectively clear senescent cells and alleviate the aging related diseases.
Assuntos
Senescência Celular , Ferroptose , Ferro , Lisossomos , Ferroptose/efeitos dos fármacos , Humanos , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Doxorrubicina/farmacologia , Coativadores de Receptor Nuclear/metabolismo , Coativadores de Receptor Nuclear/genética , Linhagem Celular , Cloroquina/farmacologia , Sobrecarga de Ferro/metabolismo , Ferritinas , OxirredutasesRESUMO
This study aimed to determine whether E prostanoid receptor-3 (EP3) is involved in prostacyclin (PGI2)-evoked vasoconstrictor activity of resistance arteries and if so, how it changes under hypertensive conditions. Mesenteric resistance arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were isolated for functional and biochemical studies. Here we show that in vessels from WKYs, PGI2 or the endothelial muscarinic agonist ACh (which stimulates in vitro PGI2 synthesis) evoked vasoconstrictor activity, which increased in SHRs. The thromboxane-prostanoid receptor (TP) antagonist SQ29548 partially removed the vasoconstrictor activity, and an increased contractile activity of PGI2 resistant to SQ29548 was observed in SHRs. Interestingly, L798106, an antagonist of EP3 (whose expression was higher in SHRs than in WKYs), not only added to the effect of SQ29548 but also caused relaxation to PGI2 more than that obtained with SQ29548. In accordance, EP3 deletion, which reduced PGI2-evoked contraction, together with SQ29548 resulted in relaxation evoked by the agonist in mouse aortas. These results thus demonstrate an explicit involvement of EP3 in PGI2-evoked vasoconstrictor activity in rat mesenteric resistance arteries and suggest that up-regulation of the receptor contributes significantly to the increased contractile activity evoked by PGI2 under hypertensive conditions.
Assuntos
Epoprostenol/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Feminino , Masculino , Ratos , Ratos Transgênicos , Receptores de Prostaglandina E Subtipo EP3/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Tromboxanos/metabolismoRESUMO
OBJECTIVE: RNAe is a new method that enhances protein expression at the post-transcriptional level. RNAe utility was further explored to improve endogenous protein expression. RESULTS: Transgenic mice were created by targeting RNAe to growth hormone gene into the C57/BL mouse genome by transposon mediated integration; the mice showed a heavier body weight and longer body length compared with normal mice. RNAe can also be used for gene therapy through the delivery of in vitro transcribed RNA. CONCLUSION: This study takes a further step towards applying RNAe in pharmaceutical approaches by transposon-based transgenic mice model construction and the use of in vitro transcribed RNA transfection assay.
Assuntos
Hormônio do Crescimento/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Terapia Genética/métodos , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , GravidezRESUMO
Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.
Assuntos
Técnicas de Inativação de Genes/métodos , HIV-1/fisiologia , Receptores CCR5/deficiência , Receptores de HIV/deficiência , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Adulto , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , HIV-1/crescimento & desenvolvimento , Humanos , Deleção de SequênciaRESUMO
MeCP2 mutations are associated with the Rett syndrome (RTT). Currently, there is an urgent need for new animal models for RTT as the existing MeCP2 knockout mouse models fail to fully mimic the pathogenesis and symptoms of RTT patients. In order to investigate the role of MeCP2 in brain development and RTT pathogenesis, we aimed to set up the MeCP2-null rat model using the CRISPR/Cas9 technology. Firstly we constructed the MeCP2 targeting vector and then microinjected Cas9 mRNA and sgRNA mixtures into fertilized ova of SD rats. The sgRNA was designed to target the exon 2 of MeCP2. Next, knockout rats were confirmed using DNA sequencing and Western blotting. Lastly, phenotypes including growth and behaviors of MeCP2 knockout rats were analyzed. The results indicated that the MeCP2 knockout rats showed body weight loss, anxiety tendency and cognitive deficits. The MeCP2-null rat model established in this study recapitulates the major symptoms of RTT patients and provides an alternative tool for future studies of MeCP2 functions.
Assuntos
Modelos Animais de Doenças , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Animais , Sequência de Bases , Técnicas de Inativação de Genes , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Síndrome de Rett/metabolismoRESUMO
Gene editing in non-human primates may lead to valuable models for exploring the etiologies and therapeutic strategies of genetically based neurological disorders in humans. However, a monkey model of neurological disorders that closely mimics pathological and behavioral deficits in humans has not yet been successfully generated. Microcephalin 1 (MCPH1) is implicated in the evolution of the human brain, and MCPH1 mutation causes microcephaly accompanied by mental retardation. Here we generated a cynomolgus monkey (Macaca fascicularis) carrying biallelic MCPH1 mutations using transcription activator-like effector nucleases. The monkey recapitulated most of the important clinical features observed in patients, including marked reductions in head circumference, premature chromosome condensation (PCC), hypoplasia of the corpus callosum and upper limb spasticity. Moreover, overexpression of MCPH1 in mutated dermal fibroblasts rescued the PCC syndrome. This monkey model may help us elucidate the role of MCPH1 in the pathogenesis of human microcephaly and better understand the function of this protein in the evolution of primate brain size.
Assuntos
Microcefalia/patologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Animais , Sequência de Bases , Comportamento Animal , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Genótipo , Humanos , Macaca fascicularis , Imageamento por Ressonância Magnética , Mutação , FenótipoRESUMO
Liver cancer is one of the most lethal cancer types in humans, but our understanding of the molecular mechanisms underlying this process remains insufficient. Here, we conducted high-content screening of the potential genes involved in liver cancer metastasis, which we selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, based on the SAMcell method and RNA interference technology. We identified two powerful genes in the liver cancer metastasis process, AEG-1 and AKR1C2, both of which proved to be positive regulators in promoting metastasis in liver cancer. Further clinical results verified their roles in liver cancer. In summary, these findings could provide new insight into the liver cancer mechanism and potentially therapeutic novel targets for liver cancer therapies in the future.
Assuntos
Moléculas de Adesão Celular/genética , Hidroxiesteroide Desidrogenases/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fígado/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana , Interferência de RNA/fisiologia , Proteínas de Ligação a RNARESUMO
Age-related macular degeneration (AMD) is a leading cause of irreversible central blindness among the elderly worldwide. We use exome sequencing to analyse nonsynonymous single-nucleotide variants (SNVs) across the whole genome of 216 neovascular AMD cases and 1,553 controls. As a follow-up validation, we evaluate 3,772 neovascular AMD cases and 6,942 controls from five independent cohorts in the East Asian population. Here we show strong evidence of an association at a novel, missense SNV, rs7739323, which is located in the ubiquitin protein ligase E3D (UBE3D) gene (Pmeta=1.46 × 10(-9), odds ratio (OR)=0.74, 95% confidence interval (CI): 0.63-0.88). Furthermore, ablation of the UBE3D protein lead to an abnormal amount of pigment granules deposited in retinal pigment epithelium microvilli area and an abnormal response on electroretinography (ERG) in UBE3D(+/-) heterozygous mice. Our findings indicate that the ubiquitin-proteasome system may play a role in the pathogenesis of neovascular AMD.
Assuntos
Povo Asiático/genética , Degeneração Macular/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Angiografia , Animais , Estudos de Casos e Controles , China , Corantes , Eletrorretinografia , Exoma/genética , Feminino , Predisposição Genética para Doença , Hong Kong , Humanos , Verde de Indocianina , Japão , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Epitélio Pigmentado da Retina/patologia , Análise de Sequência de DNA , Singapura , Tomografia de Coerência ÓpticaRESUMO
Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE-DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE-TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Movimento Celular , Células HeLa , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Fosfotransferases/genética , Engenharia de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
AIM: To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells. METHODS: Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deï¬cient mice (Msx2(-/-) ) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis. RESULTS: After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti-phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real-time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2 overexpressed cell. CONCLUSION: Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells.
RESUMO
A TALE of two assays: Transcription activator-like effectors (TALEs) are programmable proteins that can specifically recognize a DNA sequence. Previous strategies for the synthesis of TALEs were complicated and time-consuming. The solid-phase synthesis strategy demonstrated here allows quick and simple purification of the ligation product.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Transativadores/síntese química , Fatores de Transcrição/síntese química , Células HeLa , Humanos , Engenharia de Proteínas , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The different cell types in an animal are often considered to be specified by combinations of transcription factors, and defined by marker gene expression. This paradigm is challenged, however, in stem cell research and application. Using a mouse embryonic stem cell (mESC) culture system, here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4, Sox2 and Nanog failed to monitor cell status transition during mESC differentiation. On the other hand, the response patterns of cell signalling network to external stimuli, as monitored by the dynamics of protein phosphorylation, changed dramatically. Our results also suggest that an irreversible alternation in cell signalling network precedes the adjustment of transcription factor levels. This is consistent with the notion that signal transduction events regulate cell fate specification. We propose that interrogating cell signalling network can assess the cell property more precisely, and provide a sensitive measurement for the early events in cell fate transition. We wish to bring up attention to the potential problem of cell identification using a few marker genes, and suggest a novel methodology to address this issue.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 8 de Crescimento de Fibroblasto/farmacologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Fator Inibidor de Leucemia/farmacologia , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Homeodomínio/genética , Fator de Transcrição MSX1/genética , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Elementos de RespostaRESUMO
Kinases control many important aspects of cell behavior, such as signal transduction, growth/differentiation, and tumorogenesis. Current methods for assessing kinase activity often require specific antibodies, and/or radioactive labeling. Here we demonstrated a novel detection method to assess kinase activity based on surface enhanced Raman spectroscopy (SERS). Raman signal was obtained after amplification by silver nanoparticles. The sensitivity of this method was comparable to fluorescence measurement of peptide concentration. When purified kinase enzyme was used, the detection limit was comparable to conventional radio-labeling method. We further demonstrated the feasibility to measure kinase activity in crude cell lysate. We suggested this SERS-based kinase activity assay could be a new tool for biomedical research and application.
Assuntos
Bioensaio/métodos , Proteínas Quinases/metabolismo , Análise Espectral Raman/métodos , Animais , Linhagem Celular , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Nanopartículas Metálicas/química , Camundongos , Microscopia de Força Atômica , Peptídeos/química , Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Prata/química , Propriedades de SuperfícieRESUMO
Eaf2 encodes a tumor suppressor that plays multiple functions in transcriptional activation, apoptosis, and embryonic development. In this study, we utilized GFP-EAF2 fusion protein to describe the dynamic subcellular movement of Eaf2. GFP-EAF2 is preferentially localized to the nucleus, and in the presence of ELL, it accumulates in nuclear speckles. However, Eaf2 is an unstable nuclear protein whose stability is affected by serum. Further, we provided first evidence that nuclear distribution of Eaf2 is responsive to DNA damage. Following UV irradiation, Eaf2 is relocalized to the nucleolus, suggesting a possible functional involvement of Eaf2 in DNA damage response.
Assuntos
Dano ao DNA/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Raios Ultravioleta , Animais , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional/efeitos da radiação , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribuição Tecidual/efeitos da radiação , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , TransfecçãoRESUMO
Small interefence RNA (siRNA) has revamped the technology of gene silencing in cultured mammalian cells after its first demonstration by Tushl et al. 4 yr ago. To circumvent the cost and the inconvenience in identifying a unique siRNA duplex that can quench target gene expression, we devised a reporter-based system to test knockdown efficiency of selected siRNAs. We demonstrated that this luciferase-based siRNA testing system can be used to evaluate the knockdown efficiency of a directly transfected siRNA duplex or an siRNA expressed from a lentiviral vector.
Assuntos
RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular , Inativação Gênica , Genes Reporter , Vetores Genéticos , Lentivirus , Luciferases/metabolismo , Camundongos , Mioblastos/metabolismo , Plasmídeos , RNA Interferente Pequeno/análise , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , TransfecçãoRESUMO
Bone morphogenetic protein 4 (BMP4) is a potent growth factor that is involved in many important biological processes. Regulation of the level of secreted mature BMP4 determines the biological effects of BMP4 on cells in the local microenvironment. Previous studies suggested that Gremlin, a member of DAN family proteins, antagonizes BMP4 activity by sequestering extracellular BMP4. Herein, we report a novel intracellular regulatory mechanism by which Gremlin interacts with BMP4 precursor, prevents secretion of mature BMP4, and therefore inhibits BMP4 activity more efficiently. Furthermore, we also defined a 30-amino acid peptide sequence within the Gremlin DAN domain that is essential for BMP4 interaction. This novel Gremlin-mediated BMP4 posttranslational regulatory mechanism implies that the level of BMP4 mRNA expression does not truly reflect BMP4 activity when Gremlin and BMP4 are coexpressed within the same cell. Similar regulatory mechanisms may be utilized by other DAN family proteins.