Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 14.593
Filtrar
1.
PLoS Comput Biol ; 16(9): e1008194, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936799

RESUMO

CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.


Assuntos
Sistemas CRISPR-Cas/genética , Genômica/métodos , Análise de Sequência de DNA/métodos , Software , Teorema de Bayes , Humanos , Células Jurkat , RNA Guia/genética
2.
PLoS Pathog ; 16(9): e1008834, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956422

RESUMO

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Transcrição Genética , Latência Viral , Linfócitos T CD4-Positivos/virologia , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
3.
PLoS Pathog ; 16(9): e1008879, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32997728

RESUMO

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.


Assuntos
Moléculas de Adesão Celular , Adesões Focais , Produtos do Gene gag , Vírus Linfotrópico T Tipo 1 Humano , Proteínas dos Microfilamentos , Fosfoproteínas , Linfócitos T , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Adesões Focais/química , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/virologia , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos , Linfócitos T/química , Linfócitos T/metabolismo , Linfócitos T/virologia
4.
PLoS One ; 15(8): e0235753, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745080

RESUMO

CD154 plays a major role in the pathogenesis of several autoimmune and inflammatory diseases. In addition to CD40, soluble CD154 (sCD154) binds to other receptors namely αIIbß3, αMß2, α5ß1 and αvß3 integrins. We have previously reported that binding of sCD154 to α5ß1 integrin expressed on several human T cell lines is capable of inhibiting Fas-induced cell death. In the current study, we show that such effect of the sCD154/α5ß1 interaction is not restricted to the cell death response induced by Fas but could also be exhibited toward other death signals such as TRAIL and TNF- α. We also demonstrate that sCD154 is capable of inhibiting Fas-mediated death of human activated T cells, more importantly of CD4+ than CD8+ T ones. Our data also show that membrane-bound CD154 and α5ß1 integrin expressed on the surface of distinct cells failed to influence cell death responses. However, when membrane-bound CD154 and α5ß1 are expressed on the surface of same cell, their interaction was capable of down regulating cell death. CD154 was shown to co-localize with the α5ß1 integrin on the surface of these cells. These data strongly suggest a cis-type of interaction between CD154 and α5ß1 when both are expressed on the same cell surface, rather than a trans-interaction which usually implicates the ligand and its receptor each expressed on the surface of a distinct cell. Taken together, these findings add to the list of roles through which CD154 is contributing to the pathogenesis of autoimmune-inflammatory diseases, i.e. by protecting T cells from death and enhancing their survival.


Assuntos
Ligante de CD40/metabolismo , Integrina alfa5beta1/metabolismo , Linfócitos T/citologia , Ligante de CD40/análise , Morte Celular , Células HEK293 , Humanos , Inflamação/metabolismo , Integrina alfa5beta1/análise , Células Jurkat , Mapas de Interação de Proteínas , Linfócitos T/metabolismo
5.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia
6.
Cardiovasc Ther ; 2020: 1389312, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32788926

RESUMO

Inflammation plays a major role in the development of myocardial ischemia-reperfusion (IR) injury. Recombinant human brain natriuretic peptide (rhBNP), a man-made version of a peptide that is elevated in heart failure, exhibits anti-inflammatory effects in various tissues. However, its role in myocardial IR injury remains unclear. In this study, we demonstrate that treatment with rhBNP provided protection for mice against myocardial IR injury as manifested by reduced infarct size and well-preserved myocardial, attenuated inflammatory infiltration and CD4+ T cell proliferation function, and inhibited expression of proinflammatory related genes. Furthermore, mechanistic studies revealed that rhBNP inhibited Jurkat T proliferation by promoting PI3K/AKT/mTOR phosphorylation. Collectively, our data suggest that the administration of rhBNP during IR injury could expand our understanding of the cardioprotective effects of rhBNP.


Assuntos
Anti-Inflamatórios/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Peptídeo Natriurético Encefálico/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Células Jurkat , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/imunologia , Miocárdio/patologia , Fosforilação , Proteínas Recombinantes/farmacologia , Transdução de Sinais
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1144-1151, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798389

RESUMO

OBJECTIVE: To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid (GC) resistance. METHODS: CCRF CEM and Jurkat cells were treated with dexamethasone (DEX) at concentrations of 100、10、1、0.1、0.01 and 0.001 µmol/L, and cultured for 24、48 and 72 h. The proliferation inhibition rate of Jurkat cell was detected by CCK-8. A20 plasmid was constructed, A20-siRNA was designed and synthesized, and transfected into Jurkat cells by liposome. CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group, DEX combined with A20 plasmid group and A20-siRNA group. The mRNA expression level of NF-κB was detected by RT-qPCR, the protein expression level of NF-κB was detected by Western blot, and the apoptosis of Jurkat cells was examined by flow cytometry. RESULTS: The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent (r=0.984, P<0.05) and concentration-dependent (r=0.966, P<0.05). At the point of 24 hour, the IC50 approached 1 µmol/L in CCRF CEM cells. Great large differences began to appear between 1 and 10 µmol/L, the proliferation rate of Jurkat cells treated with 1 µmol/L DEX did not show a significant change. Therefore, 1 µmol/L was selected as control group. The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group. After transfection of A20 plasmid, the expression level of NF-κB was significantly lower than that of control group (P<0.05), and the apoptotic rate was significantly higher than that of control group (P<0.05). After transfection of Jurkat cells with A20-siRNA, the expression level of NF-κB was significantly higher than that of control group (P<0.05). The apoptotic rate of cells in A20-siRNA group was not significantly changed (P>0.05). CONCLUSION: Jurkat cells are resistant to DEX. A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells, down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.


Assuntos
Apoptose , NF-kappa B , Proliferação de Células , Humanos , Células Jurkat , RNA Interferente Pequeno , Transfecção , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
8.
Nat Commun ; 11(1): 3294, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620744

RESUMO

Systemic lupus erythematosus (SLE) is mediated by autoreactive antibodies that damage multiple tissues. Genome-wide association studies (GWAS) link >60 loci with SLE risk, but the causal variants and effector genes are largely unknown. We generated high-resolution spatial maps of SLE variant accessibility and gene connectivity in human follicular helper T cells (TFH), a cell type required for anti-nuclear antibodies characteristic of SLE. Of the ~400 potential regulatory variants identified, 90% exhibit spatial proximity to genes distant in the 1D genome sequence, including variants that loop to regulate the canonical TFH genes BCL6 and CXCR5 as confirmed by genome editing. SLE 'variant-to-gene' maps also implicate genes with no known role in TFH/SLE disease biology, including the kinases HIPK1 and MINK1. Targeting these kinases in TFH inhibits production of IL-21, a cytokine crucial for class-switched B cell antibodies. These studies offer mechanistic insight into the SLE-associated regulatory architecture of the human genome.


Assuntos
Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Lúpus Eritematoso Sistêmico/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Células Cultivadas , Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Interferência de RNA , Receptores CXCR5/genética , Linfócitos T Auxiliares-Indutores/imunologia
9.
Nat Commun ; 11(1): 3301, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620849

RESUMO

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação/genética , Células Cultivadas , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética
10.
Anticancer Res ; 40(8): 4471-4479, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727777

RESUMO

BACKGROUND/AIM: Adult T-cell leukemia (ATL) is a hematological malignancy caused by infection with human T-cell leukemia virus type 1 (HTLV-1). Chemotherapy, antibody therapy, and bone marrow transplantation are used to treat this disease, however, median survival time has not been significantly improved. Our aim was to develop and evaluate a novel antibody-drug conjugate (ADC) with regards to cell cytotoxicity and target specificity. MATERIALS AND METHODS: In this study, we have constructed a novel ADC, which is composed of an anti-CD70 single chain Fv-Fc antibody conjugated with the anticancer agent emtansine using a novel antibody modification method. Cell cytotoxicity and target specificity were assessed using a cell proliferation assay. RESULTS: The anti-CD70 ADC selectively killed HTLV-1-infected cells and ATL cells without affecting other cells. CONCLUSION: The anti-CD70 ADC offers some chemotherapeutic potential for the treatment of ATL.


Assuntos
Ligante CD27/antagonistas & inibidores , Imunoconjugados/farmacologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Maitansina/farmacologia , Anticorpos de Cadeia Única/farmacologia , Adulto , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/terapia , Masculino , Pessoa de Meia-Idade
11.
PLoS One ; 15(7): e0235563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645092

RESUMO

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.


Assuntos
Western Blotting/métodos , Proteínas de Transporte de Cátions/análise , Fracionamento Celular/métodos , Células A549 , Anticorpos/imunologia , Células CACO-2 , Proteínas de Transporte de Cátions/imunologia , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Limite de Detecção , Células MCF-7
12.
Mol Immunol ; 125: 115-122, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659596

RESUMO

Genome-wide association studies have established that human REL is a susceptibility gene for lymphoid cancers and inflammatory diseases. REL is the hematopoietic member of the nuclear factor-κB (NF-κB) family and is frequently amplified in human lymphomas. However, the mechanism through which REL and its encoded protein c-Rel affect human lymphoma is largely unknown. Using both loss-of-function and gain-of-function approaches, we studied the roles of REL gene in human Jurkat leukemia cells. Compared with control Jurkat cells, REL knockout cells exhibited significant defects in cell growth and mitochondrial respiration. Genome-wide transcriptome analyses revealed that T cells lacking c-Rel had selective defects in the expression of inflammatory and metabolic genes including c-Myc. We found that c-Rel controlled the expression of c-Myc through its promotor, and expressing c-Myc in c-Rel-deficient lymphoma cells rescued their proliferative and metabolic defects. Thus, the human c-Rel-c-Myc axis controls lymphoma growth and metabolism and could be a therapeutic target for lymphomas.


Assuntos
Proliferação de Células/fisiologia , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Técnicas de Inativação de Genes , Humanos , Células Jurkat
13.
Proc Natl Acad Sci U S A ; 117(31): 18701-18710, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690679

RESUMO

Yin Yang 1 (YY1) is a DNA-binding transcription factor that either activates or represses gene expression. YY1 has previously been implicated in the transcriptional silencing of many retroviruses by binding to DNA sequences in the U3 region of the viral long terminal repeat (LTR). We here show that YY1 overexpression leads to profound activation, rather than repression, of human T lymphotropic virus type 1 (HTLV-1) expression, while YY1 down-regulation reduces HTLV-1 expression. The YY1 responsive element mapped not to YY1 DNA-binding sites in the HTLV-1 LTR but to the R region. The HTLV-1 R sequence alone is sufficient to provide YY1 responsiveness to a nonresponsive promoter, but only in the sense orientation and only when included as part of the mRNA. YY1 binds to the R region of HTLV-1 RNA in vitro and in vivo, leading to increased transcription initiation and elongation. The findings indicate that YY1 is a potent transactivator of HTLV-1 gene expression acting via binding viral RNA, rather than DNA.


Assuntos
Regulação Viral da Expressão Gênica/genética , Vírus Linfotrópico T Tipo 1 Humano , RNA/metabolismo , Sequências Repetidas Terminais/genética , Fator de Transcrição YY1 , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Ligação Proteica/genética , RNA/genética , Ativação Transcricional/genética , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
14.
PLoS One ; 15(6): e0234867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569300

RESUMO

Different modes of exogenous electrical stimulation at physiological strength has been applied to various diseases. Previously, we extensively demonstrated the usability of mild electrical stimulation (MES) with low frequency pulse current at 55 pulses per second (MES55) for several disease conditions. Here we found that MES with high frequency pulse-current (5500 pulse per second; MES5500) suppressed the overproduction of pro-inflammatory cytokines induced by phorbol myristate acetate/ionomycin in Jurkat T cells and primary splenocytes. MES5500 also suppressed the overproduction of inflammatory cytokines, improved liver damage and reduced mouse spleen enlargement in concanavalin-A-treated BALB/c mice. The molecular mechanism underlying these effects included the ability of MES5500 to induce modest amount of hydrogen peroxide and control multiple signaling pathways important for immune regulation, such as NF-κB, NFAT and NRF2. In the treatment of various inflammatory and immune-related diseases, suppression of excessive inflammatory cytokines is key, but because immunosuppressive drugs used in the clinical setting have serious side effects, development of safer methods of inhibiting cytokines is required. Our finding provides evidence that physical medicine in the form of MES5500 may be considered as a novel therapeutic tool or as adjunctive therapy for inflammatory and immune-related diseases.


Assuntos
Citocinas/imunologia , Terapia por Estimulação Elétrica/métodos , Peróxido de Hidrogênio/imunologia , Imunossupressão/métodos , Inflamação/imunologia , Inflamação/terapia , Animais , Concanavalina A , Feminino , Humanos , Inflamação/induzido quimicamente , Células Jurkat , Fígado/imunologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Baço/patologia
15.
Nat Commun ; 11(1): 3244, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591520

RESUMO

Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.


Assuntos
Organofosfonatos/metabolismo , Biossíntese de Proteínas , Animais , Biotina/metabolismo , Bovinos , Células HeLa , Humanos , Interferon gama/farmacologia , Células Jurkat , Organofosfonatos/química , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Coloração e Rotulagem , Estreptavidina/metabolismo
16.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(3): 753-757, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32552932

RESUMO

OBJECTIVE: To investigate the effects of dihydroartemisinin (DHA) on the proliferation and apoptosis of human T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cell. METHODS: The effects of DHA on the proliferation of Jurkat cells and the recovery of DHA-inhibited cell viability by N-acetyl-L-cysteine (NAC) were examined by CCK-8 assay. Flow cytometry was performed to analyze the cell apoptosis and generation of reactive oxygen species (ROS). Western-blot was used to detected protein expression of DNA damage-related genes, as well as apoptosis-associated genes, respectively. RESULTS: DHA inhibited the proliferation of Jurkat cells, and shows a concentration-dependent manner(r =0.936), and NAC could partially restore the activity of DHA on cell proliferation inhibition. With the increase of drug concentration, the apoptosis rate (r =0.946) and ROS accumulation was increased (r =0.965). Western blot showed that the protein expressions of DNA damage-related gene γ-H2AX and apoptosis-related genes p53, c-Caspase3, BAX and cPARP were significantly increased, and BCL-2 protein expression was decreased. CONCLUSION: DHA can induce ROS production in Jurkat cells, which can cause DNA damage, activate the P53 apoptotic pathway, and promote apoptosis of cells.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Apoptose , Artemisininas , Humanos , Células Jurkat , Estresse Oxidativo , Espécies Reativas de Oxigênio
17.
Clin Sci (Lond) ; 134(12): 1475-1489, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32538435

RESUMO

Sphingosine-1-phosphate (S1P) is a pleiotropic lysosphingolipid derived from the metabolism of plasma membrane lipids. The interaction between S1P and its ubiquitously expressed G-protein-coupled receptors (S1PR1-5) is crucial in many pathophysiological processes. Emerging evidence suggested a potential role for S1P receptors in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In the present study, we investigated the effects of three different S1P receptors modulators (FTY720, SEW2871 and TY52156) in a recognized rat model of experimental autoimmune vasculitis (EAV). The effects of treatments were evaluated with clinico-pathological parameters including hematuria, proteinuria, crescent formation, pulmonary hemorrhage, etc. In vitro functional studies were performed in a Jurkat T-cell line following stimulations of serum from myeloperoxidase-AAV patients. We found that only the FTY720 treatment significantly alleviated hematuria and proteinuria, and diminished glomerular crescent formation, renal tubulointerstitial lesions and pulmonary hemorrhage in EAV. The attenuation was accompanied by less renal T-cell infiltration, up-regulated mRNA of S1PR1 and down-regulated IL-1ß in kidneys, but not altered circulating ANCA levels, suggesting that the therapeutic effects of FTY720 were B-cell independent. Further in vitro studies demonstrated that FTY720 incubation could significantly inhibit the proliferation, adhesion, and migration, and increase apoptosis of T cells. In conclusion, the S1P modulator FTY720 could attenuate EAV through the reduction and inhibition of T cells, which might become a novel treatment of ANCA-associated vasculitis.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Cloridrato de Fingolimode/uso terapêutico , Peroxidase/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/urina , Anticorpos/imunologia , Apoptose , Feminino , Cloridrato de Fingolimode/farmacologia , Hematúria/complicações , Humanos , Células Jurkat , Rim/patologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proteinúria/complicações , Ratos Endogâmicos WKY , Transdução de Sinais
18.
Cell Rep ; 31(11): 107772, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32553163

RESUMO

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.


Assuntos
Citocinas/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismo , Animais , Células HEK293 , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Padrões Moleculares Associados a Patógenos , Febre Tifoide/imunologia , Febre Tifoide/metabolismo , Proteínas não Estruturais Virais/metabolismo
19.
Mol Carcinog ; 59(7): 713-723, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32391973

RESUMO

Chimeric antigen receptor (CAR) T-cells treatment demonstrate the increasing and powerful potential of immunotherapeutic strategies, as seen mainly for hematological malignancies. Still, efficient CAR-T cell approaches for the treatment of a broader spectrum of tumors are needed. It has been shown that cancer cells can implement strategies to evade immune response that include the expression of inhibitory ligands, such as hypersialylated proteins (sialoglycans) on their surface. These may be recognized by sialic acid-binding immunoglobulin-type lectins (siglecs) which are surface receptors found primarily on immune cells. In this regard, siglec-7 and -9 are found on immune cells, such as natural killer cells, T-cells, and dendritic cells and they can promote immune suppression when binding to sialic acids expressed on target cells. In the present study, we hypothesized that it is possible to use genetically engineered T-cells expressing siglec-based CARs, enabling them to recognize and eliminate tumor cells, in a non-histocompatibility complex molecule restricted way. Thus, we genetically modified human T-cells with different chimeric receptors based on the exodomain of human siglec-7 and -9 molecules and selected optimal receptors. We then assessed their antitumor activity in vitro demonstrating the recognition of cell lines from different histologies. These results were confirmed in a tumor xenograft model exemplifying the potential of the present approach. Overall, this study demonstrates the benefit of targeting cancer-associated glycosylation patterns using CAR based on native immune receptors and expressed in human primary T-cells.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Lectinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Células HeLa , Xenoenxertos/metabolismo , Humanos , Células Jurkat , Células K562 , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
20.
Nat Commun ; 11(1): 2193, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366851

RESUMO

Innate immunity to nucleic acids forms the backbone for anti-viral immunity and several inflammatory diseases. Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type I interferon (IFN-I). TRAF3-interacting protein 3 (TRAF3IP3, T3JAM) is essential for T and B cell development. It is also well-expressed by myeloid cells, where its role is unknown. Here we report that TRAF3IP3 suppresses cytosolic poly(I:C), 5'ppp-dsRNA, and vesicular stomatitis virus (VSV) triggers IFN-I expression in overexpression systems and Traf3ip3-/- primary myeloid cells. The mechanism of action is through the interaction of TRAF3IP3 with endogenous TRAF3 and TBK1. This leads to the degradative K48 ubiquitination of TBK1 via its K372 residue in a DTX4-dependent fashion. Mice with myeloid-specific gene deletion of Traf3ip3 have increased RNA virus-triggered IFN-I production and reduced susceptibility to virus. These results identify a function of TRAF3IP3 in the regulation of the host response to cytosolic viral RNA in myeloid cells.


Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Interferon Tipo I/genética , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Viral/genética , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Citosol/virologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Células Jurkat , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/virologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Viral/metabolismo , Células THP-1 , Ubiquitinação , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA