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1.
Nat Commun ; 11(1): 5079, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033234

RESUMO

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Assuntos
Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/metabolismo , Ontologia Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteína Smad3/metabolismo , Transativadores/metabolismo , Regulação para Cima
2.
Nat Commun ; 11(1): 4997, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020472

RESUMO

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Interleucina-33/metabolismo , Animais , Neoplasias Encefálicas/mortalidade , Carcinogênese , Núcleo Celular/metabolismo , Citocinas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioma/mortalidade , Humanos , Inflamação , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos SCID , Microglia , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Microambiente Tumoral/imunologia
3.
Mol Cell ; 80(1): 140-155.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007254

RESUMO

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Neurônios/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas ELAV/química , Larva/metabolismo , Mutação/genética , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
5.
Nat Commun ; 11(1): 4869, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978394

RESUMO

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.


Assuntos
Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas , Glicosídeo Hidrolases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional
6.
Nat Commun ; 11(1): 4458, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895383

RESUMO

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.


Assuntos
Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Hipotálamo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteína Relacionada com Agouti/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Glicemia/análise , Comunicação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Humanos , Hipotálamo/citologia , Hipotálamo/patologia , Injeções Intraventriculares , Leptina/genética , Masculino , Melanocortinas/metabolismo , Hormônios Estimuladores de Melanócitos/administração & dosagem , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA-Seq , Receptor Tipo 4 de Melanocortina/genética , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismo , Indução de Remissão/métodos , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Técnicas Estereotáxicas , Transcriptoma/efeitos dos fármacos
7.
Nat Commun ; 11(1): 4581, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917893

RESUMO

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética
8.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
9.
Molecules ; 25(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899754

RESUMO

The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.


Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Chlamydomonas reinhardtii/genética , Infecções por Coronavirus/tratamento farmacológico , Lectinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Polifenóis/farmacologia , Polissacarídeos/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/metabolismo , Infecções por Coronavirus/prevenção & controle , Engenharia Genética/métodos , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Pandemias , Polifenóis/química , Polifenóis/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Vacinas Virais/biossíntese , Vacinas Virais/farmacologia
10.
Nat Commun ; 11(1): 4868, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985517

RESUMO

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ubiquitina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição TFIIH/genética , Ubiquitinação
11.
Nat Commun ; 11(1): 4828, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973141

RESUMO

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismo
12.
Nat Commun ; 11(1): 4755, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958772

RESUMO

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.


Assuntos
Homeostase , Oxigênio/metabolismo , RNA Longo não Codificante/fisiologia , Esteróis/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Colesterol/metabolismo , Estrogênios/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Células MCF-7 , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
13.
Virology ; 548: 31-38, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838944

RESUMO

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-ß, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1ß. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation.


Assuntos
Núcleo Celular/metabolismo , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Doenças dos Suínos/metabolismo , alfa Carioferinas/metabolismo , Animais , Núcleo Celular/genética , Infecções por Circoviridae/genética , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/genética , Interações Hospedeiro-Patógeno , Interleucina-10/genética , Interleucina-10/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transporte Proteico , Transdução de Sinais , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , alfa Carioferinas/genética
14.
PLoS One ; 15(8): e0236896, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745119

RESUMO

Single-stranded DNA binding protein 2 (SSBP2) is ubiquitously expressed, with several studies reporting it to be a tumor suppressor. We investigated SSBP2 expression and its clinicopathological significance in gastric cancer. SSBP2 expression was examined by immunohistochemistry in 539 gastric cancer sections. The cases were divided into three subtypes, namely, Epstein-Barr virus-associated (EBV), microsatellite unstable, and others (microsatellite stable and EBV negative), based on the molecular classification of The Cancer Genome Atlas (TCGA). Cases were also divided into two subgroups according to the amplification status of human epidermal growth factor receptor 2 (HER2). Most cases showed SSBP2 positivity, and only 24 (4.5%) cases displayed negative nuclear expression. Loss of nuclear expression correlated significantly with high pT category (P = 0.001), nodal metastasis (P = 0.002), and stage of progression (P = 0.005), with no correlation between molecular characteristics and SSBP2 expression. All HER2 amplification cases displayed positive SSBP2 expression. Negative SSBP2 cases showed significantly shorter recurrence-free survival (RFS) compared to positive SSBP2 cases (P = 0.008). Loss of nuclear expression of SSBP2 was significantly associated with shorter RFS in the microsatellite stable and EBV negative groups (P = 0.002), as well as the HER2 negative group (P = 0.007). However, there were no statistically significant differences in multivariate analyses. Loss of nuclear expression of SSBP2 was a poor prognostic factor, associated with stage of progression and recurrence, and showed no significant difference in molecular characteristics, including TCGA subtype and HER2 status.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptor ErbB-2/genética , Neoplasias Gástricas , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/classificação , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
PLoS Pathog ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760121

RESUMO

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Núcleo Celular/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/genética , DNA Viral/genética , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Células Hep G2 , Humanos , Imunidade Inata/imunologia , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Nucleares/genética , Fator de Transcrição Sp1/genética , Replicação Viral
16.
Nat Commun ; 11(1): 4305, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855391

RESUMO

Oligomeric assemblies of tau and the RNA-binding proteins (RBPs) Musashi (MSI) are reported in Alzheimer's disease (AD). However, the role of MSI and tau interaction in their aggregation process and its effects are nor clearly known in neurodegenerative diseases. Here, we investigated the expression and cellular localization of MSI1 and MSI2 in the brains tissues of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as in the wild-type mice and tau knock-out and P301L tau mouse models. We observed that formation of pathologically relevant protein inclusions was driven by the aberrant interactions between MSI and tau in the nuclei associated with age-dependent extracellular depositions of tau/MSI complexes. Furthermore, tau and MSI interactions induced impairment of nuclear/cytoplasm transport, chromatin remodeling and nuclear lamina formation. Our findings provide mechanistic insight for pathological accumulation of MSI/tau aggregates providing a potential basis for therapeutic interventions in neurodegenerative proteinopathies.


Assuntos
Núcleo Celular/patologia , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas tau/metabolismo , Transporte Ativo do Núcleo Celular , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Lobo Frontal/citologia , Lobo Frontal/patologia , Células HEK293 , Humanos , Corpos de Inclusão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Agregados Proteicos , Ligação Proteica , Proteínas tau/genética
17.
Medicine (Baltimore) ; 99(34): e21841, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32846831

RESUMO

RATIONALE: Ovarian microcystic stromal tumor is a relatively rare tumor type, which is characterized by morphology with microcyst structure, solid cellular areas, and hyalinized fibrous stroma. The most reported tumors were stage I with good prognosis. PATIENT CONCERNS: We report a case of a 33-year-old woman with primary ovarian microcystic stromal tumor with significant bizarre nuclei. We describe the clinical, histopathological, and immunohistochemical findings and review the English literatures. So far, as we know, the patient presented here is a rare case of ovarian microcystic stromal tumor with prominent bizarre nuclei accounting for about 50% of the tumor cells. DIAGNOSES: She was diagnosed with ovarian microcystic stromal tumor with significant bizarre nuclei. INTERVENTIONS: The right ovarian tumor was resected laparoscopically on October 19, 2018. OUTCOMES: Up to now, the patient is free of disease at 19 months of follow-up. LESSONS: This is a rare case of ovarian microcystic stromal tumor with obvious bizarre nuclei. This report will contribute to expand the morphological spectrum of ovarian microcystic stromal tumor.


Assuntos
Núcleo Celular/patologia , Neoplasias Ovarianas/patologia , Tumores do Estroma Gonadal e dos Cordões Sexuais/cirurgia , beta Catenina/metabolismo , Adulto , Assistência ao Convalescente , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica/métodos , Laparoscopia/métodos , Resultado do Tratamento
18.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
19.
Science ; 369(6505): 823-828, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792394

RESUMO

The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/imunologia , Imunidade Inata , Nucleotidiltransferases/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Membrana Nuclear/metabolismo
20.
Nat Cell Biol ; 22(8): 973-985, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32753672

RESUMO

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/fisiologia , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Calcineurina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/fisiologia , Transporte Proteico , Proteínas Qa-SNARE/metabolismo
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