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1.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198485

RESUMO

Brain microvascular endothelial cells (BMECs) constitute the structural and functional basis for the blood-brain barrier (BBB) and play essential roles in bacterial meningitis. Although the BBB integrity regulation has been under extensive investigation, there is little knowledge regarding the roles of long non-coding RNAs (lncRNAs) in this event. The present study aimed to investigate the roles of one potential lncRNA, lncRSPH9-4, in meningitic E. coli infection of BMECs. LncRSPH9-4 was cytoplasm located and significantly up-regulated in meningitic E. coli-infected hBMECs. Electrical cell-substrate impedance sensing (ECIS) measurement and Western blot assay demonstrated lncRSPH9-4 overexpression in hBMECs mediated the BBB integrity disruption. By RNA-sequencing analysis, 639 mRNAs and 299 miRNAs were significantly differentiated in response to lncRSPH9-4 overexpression. We further found lncRSPH9-4 regulated the permeability in hBMECs by competitively sponging miR-17-5p, thereby increasing MMP3 expression, which targeted the intercellular tight junctions. Here we reported the infection-induced lncRSPH9-4 aggravated disruption of the tight junctions in hBMECs, probably through the miR-17-5p/MMP3 axis. This finding provides new insights into the function of lncRNAs in BBB integrity during meningitic E. coli infection and provides the novel nucleic acid targets for future treatment of bacterial meningitis.


Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Escherichia coli/fisiologia , Metaloproteinase 3 da Matriz/metabolismo , Meningites Bacterianas/genética , Meningites Bacterianas/microbiologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Sequência de Bases , Citoplasma/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , Microvasos/patologia , Modelos Biológicos , Permeabilidade , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Junções Íntimas/metabolismo , Transcrição Genética , Regulação para Cima/genética
2.
Anticancer Res ; 41(7): 3401-3407, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230135

RESUMO

BACKGROUND/AIM: Plakophilin 1 (PKP1) expression is inversely related to cancer grade. This study aimed to evaluate whether PKP1 is a prognostic marker for esophageal cancer (EC). MATERIALS AND METHODS: We tested immunohistochemically for PKP1 in squamous cell carcinoma EC specimens from 99 patients, including cytoplasmic (C), membrane (M), and nuclear (N) cellular areas, and analyzed their relationships with clinicopathological factors. RESULTS: PKP1stains were stratified into strong and weak for all three cellular areas. Staining was inversely related to tumor depth (C: p=0.002, M: p=0.00007, N: p=0.02), lymph node metastasis (C: p=0.003, M: p=0.001, N: p=0.004) and pathological stage (C: p=0.0004, M: p=0.0001, N: p=0.006). Cytoplasmic and membrane staining were inversely related to vessel invasion. Patients with strong C stain had a better overall survival than those with weak C stains (p=0.01). Disease-free survival of patients with strong M stains was better than that of those with weak staining (p=0.01). CONCLUSION: Cytoplasmic and membrane PKP1 expression is a possible prognostic marker for EC.


Assuntos
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Placofilinas/metabolismo , Idoso , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Intervalo Livre de Doença , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Metástase Linfática/patologia , Masculino , Prognóstico
3.
Molecules ; 26(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203597

RESUMO

We used Raman micro-spectroscopy technique to analyze the molecular changes associated with oral squamous cell carcinoma (SCC) cells in the form of frozen tissue. Previously, Raman micro-spectroscopy technique on human tissue was mainly based on spectral analysis, but we worked on imaging of molecular structure. In this study, we evaluated the distribution of four components at the cell level (about 10 µm) to describe the changes in protein and molecular structures of protein belonging to malignant tissue. We analyzed ten oral SCC samples of five patients without special pretreatments of the use of formaldehyde. We obtained cell level images of the oral SCC cells at various components (peak at 935 cm-1: proline and valine, 1004 cm-1: phenylalanine, 1223 cm-1: nucleic acids, and 1650 cm-1: amide I). These mapping images of SCC cells showed the distribution of nucleic acids in the nuclear areas; meanwhile, proline and valine, phenylalanine, and amide I were detected in the cytoplasm areas of the SCC cells. Furthermore, the peak of amide I in the cancer area shifts to the higher wavenumber side, which indicates the α-helix component may decrease in its relative amounts of protein in the ß-sheet or random coil conformation. Imaging of SCC cells with Raman micro-spectroscopy technique indicated that such a new observation of cancer cells is useful for analyzing the detailed distribution of various molecular conformation within SCC cells.


Assuntos
Análise Espectral Raman/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Diagnóstico por Imagem/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Japão , Conformação Molecular , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
4.
Nat Commun ; 12(1): 4229, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244477

RESUMO

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pinças Ópticas , Paxilina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Respiração , Organismos Livres de Patógenos Específicos , Talina/genética , Talina/metabolismo
5.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067970

RESUMO

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.


Assuntos
Citoesqueleto de Actina/metabolismo , Processamento Alternativo , Citoplasma/metabolismo , Éxons , Tropomiosina/metabolismo , Humanos , Isoformas de Proteínas , Tropomiosina/química , Tropomiosina/genética
6.
Nat Commun ; 12(1): 3296, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075043

RESUMO

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.


Assuntos
Endotélio Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Células Receptoras Sensoriais/metabolismo , Vasodilatação/fisiologia , Zinco/metabolismo , Idoso , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Canais de Cálcio Tipo N/metabolismo , Quelantes/farmacologia , Citoplasma/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/inervação , Etilenodiaminas/farmacologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Ratos , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Vasodilatação/efeitos dos fármacos
7.
Gene ; 793: 145749, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34077776

RESUMO

Maize ZmGS5 was reported to be positively associated with kernel-related traits, however, its regulatory mechanism on plant development and seed size remains unknown. In this study, ZmGS5 was demonstrated to be widely expressed in various maize tissues with the highest expression level in developing embryos, indicating its critical roles in early kernel development process. The ZmGS5 protein was subcellularly localized to both the nucleus and cytoplasm. Transgenic Arabidopsis plants overexpressing ZmGS5 under the control of either the constitutive maize Ubiquitin1 promotor or native ZmGS5 promoter resulted in increased plant size, biomass, seed size and weight, although no significant difference was observed between transgenic lines harboring the two constructs. In contrast, the antisense-ZmGS5 transgene resulted in opposite phenotypes. Our cytological data suggested that ZmGS5 enlarged petal size through enhancing cell expansion. Quantitative RT-PCR analysis indicated that ZmGS5 might enhance cell expansion and grain filling by upregulating expression levels of particular EXPA or SWEET genes. Collectively, these findings help us further understand the biological function and regulatory mechanism of ZmGS5 in improving organ size and seed weight, which imply its great potential for high-yield breeding in the future.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Sementes/genética , Transgenes , Zea mays/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Humanos , Tamanho do Órgão , Melhoramento Vegetal/métodos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sementes/anatomia & histologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
8.
Mol Cell Biol ; 41(7): e0010321, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941617

RESUMO

The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full-length ECD but not an ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publicly available BC mRNA The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets. In both data sets, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In the METABRIC data set, ECD overexpression also correlated with poor patient survival in triple-negative breast cancer (TNBC). Furthermore, ECD KD in ErbB2+ BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export and was associated with impairment of oncogenic traits. These findings provide novel mechanistic insight into the physiological and pathological functions of ECD.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , RNA Helicases DEAD-box/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Expressão Gênica/genética , Humanos , Splicing de RNA/genética , Transporte de RNA/genética , Neoplasias de Mama Triplo Negativas/metabolismo
9.
Methods Mol Biol ; 2255: 21-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033091

RESUMO

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.


Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Western Blotting , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
10.
Nat Commun ; 12(1): 2616, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972548

RESUMO

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno , Retina/metabolismo , Fator de Resposta Sérica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
12.
Nat Commun ; 12(1): 3028, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021132

RESUMO

Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Citoplasma/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Sinapses/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Mutação , Fenótipo , Transmissão Sináptica/fisiologia
13.
Methods Mol Biol ; 2262: 185-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977477

RESUMO

Ras proteins are non-integral membrane proteins, which bind to the plasma membrane by virtue of farnesylation and palmitoylation or a positively charged polybasic cluster at their C-terminus. Their membrane interactions and/or localization to membrane microdomains, which play important roles in signaling, are regulated by their lateral diffusion at the plasma membrane and their ability to exchange between the membrane and the cytoplasm (binding/unbinding kinetics). Here, using N-Ras as an example, we describe the use of variations of fluorescence recovery after photobleaching (FRAP) to measure the dynamics of the association of N-Ras with the plasma membrane of living cells and their dependence on several parameters (cholesterol, clustering of raft proteins, and palmitoylation/depalmitoylation).


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Fluorescência Verde/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas ras/metabolismo , Difusão , Humanos , Transporte Proteico , Transdução de Sinais
14.
Nucleic Acids Res ; 49(10): 5867-5880, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048556

RESUMO

Mammalian oocyte maturation is driven by strictly regulated polyadenylation and translational activation of maternal mRNA stored in the cytoplasm. However, the poly(A) polymerase (PAP) that directly mediates cytoplasmic polyadenylation in mammalian oocytes has not been determined. In this study, we identified PAPα as the elusive enzyme that catalyzes cytoplasmic mRNA polyadenylation implicated in mouse oocyte maturation. PAPα was mainly localized in the germinal vesicle (GV) of fully grown oocytes but was distributed to the ooplasm after GV breakdown. Inhibition of PAPα activity impaired cytoplasmic polyadenylation and translation of maternal transcripts, thus blocking meiotic cell cycle progression. Once an oocyte resumes meiosis, activated CDK1 and ERK1/2 cooperatively mediate the phosphorylation of three serine residues of PAPα, 537, 545 and 558, thereby leading to increased activity. This mechanism is responsible for translational activation of transcripts lacking cytoplasmic polyadenylation elements in their 3'-untranslated region (3'-UTR). In turn, activated PAPα stimulated polyadenylation and translation of the mRNA encoding its own (Papola) through a positive feedback circuit. ERK1/2 promoted Papola mRNA translation in a 3'-UTR polyadenylation signal-dependent manner. Through these mechanisms, PAPα activity and levels were significantly amplified, improving the levels of global mRNA polyadenylation and translation, thus, benefiting meiotic cell cycle progression.


Assuntos
Meiose , Oócitos/metabolismo , Oogênese , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro Estocado/metabolismo , Animais , Ciclo Celular , Citoplasma/metabolismo , Vesículas Citoplasmáticas/metabolismo , Células HeLa , Humanos , Meiose/genética , Camundongos , Camundongos Endogâmicos ICR , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oogênese/genética , Fosforilação , Poliadenilação , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Polinucleotídeo Adenililtransferase/genética , Biossíntese de Proteínas , RNA Mensageiro Estocado/genética , RNA Interferente Pequeno , Fuso Acromático/genética , Fuso Acromático/metabolismo , Regulação para Cima
15.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827951

RESUMO

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.


Assuntos
Enterovirus Humano B/imunologia , Enterovirus Humano B/fisiologia , Imunidade Inata , Proteína FUS de Ligação a RNA/metabolismo , Proteases Virais 3C/metabolismo , Animais , Antivirais/farmacologia , Autofagia , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Citocinas/biossíntese , Citocinas/genética , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Genoma Viral , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/genética , Sítios Internos de Entrada Ribossomal , Camundongos , Neurônios Motores/virologia , Poli I-C/farmacologia , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteína FUS de Ligação a RNA/genética , Estresse Fisiológico , Transcrição Genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
16.
Nat Commun ; 12(1): 2166, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846308

RESUMO

Crh proteins catalyze crosslinking of chitin and glucan polymers in fungal cell walls. Here, we show that the BcCrh1 protein from the phytopathogenic fungus Botrytis cinerea acts as a cytoplasmic effector and elicitor of plant defense. BcCrh1 is localized in vacuoles and the endoplasmic reticulum during saprophytic growth. However, upon plant infection, the protein accumulates in infection cushions; it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids are sufficient for protein uptake and cell death induction, respectively. BcCrh1 mutant variants that are unable to dimerize lack transglycosylation activity, but are still able to induce plant cell death. Furthermore, Arabidopsis lines expressing the bccrh1 gene exhibit reduced sensitivity to B. cinerea, suggesting a potential use of the BcCrh1 protein in plant immunization against this necrotrophic pathogen.


Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Botrytis/enzimologia , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Glicosiltransferases/metabolismo , Células Vegetais/microbiologia , Agrobacterium/metabolismo , Botrytis/crescimento & desenvolvimento , Botrytis/patogenicidade , Morte Celular , Resistência à Doença , Proteínas Fúngicas/química , Doenças das Plantas/microbiologia , Imunidade Vegetal , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Tabaco/microbiologia
17.
Nucleic Acids Res ; 49(8): 4613-4628, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33836077

RESUMO

Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.


Assuntos
Epigênese Genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Oryza/metabolismo , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Acetilação , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatografia Líquida , Citoplasma/genética , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Histona Desacetilases/genética , Mutação , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteoma/genética , Proteômica , RNA-Seq , Espectrometria de Massas em Tandem
18.
J Biosci Bioeng ; 132(1): 56-63, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33896701

RESUMO

Dissolved oxygen (DO)-stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was applied to anti-CRP single-chain variable fragment (scFv) production using recombinant Escherichia coli. The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5 to 0.8 g/L by flask and batch cultures to 2.8-3.0 g/L by the fed-batch culture under the conditions of 5-40% of DO saturation. The formation of inclusion bodies was effectively depressed during DO-stat fed-batch operation; consequently, the solubility of anti-CRP scFv was significantly improved from 36-43% by the flask and batch cultures to 96-98% by the DO-stat fed-batch culture under a wide range of DO conditions. From the kinetic analysis of fed-batch experiments, it was also found that the successful folding of anti-CRP scFv in the cytoplasm occurred when metabolic rates, such as the specific growth rate and specific glucose consumption rate, were relatively low. These results show that the fed-batch culture operated by the DO-stat feeding strategy was effective for the enhanced production of anti-CRP scFv with high solubility.


Assuntos
Técnicas de Cultura Celular por Lotes , DNA Recombinante/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Anticorpos de Cadeia Única/biossíntese , Citoplasma/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Fermentação , Corpos de Inclusão/metabolismo , Cinética , Oxigênio/metabolismo
19.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925234

RESUMO

Cytoplasmic male sterility (CMS) is important for large-scale hybrid seed production. Rearrangements in the mitochondrial DNA (mtDNA) for the cotton (Gossypium hirsutum L.) CMS line J4A were responsible for pollen abortion. However, the expression patterns of nuclear genes associated with pollen abortion and the molecular basis of CMS for J4A are unknown, and were the objectives of this study by comparing J4A with the J4B maintainer line. Cytological evaluation of J4A anthers showed that microspore abortion occurs during meiosis preventing pollen development. Changes in enzyme activity of mitochondrial respiratory chain complex IV and mitochondrial respiratory chain complex V and the content of ribosomal protein and ATP during anther abortion were observed for J4A suggesting insufficient synthesis of ATP hindered pollen production. Additionally, levels of sucrose, starch, soluble sugar, and fructose were significantly altered in J4A during the meiosis stage, suggesting reduced sugar metabolism contributed to sterility. Transcriptome and miRNAomics analyses identified 4461 differentially expressed mRNAs (DEGs) and 26 differentially expressed microRNAs (DEMIs). Pathway enrichment analysis indicated that the DEMIs were associated with starch and sugar metabolism. Six deduced target gene regulatory pairs that may participate in CMS were identified, ghi-MIR7484-10/mitogen-activated protein kinase kinase 6 (MAPKK6), ghi-undef-156/agamous-like MADS-box protein AGL19 (AGL19), ghi-MIR171-1-22/SNF1-related protein kinase regulatory subunit gamma-1 and protein trichome birefringence-like 38, and ghi-MIR156-(8/36)/WRKY transcription factor 28 (WRKY28). Overall, a putative CMS mechanism involving mitochondrial dysfunction, the ghi-MIR7484-10/MAPKK6 network, and reduced glucose metabolism was suggested, and ghi-MIR7484-10/MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers.


Assuntos
Gossypium/genética , MicroRNAs/genética , Infertilidade das Plantas/genética , Citoplasma/metabolismo , Citosol/metabolismo , Flores/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Ontologia Genética , Pólen/genética , Transcriptoma/genética
20.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926037

RESUMO

The 20S proteasome, which is composed of layered α and ß heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Microscopia Crioeletrônica/métodos , Citoplasma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Multimerização Proteica/fisiologia , Subunidades Proteicas/metabolismo
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