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1.
Life Sci ; 259: 118375, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891612

RESUMO

OBJECTIVE: Short-chain fatty acids were reported to be the precursors of milk fat and can stimulate the de novo synthesis of fatty acids in bovine mammary epithelial cells (bMECs). However, the mechanism has not been elucidated. The purpose of this study was to investigate the effects of sodium butyrate (NaB) on milk fat synthesis in bMECs and explore its potential mechanism. METHODS: Bovine mammary epithelial cells (bMECs) were isolated for subsequent experimental uses. BODIPY staining and triglyceride kit were used to detect the milk fat synthesis in bMECs. Western blotting and RT-PCR assays were performed to detect the expression of related genes in bMECs. Immunoprecipitation was used to detect the acetylation of SREBP1 in bMECs. RESULTS: The results showed that NaB significantly promoted milk fat synthesis, promoted the activity of mechanistic target of rapamycin (mTOR) and S6 kinase (S6K), inhibited the activity of AMP-activated protein kinase (AMPK), and promoted the gene expression of G protein-coupled receptor 41 (GPR41). Knockdown of GPR41 and sterol regulatory element binding protein 1 (SREBP1) and overexpression of sirtuin1 (SIRT1), mTOR inhibitor (rapamycin), and AMPK activator (AICIR) eliminated these effects. These results indicated that NaB increased the nuclear translocation of SREBP1 via the GPR41/AMPK/mTOR/S6K signalling pathway, promoted the acetylation of mature SREBP1a via GPR41/AMPK/SIRT1, and then promoted milk fat synthesis. CONCLUSION: Taken together, these results demonstrated that NaB increased nuclear translocation and acetylation of SREBP1 to promote milk fat synthesis by activating GPR41 and its downstream signalling pathways.


Assuntos
Ácido Butírico/farmacologia , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Glândulas Mamárias Animais/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Carbazóis , Bovinos , Células Cultivadas , Feminino , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Naftalenos , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
2.
Life Sci ; 259: 118383, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896555

RESUMO

AIMS: Previous studies have shown that the widespread use of estrogen preparations can cause adverse outcomes such as thrombosis and cardiovascular disease. Autophagy is a biochemical process necessary to maintain cell homeostasis. The present study investigated whether E-2 mediates autophagy-induced endothelial cell dysfunction. The role of aspirin in this process was then studied. MAIN METHODS: Western blot, fluorescence microscopy, electron transmission microscopy, plasma construction and transfection, vasoreactivity study in wire myograph are all used in this study. KEY FINDINGS: We found that E-2 activated the PI3K/mTOR signaling pathway and inhibited the formation of the Atg14L-Beclin1-Vps34-Vps15 complex, thereby inhibiting autophagy. Aspirin promoted Beclin1 phosphorylation in autophagy initiation complexes and enhanced autophagy. Furthermore, E-2 treatment of HAECs resulted in endothelial dysfunction by inhibiting autophagy and leading to accumulation of α-smooth muscle actin (α-SMA). E-2 inhibited the activation of eNOS and reduced the expression of eNOS protein. In the mouse aortic vascular function test, E-2 disrupted endothelium-dependent vasodilation. An α-SMA-shRNA lentivirus eliminated the disruption to endothelium-dependent vasodilation by E-2. Aspirin inhibited α-SMA accumulation by enhancing autophagy, reversed endothelial functional impairment caused by E-2, and promoted endothelium-dependent vasodilation. SIGNIFICANCE: This study provides new evidence that E-2 inhibits autophagy and induces abnormal accumulation of α-SMA, resulting in endothelial cell dysfunction and affecting vasodilation. Aspirin can effectively restore the endothelial cell function disrupted E-2.


Assuntos
Actinas/metabolismo , Aspirina/farmacologia , Autofagia/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Estradiol/metabolismo , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fosforilação/efeitos dos fármacos
4.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32817357

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Médica Translacional , Adulto Jovem
5.
PLoS Genet ; 16(8): e1008941, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760060

RESUMO

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.


Assuntos
Proteínas de Transporte/genética , Lipídeos/genética , Lipoproteínas/genética , Triglicerídeos/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Transporte Proteico/genética , Triglicerídeos/metabolismo , Peixe-Zebra/genética
6.
PLoS Genet ; 16(8): e1008955, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776921

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by excess lipid accumulation in the liver without significant consumption of alcohol. The transmembrane 6 superfamily member 2 (TM6SF2) E167K missense variant strongly associates with NAFLD in humans. The E167K mutation destabilizes TM6SF2, resulting in hepatic lipid accumulation and low serum lipid levels. However, the molecular mechanism by which TM6SF2 regulates lipid metabolism remains unclear. By using tandem affinity purification in combination with mass spectrometry, we found that apolipoprotein B (APOB), ER lipid raft protein (ERLIN) 1 and 2 were TM6SF2-interacting proteins. ERLINs and TM6SF2 mutually bound and stabilized each other. TM6SF2 bound and stabilized APOB via two luminal loops. ERLINs did not interact with APOB directly but still increased APOB stability through stabilizing TM6SF2. This APOB stabilization was hampered by the E167K mutation that reduced the protein expression of TM6SF2. In mice, knockout of Tm6sf2 and knockdown of Tm6sf2 or Erlins decreased hepatic APOB protein level, causing lipid accumulation in the liver and lowering lipid levels in the serum. We conclude that defective APOB stabilization, as a result of ERLINs or TM6SF2 deficiency or E167K mutation, is a key factor contributing to NAFLD.


Assuntos
Apolipoproteína B-100/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Colesterol/genética , Colesterol/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Imunoprecipitação , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Lipídeos/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/genética , Transfecção
7.
PLoS Genet ; 16(7): e1008923, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735630

RESUMO

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1LV1, RCC1LV2 and RCC1LV3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1LV1 and RCC1LV3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1LV2. Overexpression and silencing of RCC1LV1 or RCC1LV3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas Mitocondriais/genética , Isoformas de Proteínas/genética , Proteínas Ribossômicas/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Biossíntese de Proteínas/genética , RNA/genética , Proteínas de Ligação a RNA/genética
8.
Nat Commun ; 11(1): 4053, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792481

RESUMO

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequenciamento Completo do Exoma/métodos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/uso terapêutico , Piridinas/uso terapêutico
9.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
10.
Nat Commun ; 11(1): 4060, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792512

RESUMO

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Drosophila , Epigênese Genética/genética , Citometria de Fluxo , Imunofluorescência , Histona-Lisina N-Metiltransferase/genética , Imunoprecipitação , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética
11.
Nucleic Acids Res ; 48(15): e90, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32609809

RESUMO

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional
12.
Vet Microbiol ; 246: 108729, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32605758

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes severe diarrhea in pigs of all ages and a high fatality rate in neonates. The PEDV membrane protein (M) plays crucial roles in viral assembly, viral budding and host immune regulation, most likely by interacting with host cell proteins that have yet to be identified. In this study, co-immunoprecipitation (Co-IP) using an M-specific monoclonal antibody, coupled with LC-MS/MS, was employed to identify M protein-interacting proteins in PEDV-infected cells. Three viral proteins (S, E and ORF3) and 218 host cell proteins were identified as putative M-interacting partners. Bioinformatic analysis showed that the identified host cell proteins were related to 131 signal pathways and 10 biological processes. In addition, interaction between translation initiation factor 3(eIF3L) and M protein was validated by Co-IP. Down-regulation of eIF3L expression significantly increased viral production, which suggests that eIF3L could be a negative regulator in PEDV replication. This interactome study of the PEDV M protein will serve to clarify its function during viral replication.


Assuntos
Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas da Matriz Viral/fisiologia , Animais , Chlorocebus aethiops , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Imunoprecipitação , Interferência de RNA , Células Vero
13.
J Vis Exp ; (159)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32478728

RESUMO

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.


Assuntos
5-Metilcitosina/metabolismo , Arabidopsis/metabolismo , Imunoprecipitação/métodos , RNA de Plantas/metabolismo , Arabidopsis/genética , Sequência de Bases , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , Análise de Sequência de RNA/métodos , Transcrição Genética
14.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510481

RESUMO

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Imunoprecipitação/métodos , Animais , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Extratos de Tecidos/metabolismo
15.
Nucleic Acids Res ; 48(14): e84, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32544226

RESUMO

R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA-DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA-RNA immunoprecipitation (qDRIP), a method combining synthetic RNA-DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA-DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA-DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.


Assuntos
DNA/metabolismo , Imunoprecipitação/métodos , Hibridização de Ácido Nucleico , Estruturas R-Loop , RNA/metabolismo , Animais , Linhagem Celular , Drosophila/citologia , Biblioteca Gênica , Genoma , Meia-Vida , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Ribonuclease H , Sonicação , Transcrição Genética
16.
Nat Commun ; 11(1): 2954, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528069

RESUMO

Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.


Assuntos
Cálcio/metabolismo , Ebolavirus/patogenicidade , Neurônios/metabolismo , Western Blotting , Proteínas de Transporte/metabolismo , Sobrevivência Celular/fisiologia , Ebolavirus/genética , Células HEK293 , Células HeLa , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunoprecipitação , Interferons/metabolismo , Cinética , Ultrassonografia
17.
Nat Commun ; 11(1): 2919, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522980

RESUMO

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.


Assuntos
Proteína Centromérica A/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proteína Centromérica A/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila , Proteínas de Drosophila/genética , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Mitose/genética , Mitose/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética
18.
Nat Commun ; 11(1): 2953, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528005

RESUMO

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.


Assuntos
Proteínas de Transporte/metabolismo , Ebolavirus/patogenicidade , Interferons/farmacologia , Animais , Western Blotting , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Ebolavirus/metabolismo , Imunofluorescência , Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Proteínas Virais/metabolismo , Internalização do Vírus , Replicação Viral/genética , Replicação Viral/fisiologia
19.
Emerg Microbes Infect ; 9(1): 1497-1505, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529906

RESUMO

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Vírus da SARS/imunologia , Testes Sorológicos/métodos , Síndrome Respiratória Aguda Grave/diagnóstico , Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Reações Cruzadas , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Células HEK293 , Humanos , Imunoprecipitação , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Domínios Proteicos/imunologia , Vírus da SARS/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia
20.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
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