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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544877

RESUMO

Necroptosis is a form of regulated necrosis mediated by the formation of the necrosome, composed of the RIPK1/RIPK3/MLKL complex. Here, we developed a proximity ligation assay (PLA) that allows in situ visualization of necrosomes in necroptotic cells and in vivo. Using PLA assay, we show that necrosomes can be found in close proximity to the endoplasmic reticulum (ER). Furthermore, we show that necroptosis activates ER stress sensors, PERK, IRE1α, and ATF6 in a RIPK1-RIPK3-MLKL axis-dependent manner. Activated MLKL can be translocated to the ER membrane to directly initiate the activation of ER stress signaling. The activation of IRE1α in necroptosis promotes the splicing of XBP1, and the subsequent incorporation of spliced XBP1 messenger RNA (mRNA) into extracellular vesicles (EVs). Finally, we show that unlike that of a conventional ER stress response, necroptosis promotes the activation of unfolded protein response (UPR) sensors without affecting their binding of GRP78. Our study reveals a signaling pathway that links MLKL activation in necroptosis to an unconventional ER stress response.


Assuntos
Endorribonucleases/metabolismo , Proteínas de Choque Térmico/metabolismo , Necroptose , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo , Apoptose , Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Células HT29 , Proteínas de Choque Térmico/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína 1 de Ligação a X-Box/genética , eIF-2 Quinase/genética
2.
mBio ; 12(4): e0178121, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34372695

RESUMO

The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Nucleotídeos de Adenina/metabolismo , Endorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Vírus da Hepatite Murina/enzimologia , Oligorribonucleotídeos/metabolismo , Rotavirus/enzimologia , Animais , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Camundongos
4.
ACS Chem Neurosci ; 12(16): 3101-3111, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34338497

RESUMO

The IRE1/XBP1 signaling pathway is the most conserved component of the endoplasmic reticulum unfolded protein response (UPRER). Activating this branch to correct defects in ER proteostasis is regarded as a promising anti-Parkinson's disease (PD) strategy. P-53 is a marine-derived xyloketal B analog which exhibited potential neuroprotective activities in previous research studies; however, the molecular mechanism underneath its protective effect remains unknown. Herein, a transcriptomic approach was introduced to explore the protective mechanism of P-53. RNA microarray profiling was conducted based on an MPP+-induced C. elegans PD model, and bioinformatics analyses including GO enrichment and PPI network analysis were subsequently performed. In particular, the recovery of the impaired UPRER was highlighted as a main physiological change caused by P-53, and a cluster of genes including abu and hsp family genes which are involved in the IRE1/XBP1 branch of the UPRER were identified as the key genes related to its neuroprotective effect. The transcription levels of these key genes were validated by RT-qPCR assays. Further results showed that P-53 enhanced the phosphorylation of IRE1, the splicing of xbp-1 mRNA, and the translation of XBP1S and boosted the expression level of the downstream targets of the IRE1/XBP1 signaling pathway. Moreover, it was also demonstrated that P-53 accelerated the scavenging of misfolded α-synuclein and attenuated the correlative mitochondrial dysfunction. Finally, the protective effect of P-53 against MPP+-induced dopaminergic neuronal loss was assessed. Taken together, these results revealed that P-53 plays its neuroprotective role through regulating of the IRE1/XBP1 signaling pathway and laid the foundation for its further development as an ER proteostasis-regulating agent.


Assuntos
Caenorhabditis elegans , Endorribonucleases , Proteínas Serina-Treonina Quinases , Proteína 1 de Ligação a X-Box , Animais , Proteínas de Caenorhabditis elegans , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
5.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
6.
Nat Commun ; 12(1): 4105, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215755

RESUMO

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Dermatite/metabolismo , Endorribonucleases/metabolismo , Inflamação/metabolismo , Células Mieloides/metabolismo , Ribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CCL2 , Células Dendríticas , Endorribonucleases/deficiência , Endorribonucleases/genética , Epigenômica , Humanos , Imiquimode , Inflamação/patologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Psoríase , Ribonucleases/deficiência , Ribonucleases/genética , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
7.
Biochem J ; 478(13): 2465-2479, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198324

RESUMO

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Endorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Chlorocebus aethiops , Endorribonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Ensaios Enzimáticos , Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Cinética , Naftoquinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Soluções , Células Vero , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo
8.
Am J Physiol Lung Cell Mol Physiol ; 321(3): L576-L594, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34318710

RESUMO

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.


Assuntos
Endorribonucleases/metabolismo , Células Epiteliais/metabolismo , Hexosaminas/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Resposta a Proteínas não Dobradas , Replicação Viral , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular Transformada , Endorribonucleases/genética , Células Epiteliais/patologia , Células Epiteliais/virologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hexosaminas/genética , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/patologia , Proteína 1 de Ligação a X-Box/genética
9.
Angew Chem Int Ed Engl ; 60(40): 21662-21667, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34278671

RESUMO

There is an urgent need to develop antiviral drugs and alleviate the current COVID-19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2'-OMe-modified antisense oligonucleotide and a 5'-phosphorylated 2'-5' poly(A)4 (4A2-5 ) to degrade envelope and spike RNAs of SARS-CoV-2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A2-5 was used for guided RNase L activation to sequence-specifically degrade viral RNAs. Since RNase L can potently cleave single-stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS-CoV-2 RNA targets. In pseudovirus infection models, chimera-S4 achieved potent and broad-spectrum inhibition of SARS-CoV-2 and its N501Y and/or ΔH69/ΔV70 mutants, indicating a promising antiviral agent based on the nucleic acid-hydrolysis targeting chimera (NATAC) strategy.


Assuntos
Antivirais/farmacologia , Endorribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Chlorocebus aethiops , Proteínas do Envelope de Coronavírus/genética , Desenho de Fármacos , Células HEK293 , Humanos , Hidrólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
10.
PLoS Pathog ; 17(6): e1009644, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138976

RESUMO

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vírus da Hepatite Murina/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Células Vero , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
11.
Nucleic Acids Res ; 49(12): 7035-7052, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125915

RESUMO

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.


Assuntos
Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , Pequeno RNA não Traduzido/metabolismo , Rhodobacter sphaeroides/genética , Pareamento de Bases , Divisão Celular/genética , Endorribonucleases/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/crescimento & desenvolvimento , Rhodobacter sphaeroides/metabolismo , Fator sigma/fisiologia , Estresse Fisiológico/genética
12.
Nucleic Acids Res ; 49(12): 6925-6940, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34151378

RESUMO

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.


Assuntos
RNA Helicases DEAD-box/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Aclimatação , Adenosina Trifosfatases/metabolismo , Animais , Temperatura Baixa , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/classificação , RNA Helicases DEAD-box/fisiologia , Endorribonucleases/metabolismo , Mariposas/microbiologia , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , RNA/metabolismo , Estabilidade de RNA , Análise de Sequência de RNA , Virulência
13.
Arch Insect Biochem Physiol ; 107(4): e21822, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34155698

RESUMO

RNAi efficiency in insects is different from species to species; some species in Coleoptera are relatively more amenable to RNA interference (RNAi) than other species. One of the major factors is the presence of dsRNA-degrading enzymes, called dsRNases, in saliva, gut, or hemolymph in insects, which degrade the double-stranded RNA (dsRNA) introduced, resulting in the low efficacy of RNAi. In this study, we report a dsRNA-degrading activity in the gut homogenates from the spotted-wing drosophila, Drosophila suzukii, by ex vivo assay. Then, we identified two Drosophila suzukii dsRNase genes, named DrosudsRNase1 and DrosudsRNase2. In silico analysis shows that the gene structures are similar to dsRNases found in other insects. When dsRNases expressed in Sf9 cells were compared for their dsRNA degrading activities, dsRNase1 was more vital than dsRNase2. Both dsRNases were expressed highly and exclusively in the gut compared to the rest of body. Also, they were highly expressed during larval and adult stages but not in embryonic and pupal stages, suggesting the dsRNases protect foreign RNA molecules received during the feeding periods. DsRNase1 was expressed at a higher level in adults, whereas dsRNase2 showed more expression in early larvae. Our study on the tissue and development-specific patterns of dsRNases provides an improved understanding of the RNAi application for the management of D. suzukii.


Assuntos
Drosophila/enzimologia , Endorribonucleases/metabolismo , Proteínas de Insetos/metabolismo , RNA de Cadeia Dupla/metabolismo , Sequência de Aminoácidos , Animais , Simulação por Computador , Drosophila/genética , Embrião não Mamífero/enzimologia , Endorribonucleases/genética , Feminino , Trato Gastrointestinal/enzimologia , Proteínas de Insetos/genética , Larva/enzimologia , Masculino , Pupa/enzimologia , Células Sf9
14.
Nat Commun ; 12(1): 3392, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099666

RESUMO

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.


Assuntos
Efeito Espectador/genética , Fator de Transcrição E2F1/metabolismo , MicroRNAs/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Efeito Espectador/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Fator de Transcrição E2F1/genética , Estresse do Retículo Endoplasmático/imunologia , Endorribonucleases/metabolismo , Proteína HMGB1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Listeria monocytogenes/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Shigella flexneri/imunologia , Suínos
15.
Cancer Sci ; 112(7): 2803-2820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34109710

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.


Assuntos
Calreticulina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Técnicas de Inativação de Genes , Inativação Gênica , Proteínas de Choque Térmico HSP47/genética , Xenoenxertos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas
16.
Nat Commun ; 12(1): 3655, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135341

RESUMO

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.


Assuntos
Núcleo Celular/metabolismo , Espaço Extracelular/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Meios de Cultivo Condicionados/metabolismo , Endorribonucleases/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Proteínas de Ligação a RNA/metabolismo
17.
J Virol ; 95(15): e0036121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980594

RESUMO

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Endopeptidases/metabolismo , Endorribonucleases/metabolismo , Vírus da Febre Aftosa/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Animais , Apoptose/imunologia , Linhagem Celular , Cricetinae , Cães , Endopeptidases/genética , Endopeptidases/imunologia , Endorribonucleases/genética , Febre Aftosa/imunologia , Febre Aftosa/virologia , Células HEK293 , Haplorrinos , Humanos , Evasão da Resposta Imune/genética , Células Madin Darby de Rim Canino , Domínios Proteicos , Suínos
18.
Environ Pollut ; 286: 117238, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33984781

RESUMO

Ingestion of food or cereal products contaminated by deoxynivalenol (DON) and related derivatives poses a threat to the health of humans and animals. However, the toxicity and underlying mechanisms of 3-acetyldeoxynivalenol (3-Ac-DON), an acetylated form of deoxynivalenol, have not been fully elucidated. In the present study, we showed that 3-Ac-DON caused significant oxidative damage, as shown by elevated aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) in serum, increased lipid peroxidation products, such as hydrogen peroxide (H2O2) and malondialdehyde (MDA), decreased activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). In addition, 3-Ac-DON exposure led to elevated infiltrations of immune cell, increased apoptosis and autophagy in the liver. Interestingly, 3-Ac-DON-resulted apoptosis and liver injury were partially reduced by autophagy inhibitors. Further study showed that 3-Ac-DON-treated mice had altered ultrastructural changes of endoplasmic reticulum (ER), as well as enhanced protein levels of p-IRE1α, p-PERK, and downstream targets, indicating activation of unfolded protein response (UPR) in the liver. Importantly, 3-Ac-DON induced ER stress, oxidative damage, cell death, infiltration of immune cells, and increased mRNA levels of inflammatory cytokines were significantly abolished by 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, indicating a critical role of UPR signaling for the cellular damage of the liver in response to 3-Ac-DON exposure. In conclusion, using mice as an animal model, we showed that 3-Ac-DON exposure impaired the function of liver, as shown by oxidative damage, cell death, and infiltration of immune cell, in which ER stress played an important role. Restoration of the ER function might be a preventive strategy to reduce the deleterious effect of 3-Ac-DON on the liver of animals.


Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Animais , Apoptose , Endorribonucleases/metabolismo , Peróxido de Hidrogênio/metabolismo , Fígado/metabolismo , Camundongos , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Tricotecenos
19.
Biochimie ; 187: 67-74, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022290

RESUMO

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.


Assuntos
Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética
20.
Cancer Sci ; 112(7): 2739-2752, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33939241

RESUMO

DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dano ao DNA , Fator de Transcrição E2F1/genética , Endorribonucleases/metabolismo , Histonas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Células Cultivadas , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Endorribonucleases/deficiência , Endorribonucleases/isolamento & purificação , Repressão Epigenética , Regulação da Expressão Gênica , Humanos , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/isolamento & purificação , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Receptores de Neuropeptídeo Y/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Genética , Raios Ultravioleta
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