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1.
Proc Natl Acad Sci U S A ; 117(32): 19237-19244, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723815

RESUMO

The 5' messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3' end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3' to 5' degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.


Assuntos
Endorribonucleases/metabolismo , RNA Mensageiro/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo
2.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-595042

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
3.
PLoS Biol ; 18(6): e3000687, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520957

RESUMO

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.


Assuntos
Antígeno B7-H1/metabolismo , Polaridade Celular , Endorribonucleases/metabolismo , Macrófagos/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/patologia , Modelos Lineares , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Neoplasias/metabolismo , Fenótipo , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/metabolismo
4.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529952

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
5.
Nucleic Acids Res ; 48(14): 7609-7622, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32476018

RESUMO

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.


Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Proteínas de Drosophila/fisiologia , Éxons , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fosfotransferases/metabolismo , Fosfotransferases/fisiologia , Clivagem do RNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
6.
J Allergy Clin Immunol ; 146(2): 330-331, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593491
7.
Life Sci ; 255: 117842, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: covidwho-403331

RESUMO

The outbreak of COVID-19 caused by 2019-nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Diterpenos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melatonina/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Fator 6 Ativador da Transcrição/metabolismo , Antivirais/farmacologia , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Humanos , Terapia de Alvo Molecular , Pandemias , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
8.
Nat Commun ; 11(1): 2401, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409639

RESUMO

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.


Assuntos
Sobrevivência Celular/genética , Reparo do DNA , Proteínas de Drosophila/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA/genética , Animais , Dano ao DNA , Proteínas de Drosophila/genética , Drosophila melanogaster , Endorribonucleases/genética , Feminino , Fibroblastos , Instabilidade Genômica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Proteostase/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , RNA Mensageiro/metabolismo
9.
Nucleic Acids Res ; 48(11): 6210-6222, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32365182

RESUMO

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.


Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ricina/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endorribonucleases/farmacologia , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ricina/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento
10.
Nucleic Acids Res ; 48(11): 6353-6366, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396195

RESUMO

Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/química , Endopeptidases/química , Endopeptidases/metabolismo , Endorribonucleases/química , Holoenzimas/química , Holoenzimas/metabolismo , Ligases/metabolismo , Modelos Moleculares , Organelas/enzimologia , Organelas/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
11.
Life Sci ; 255: 117842, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454157

RESUMO

The outbreak of COVID-19 caused by 2019-nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Diterpenos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melatonina/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Fator 6 Ativador da Transcrição/metabolismo , Antivirais/farmacologia , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Humanos , Terapia de Alvo Molecular , Pandemias , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
12.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461317

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Coronavirus/metabolismo , Cisteína Endopeptidases/metabolismo , Endorribonucleases/metabolismo , Transativadores/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Exorribonucleases/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Suínos , Doenças dos Suínos/virologia
13.
Life Sci ; 255: 117847, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32470450

RESUMO

Icariin (ICA), a flavonol glycoside isolated from Epimedium, has been considered as a potential alternative therapy for ischemic stroke. However, the protective mechanisms of ICA on cerebral ischemia-reperfusion (I/R) are not fully illuminated yet. The effects of ICA on ER stress and inflammatory response which were involved in the pathological process of cerebral I/R were investigated in vitro. Microglia and neurons were subjected to OGD/R. ICA was administrated to microglia 1 h before OGD and maintained 2 h throughout OGD. At 24 h after reoxygenation, the protein expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia was measured using ELISA assay; neuronal apoptosis was assessed by TUNEL staining; and cell viability was detected using CKK-8 assay; the expression of IRE1α, XBP1u, XBP1s, and cleaved caspase-3 in neurons was examined by western blotting and qRT-PCR; the expression of p-IRE1α in neurons was detected by western blotting. We found that OGD/R induced the expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia; OGD/R and these proinflammatory cytokines promoted the mRNA as well as protein expression of XBP1u, XBP1s and cleaved caspase-3, increased the ratio of p-IRE1α/IRE1α, as well as apoptosis, and decreased cell viability in primary cortical neurons, while ICA reversed the levels of the above factors. IRE1 overexpression enhanced ER stress as well as apoptosis, and impaired the protective effects of ICA. These results suggested that ICA can inhibit apoptosis in neurons after OGD/R through IRE1/XBP1 signaling pathway beside its anti-inflammatory effect.


Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Flavonoides/farmacologia , Neurônios/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Endorribonucleases/metabolismo , Glucose/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Complexos Multienzimáticos/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo
15.
Protein Sci ; 29(7): 1596-1605, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32304108

RESUMO

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.


Assuntos
Betacoronavirus/química , Endorribonucleases/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Oligonucleotídeos/química , Vírus da SARS/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Betacoronavirus/genética , Betacoronavirus/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Modelos Moleculares , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus da SARS/genética , Vírus da SARS/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
16.
Nucleic Acids Res ; 48(10): 5749-5765, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313945

RESUMO

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Phlebovirus/enzimologia , Proteínas Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Endorribonucleases/metabolismo , Modelos Moleculares , Phlebovirus/genética , Regiões Promotoras Genéticas , Domínios Proteicos , Vírus de RNA/enzimologia , Proteínas Virais/metabolismo , Replicação Viral
17.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
18.
Nat Commun ; 11(1): 1997, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332742

RESUMO

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.


Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/patogenicidade , Proteínas E3 de Adenovirus/metabolismo , Endorribonucleases/metabolismo , Regulação Viral da Expressão Gênica/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/genética , Doença Crônica , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Hospedeiro Imunocomprometido , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Proteínas Serina-Treonina Quinases/genética , Processamento de RNA , Latência Viral , Liberação de Vírus/genética , Proteína 1 de Ligação a X-Box/genética
19.
Nat Cell Biol ; 22(5): 559-569, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341548

RESUMO

Dynein is a microtubule motor that transports many different cargos in various cell types and contexts. How dynein is regulated to perform these activities with spatial and temporal precision remains unclear. Human dynein is regulated by autoinhibition, whereby intermolecular contacts limit motor activity. Whether this mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its role in regulating dynein function remain unclear. Here, we use a combination of negative stain electron microscopy, single-molecule assays, genetic, and cell biological techniques to show that autoinhibition is conserved in budding yeast, and plays a key role in coordinating in vivo dynein function. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast), plays an important role in regulating dynein autoinhibition. Our studies demonstrate that, rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate dynein localization and activity in cells.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Dineínas/metabolismo , Endorribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microtúbulos/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(14): 8094-8103, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: covidwho-11430

RESUMO

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Coronavirus/metabolismo , Endorribonucleases/metabolismo , Poli U/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Coronavirus/enzimologia , Coronavirus/imunologia , Endorribonucleases/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Interferons/farmacologia , Poli U/química , RNA Viral/genética , RNA Viral/metabolismo , Uridina/química , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologia
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