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1.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
2.
Biochim Biophys Acta Gene Regul Mech ; 1862(8): 786-795, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31299227

RESUMO

The Lipid A moiety of the lipopolysaccharide can be covalently modified during its transport to the outer membrane by different enzymes, among which the LpxT inner membrane protein. LpxT transfers a phosphate group from the undecaprenyl pyrophosphate to the Lipid A, a modification affecting the stability of the outer membrane and its recognition by the host immune system in Enterobacteria. We previously found that the expression of the Pseudomonas aeruginosa lpxT gene, encoding LpxT, is induced in response to a temperature upshift and we proposed that an RNA thermometer was responsible for such regulation. Here we show that the Escherichia coli lpxT orthologous gene is down-regulated upon a temperature upshift and investigated the mechanism of this regulation. We found that the LpxT protein stability is not affected by the temperature change. Conversely, the lpxT mRNA levels strongly decrease upon a shift from 28 to 42 °C. The lack of MicA sRNA, which was previously implicated in lpxT regulation, does not affect lpxT thermal regulation. We identified the lpxTp promoter and demonstrated that lpxTp has temperature-sensitive activity depending on its peculiar -10 region. Moreover, we found that RNase E-dependent degradation of the lpxT mRNA is also modulated by temperature causing a strong destabilization of the lpxT mRNA at 42 °C. In vitro data argue against the involvement of factors differentially expressed at 28 and 42 °C in the temperature-dependent modulation of lpxT mRNA stability.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , DNA Glicosilases/metabolismo , Regulação para Baixo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Lipídeo A/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Estabilidade Proteica , Estabilidade de RNA , Termodinâmica
3.
Nature ; 571(7765): 355-360, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270458

RESUMO

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.


Assuntos
Genótipo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Neoplasias/genética , Neoplasias/patologia , Transcriptoma/genética , Animais , Antígenos CD34/metabolismo , Calreticulina/genética , Linhagem Celular , Proliferação de Células , Células Clonais/classificação , Células Clonais/metabolismo , Células Clonais/patologia , Endorribonucleases/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Modelos Moleculares , Transtornos Mieloproliferativos/classificação , NF-kappa B/metabolismo , Neoplasias/classificação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Resposta a Proteínas não Dobradas/genética
4.
Nat Commun ; 10(1): 3035, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292443

RESUMO

Mycobacterium tuberculosis readily adapts to survive a wide range of assaults by modifying its physiology and establishing a latent tuberculosis (TB) infection. Here we report a sophisticated mode of regulation by a tRNA-cleaving toxin that enlists highly selective ribosome stalling to recalibrate the transcriptome and remodel the proteome. This toxin, MazF-mt9, exclusively inactivates one isoacceptor tRNA, tRNALys43-UUU, through cleavage at a single site within its anticodon (UU↓U). Because wobble rules preclude compensation for loss of tRNALys43-UUU by the second M. tuberculosis lysine tRNA, tRNALys19-CUU, ribosome stalling occurs at in-frame cognate AAA Lys codons. Consequently, the transcripts harboring these stalled ribosomes are selectively cleaved by specific RNases, leading to their preferential deletion. This surgically altered transcriptome generates concomitant changes to the proteome, skewing synthesis of newly synthesized proteins away from those rich in AAA Lys codons toward those harboring few or no AAA codons. This toxin-mediated proteome reprogramming may work in tandem with other pathways to facilitate M. tuberculosis stress survival.


Assuntos
Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Mycobacterium tuberculosis/fisiologia , Proteoma/genética , Ribossomos/metabolismo , Sistemas Toxina-Antitoxina/fisiologia , Toxinas Bacterianas/metabolismo , Tuberculose Latente/microbiologia , Mycobacterium tuberculosis/patogenicidade , Proteoma/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Transcriptoma/genética
5.
J Dairy Sci ; 102(8): 7359-7370, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155263

RESUMO

Disruption of endoplasmic reticulum (ER) homeostasis, often termed ER stress, is intrinsically linked with perturbation of lipid metabolism in humans and mice. Whether ER homeostasis is affected in cows experiencing fatty liver is unknown. The aim of this study was to investigate the potential role of ER stress in hepatic lipid accumulation in calf hepatocytes and ER stress status in dairy cows with severe fatty liver. In vitro experiments were conducted in which hepatocytes were isolated from calves and treated with different concentrations of fatty acids, tauroursodeoxycholic acid (TUDCA; a canonical inhibitor of ER stress), or both. The increase in phosphorylation level of protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α) proteins, and the cleavage of activating transcription factor-6 (ATF6) protein in response to increasing doses of fatty acids (which were reversed by TUDCA treatment) in primary hepatocytes underscored a mechanistic link between fatty acids and ER stress. In addition, fatty acid treatment increased the abundance of sterol regulatory element-binding protein 1c, acetyl-CoA carboxylase-α, fatty acid synthase, and diacylglycerol acyltransferase 1, and lipid accumulation in calf primary hepatocytes, whereas inhibition of ER stress by incubating with TUDCA significantly weakened these effects. Overall, results in vitro indicate that inhibition of ER stress in calf hepatocytes alleviates fatty acid-induced lipid accumulation by downregulating the expression of lipogenic genes. In vivo experiments, liver and blood samples were collected from cows diagnosed as healthy (n = 15) or with severe fatty liver (n = 15). The phosphorylation level of PERK and IRE1α, the cleavage of ATF6 protein, and the abundance of several unfolded protein response genes (78 kDa glucose-regulated protein, AMP-dependent transcription factor 4, and spliced X-box binding protein 1) were greater in liver of cows with severe fatty liver. The present in vivo study confirms the occurrence of ER stress in dairy cows with severe fatty liver. Considering the causative role of fatty acid-induced ER stress in hepatic lipid accumulation in calf hepatocytes, the existence of ER stress in the liver of severe fatty liver cows may presage its participation in fatty liver progression in dairy cows. However, the mechanistic relationship between ER stress and fatty liver in dairy cows remain to be determined.


Assuntos
Doenças dos Bovinos/fisiopatologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos/administração & dosagem , Fígado Gorduroso/veterinária , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Bovinos , Células Cultivadas , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lipogênese/genética , Fígado/efeitos dos fármacos , Camundongos , Fosforilação , Ácido Tauroquenodesoxicólico/administração & dosagem , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismo
6.
Cancer Sci ; 110(8): 2471-2484, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187548

RESUMO

Endoplasmic reticulum stress (ERS) plays a key role in the pathogenesis and development of tumors and protects tumor cells from radiation damage and drug-induced stress. We previously demonstrated that EGFR confers radioresistance in human papillomavirus (HPV)-negative human oropharyngeal carcinoma by activating ERS signaling through PERK and IRE1α. In addition, PERK confers radioresistance by activating the inflammatory cytokine NF-κB. However, the effect of IRE1 on radiosensitivity has not yet been fully elucidated. Here, we clarified that IRE1 overexpression was associated with poor outcome in HPV-negative patients treated with radiotherapy (P = 0.0001). In addition, a significantly higher percentage of radioresistant HPV-negative patients than radiosensitive HPV-negative patients exhibited high IRE expression (66.7% vs 27.8%, respectively; P = 0.001). Silencing IRE1 and XBP1 increased DNA double-strand break (DSB) and radiation-induced apoptosis, thereby increasing the radiosensitivity of HPV-negative oropharyngeal carcinoma cells. IRE1-XBP1 silencing also inhibited radiation-induced IL-6 expression at both the RNA and protein levels. The regulatory effect of IRE1-XBP1 silencing on DNA DSB-induced and radiation-induced apoptosis was inhibited by pretreatment with IL-6. These data indicate that IRE1 regulates radioresistance in HPV-negative oropharyngeal carcinoma through IL-6 activation, enhancing X-ray-induced DNA DSB and cell apoptosis.


Assuntos
Endorribonucleases/metabolismo , Interleucina-6/metabolismo , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Humanos , NF-kappa B/metabolismo , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
7.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 757-764, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31125617

RESUMO

Gre factors reactivate stalled elongation complexes by enhancing the intrinsic transcript cleavage activity of RNA polymerase. Previous work by us has shown that unlike in Escherichia coli (E.coli), Mycobacterium tuberculosis Gre factor is essential for its survival. Apart from their role in transcription regulation Gre factors have been implicated in stress response. A recent study has shown the role of E.coli GreA as a cellular chaperone, which inhibits aggregation of substrate proteins under heat stress condition. Moreover it was shown that GreA enables E.coli to survive heat shock and oxidative stress. In the current work, we have characterized the moonlighting chaperone activity and its plausible mechanism in Mycobacterium smegmatis Gre (MsGre) factor. We show here that MsGre prevents heat-induced aggregation of the substrate protein and also protects enzymatic activity. Interestingly Gre factor exists as a dimer in solution and does not undergo heat induced oligomerization. From the 8-anilino-1-naphthalene sulfonate (ANS) binding studies MsGre was shown to expose hydrophobic surface upon heat stress that would allow binding to unfolded or partially folded substrate protein. From Circular Dichroism (CD) studies, we also show that MsGre has a stable secondary structure under thermal stress. We propose that the presence of C-terminal FKBP-like fold in MsGre factor that might contribute to its chaperone-like function.


Assuntos
Proteínas de Bactérias/química , Endorribonucleases/química , Chaperonas Moleculares/química , Mycobacterium smegmatis/enzimologia , Dobramento de Proteína , Multimerização Proteica , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Endorribonucleases/metabolismo , Temperatura Alta , Chaperonas Moleculares/metabolismo
8.
Mol Cells ; 42(4): 301-312, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31091556

RESUMO

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.


Assuntos
Relógios Circadianos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas CLOCK/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de Sinais
9.
Virology ; 533: 34-44, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31082732

RESUMO

Coronavirus infection induces the generation of autophagosomes, and certain host proteins regulating cellular autophagy are hijacked by some coronaviruses to facilitate the formation of double membrane vesicles. However, mechanisms underlying coronavirus-induced autophagy remain largely unknown. In this study, we demonstrate that autophagosome formation and apparent autophagic flux are induced in cells infected with infectious bronchitis virus (IBV) - a gammacoronavirus. Notably, IBV-induced autophagy was dependent on autophagy related 5 (ATG5) but not beclin1 (BECN1), although both are essential proteins in the canonical autophagy pathway. Moreover, the ER stress sensor inositol requiring enzyme 1 (IRE1), but not its substrate X-box protein 1 (XBP1), was also essential for the induction of autophagy during IBV infection. Finally, the anti-apoptotic extracellular signal-regulated kinase 1/2 (ERK1/2) also contributed to IBV-induced autophagy. Our findings add new knowledge to the regulatory mechanisms governing coronavirus-induced autophagy, highlighting an extensive cross-talk among cellular signaling pathways during coronavirus infection.


Assuntos
Autofagia , Infecções por Coronavirus/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Vírus da Bronquite Infecciosa/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Humanos , Vírus da Bronquite Infecciosa/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Serina-Treonina Quinases/genética
10.
Nature ; 568(7752): 351-356, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971818

RESUMO

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.


Assuntos
Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Estresse Nitrosativo , Volume Sistólico , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Endorribonucleases/metabolismo , Insuficiência Cardíaca/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
11.
Nat Struct Mol Biol ; 26(4): 315-321, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936531

RESUMO

Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Imunidade Adaptativa/genética , Imunidade Adaptativa/fisiologia , Sistemas CRISPR-Cas/genética , Clivagem do DNA , Endorribonucleases/genética , Endorribonucleases/metabolismo , Edição de Genes/métodos , Multimerização Proteica/genética , Multimerização Proteica/fisiologia
12.
Chem Commun (Camb) ; 55(37): 5339-5342, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30973558

RESUMO

A tunable chemoenzymatic strategy provides access to the entire class of magic spot nucleotides and modified analogues. The approach combines chemoselective bisphosphorylations using phosphoramidites with regioselective ribonuclease T2 cyclo-phosphate hydrolysis, leading to flexible and simple gram-scale operations.


Assuntos
Endorribonucleases/metabolismo , Nucleotídeos/biossíntese , Biocatálise , Ciclização , Eletroforese em Gel de Poliacrilamida , Hidrólise , Nucleotídeos/química , Fosfatos/química , Fosfatos/metabolismo , Estereoisomerismo
13.
Biomed Res Int ; 2019: 7398208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941371

RESUMO

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endorribonucleases/metabolismo , Técnicas de Inativação de Genes , Herpesvirus Suídeo 1/fisiologia , Replicação Viral/fisiologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Edição de Genes , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Poli I-C/farmacologia , Pseudorraiva/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Suínos , Vacinas Virais/imunologia , Replicação Viral/efeitos dos fármacos
14.
MBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914510

RESUMO

Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Biossíntese de Proteínas , Proteoma/análise , Estresse Fisiológico , Adaptação Fisiológica , Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Hidrólise , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
15.
Nat Commun ; 10(1): 1084, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842412

RESUMO

The IRE1α/XBP1 branch of unfolded protein response (UPR) pathway has a critical function in endoplasmic reticulum (ER) expansion in plasma cells via unknown mechanisms; interestingly, another UPR branch, PERK, is suppressed during plasma cell development. Here we show that Ufbp1, a target and cofactor of the ufmylation pathway, promotes plasma cell development by suppressing the activation of PERK. By contrast, the IRE1α/XBP1 axis upregulates the expression of Ufbp1 and ufmylation pathway genes in plasma cells, while Ufbp1 deficiency impairs ER expansion in plasma cells and retards immunoglobulin production. Structure and function analysis suggests that lysine 267 of Ufbp1, the main lysine in Ufbp1 that undergoes ufmylation, is dispensable for the development of plasmablasts, but is required for immunoglobulin production and stimulation of ER expansion in IRE1α-deficient plasmablasts. Thus, Ufbp1 distinctly regulates different branches of UPR pathway to promote plasma cell development and function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/fisiologia , Diferenciação Celular , Retículo Endoplasmático/metabolismo , Plasmócitos/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Animais , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Imunidade Humoral/fisiologia , Lisina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Regulação para Cima , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/isolamento & purificação , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/metabolismo
16.
Int J Mol Med ; 43(5): 2033-2043, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864731

RESUMO

Sirtuin 1 (Sirt1) exerts its cardioprotective effects in various cardiovascular diseases via multiple cellular activities. However, the therapeutic implications of Sirt1 in hypoxic cardiomyocytes and the underlying mechanisms remain elusive. The present study investigated whether Sirt1 regulates autophagy and apoptosis in hypoxic H9C2 cardiomyocytes and in an experimental hypoxic mouse model. Right ventricular outflow tract biopsies were obtained from patients with cyanotic or acyanotic congenital heart diseases. Adenovirus Ad­Sirt1 was used to activate Sirt1 and Ad­Sh­Sirt1 was used to inhibit Sirt1 expression in H9C2 cells, in order to investigate the effect of Sirt1 on cellular autophagy and apoptosis. SRT1720, a pharmacological activator of Sirt1 and EX­527, a Sirt1 antagonist, were administered to mice to explore the role of Sirt1 in hypoxic cardiomyocytes in vivo. The levels of autophagy and apoptosis­related proteins were evaluated using western blotting. Apoptosis was investigated by TUNEL staining and Annexin V/7­aminoactinomycin D flow cytometry analysis. Heart tissue samples from cyanotic patients exhibited increased autophagy and apoptosis, as well as elevated Sirt1 levels, compared with the noncyanotic control samples. The data from the western blot analysis revealed that Sirt1 promoted autophagic flux and reduced apoptosis in hypoxic H9C2 cells. In addition, Sirt1 activated AMP­activated protein kinase (AMPK), and the AMPK inhibitor Compound C abolished the effect of Sirt1 on autophagy activation. Further exploration of the mechanism revealed that Sirt1 protects hypoxic cardiomyocytes from apoptosis, at least in part, through inositol requiring kinase enzyme 1α (IRE1α). Consistent with the in vitro results, treatment with the Sirt1 activator SRT1720 activated AMPK, inhibited IRE1α, enhanced autophagy, and decreased apoptosis in the heart tissues of normoxic mice compared with the hypoxia control group. Opposite changes were observed in hypoxic mice treated with the Sirt1 inhibitor EX­527. These results suggested that Sirt1 promoted autophagy via AMPK activation and reduced hypoxia­induced apoptosis via the IRE1α pathway, to protect cardiomyocytes from hypoxic stress.


Assuntos
Apoptose , Autofagia , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/metabolismo , Sirtuína 1/metabolismo , Estresse Fisiológico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carbazóis/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Cianose/patologia , Modelos Animais de Doenças , Endorribonucleases/metabolismo , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
17.
Arch Virol ; 164(5): 1433-1439, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30868265

RESUMO

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, infects epithelial surfaces and establishes latency in the central nervous system, where astrocytes are a major immune cell type. Here, we report changes that occur in the expression of pathogen recognition receptors, such as Toll-like receptors, DNA and RNA sensors, interferons, and interferon-stimulated genes, when astrocytes are infected with HSV-1 strain F. We observed upregulation of Toll-like receptors 2, 6 and 9, MDA5, and DAI along with an increase in the expression of type I interferons and interferon-stimulated genes such as IFIT1, IFIT3 and RNase L. These genes encode proteins that mediate the antiviral immune response.


Assuntos
Astrócitos/imunologia , Astrócitos/virologia , Herpesvirus Humano 1/imunologia , Imunidade Inata/imunologia , Animais , Proteínas de Transporte/metabolismo , Cercopithecus aethiops , Endorribonucleases/metabolismo , Helicase IFIH1 Induzida por Interferon/biossíntese , Camundongos , Proteínas/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Receptor Toll-Like 9/biossíntese , Regulação para Cima/genética , Células Vero , Replicação Viral , eIF-2 Quinase/biossíntese
18.
Arch Virol ; 164(5): 1323-1334, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877450

RESUMO

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.


Assuntos
Circovirus/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Macrófagos Alveolares/patologia , Resposta a Proteínas não Dobradas/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Infecções por Circoviridae/patologia , Infecções por Circoviridae/veterinária , Retículo Endoplasmático/virologia , Endorribonucleases/metabolismo , Macrófagos Alveolares/virologia , Microscopia Eletrônica de Transmissão , Suínos , Doenças dos Suínos , Proteínas do Envelope Viral/metabolismo , eIF-2 Quinase/metabolismo
19.
Proc Natl Acad Sci U S A ; 116(11): 5071-5076, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30814222

RESUMO

Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Azacitidina/farmacologia , Desmetilação do DNA , Endorribonucleases/metabolismo , Imunidade Inata , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Radiação Ionizante , Bibliotecas de Moléculas Pequenas/farmacologia
20.
Nat Cell Biol ; 21(3): 328-337, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778220

RESUMO

Over their lifetime, long-term haematopoietic stem cells (HSC) are exposed to a variety of stress conditions that they must endure. Many stresses, such as infection/inflammation, reactive oxygen species, nutritional deprivation and hypoxia, activate unfolded protein response signalling, which induces either adaptive changes to resolve the stress or apoptosis to clear the damaged cell. Whether unfolded-protein-response signalling plays any role in HSC regulation remains to be established. Here, we report that the adaptive signalling of the unfolded protein response, IRE1α-XBP1, protects HSCs from endoplasmic reticulum stress-induced apoptosis. IRE1α knockout leads to reduced reconstitution of HSCs. Furthermore, we show that oncogenic N-RasG12D activates IRE1α-XBP1, through MEK-GSK3ß, to promote HSC survival under endoplasmic reticulum stress. Inhibiting IRE1α-XBP1 abolished N-RasG12D-mediated survival under endoplasmic reticulum stress and diminished the competitive advantage of NrasG12D HSCs in transplant recipients. Our studies illuminate how the adaptive endoplasmic reticulum stress response is advantageous in sustaining self-renewal of HSCs and promoting pre-leukaemic clonal dominance.


Assuntos
Autorrenovação Celular/genética , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Adaptação Fisiológica , Animais , Sobrevivência Celular/genética , Endorribonucleases/genética , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Lesões Pré-Cancerosas , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Proteína 1 de Ligação a X-Box/genética
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