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1.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639036

RESUMO

Considering the current dramatic and fatal situation due to the high spreading of SARS-CoV-2 infection, there is an urgent unmet medical need to identify novel and effective approaches for prevention and treatment of Coronavirus disease (COVID 19) by re-evaluating and repurposing of known drugs. For this, tomatidine and patchouli alcohol have been selected as potential drugs for combating the virus. The hit compounds were subsequently docked into the active site and molecular docking analyses revealed that both drugs can bind the active site of SARS-CoV-2 3CLpro, PLpro, NSP15, COX-2 and PLA2 targets with a number of important binding interactions. To further validate the interactions of promising compound tomatidine, Molecular dynamics study of 100 ns was carried out towards 3CLpro, NSP15 and COX-2. This indicated that the protein-ligand complex was stable throughout the simulation period, and minimal backbone fluctuations have ensued in the system. Post dynamic MM-GBSA analysis of molecular dynamics data showed promising mean binding free energy 47.4633 ± 9.28, 51.8064 ± 8.91 and 54.8918 ± 7.55 kcal/mol, respectively. Likewise, in silico ADMET studies of the selected ligands showed excellent pharmacokinetic properties with good absorption, bioavailability and devoid of toxicity. Therefore, patchouli alcohol and especially, tomatidine may provide prospect treatment options against SARS-CoV-2 infection by potentially inhibiting virus duplication though more research is guaranteed and secured.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Endorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Sesquiterpenos/farmacologia , Tomatina/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Endorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2/efeitos dos fármacos , Tomatina/farmacologia , Proteínas não Estruturais Virais/metabolismo
2.
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
4.
Front Cell Infect Microbiol ; 11: 707107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34532300

RESUMO

Endoplasmic reticulum (ER) stress-induced autophagy is closely associated with viral infection and propagation. However, the intrinsic link between ER stress, autophagy, and viral replication during foot-and-mouth disease virus (FMDV) infection is not fully elucidated. Our previous studies demonstrated that FMDV infection activated the ER stress-associated UPR of the PERK-eIF2a and ATF6 signaling pathway, whereas the IRE1a signaling was suppressed. We found that the activated-ATF6 pathway participated in FMDV-induced autophagy and FMDV replication, while the IRE1α pathway only affected FMDV replication. Further studies indicated that Sec62 was greatly reduced in the later stages of FMDV infection and blocked the activation of the autophagy-related IRE1α-JNK pathway. Moreover, it was also found that Sec62 promoted IRE1a phosphorylation and negatively regulated FMDV proliferation. Importantly, Sec62 may interact with LC3 to regulate ER stress and autophagy balance and eventually contribute to FMDV clearance via fusing with lysosomes. Altogether, these results suggest that Sec62 is a critical molecule in maintaining and recovering ER homeostasis by activating the IRE1α-JNK pathway and delivering autophagosome into the lysosome, thus providing new insights on FMDV-host interactions and novel antiviral therapies.


Assuntos
Vírus da Febre Aftosa , Animais , Autofagia , Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Replicação Viral
5.
Nat Commun ; 12(1): 5321, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493732

RESUMO

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers.


Assuntos
Carcinoma Epitelial do Ovário/terapia , Endorribonucleases/genética , Neoplasias Ovarianas/terapia , Receptor de Morte Celular Programada 1/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Benzopiranos/farmacologia , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Himecromona/análogos & derivados , Himecromona/farmacologia , Inibidores de Checkpoint Imunológico , Camundongos , Terapia de Alvo Molecular , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Toxicon ; 200: 165-172, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34343521

RESUMO

Fumonisin B1 (FB1) is a major food-borne mycotoxin commonly found in maize and maize-based products, while cadmium is one of the most common toxic heavy metals found in food, particularly in wheat and rice. Given the possibility of co-exposure to FB1 and cadmium for consumers, we elevated combined toxicity of FB1 and cadmium using both in vitro and in vivo models. Acute toxicity setting was employed in the present study. Mouse embryonic fibroblast (MEF) and human L02 liver cells were used to determine the in vitro cytotoxicity, while C57BL/6 N mice were used to assess the in vivo toxicity. Results showed that treatment with combination of FB1 (15, 20, 25, 30, 35 µM) and cadmium (3, 4, 5, 6, 7 µM) for 24 h led to synergistic cytotoxicity in vitro, and acute treatment with the combination of FB1/cadmium (1.5 mg/kg/60 mg/kg) for 5 days increased liver damage in vivo. Mechanistically, the combined toxicity was associated with elevated activation of IRE1α-JNK pathway. Glycyrol, a representative coumarin compound isolated from licorice, was able to reduce the combination-induced toxicity both in vitro and in vivo through suppression of IRE1α-JNK axis. The combined toxicity of FB1/cadmium should be taken into consideration for performing human health risk assessment of FB1/cadmium exposure.


Assuntos
Endorribonucleases , Fumonisinas , Animais , Cádmio/toxicidade , Fibroblastos , Flavonoides , Fumonisinas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases
7.
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
8.
Microb Pathog ; 159: 105147, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34400280

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis remains a serious global public health threat. M. tuberculosis PE and PPE proteins are closely involved in pathogen-host interaction. To explore the predicted function of the M. tuberculosis PE17 (Rv1646), we heterologously expressed PE17 in a non-pathogenic Mycobacterium smegmatis strain (Ms_PE17). PE17 can reduce the survival of M. smegmatis within macrophages associated with altering the transcription levels of inflammatory cytokines IL1ß, IL6, TNFα, and IL10 in Ms_PE17 infected macrophages through JNK signaling. Furthermore, macrophages apoptosis was increased upon Ms_PE17 infection in a caspases-dependent manner, accompanied by the activation of the Endoplasmic Reticulum stress IRE1α/ASK1/JNK signaling pathway. This can be largely interpreted by the epigenetic changes through reduced H3K9me3 chromatin occupancy post Ms_PE17 infection. To our knowledge, this is the first report that PE17 altered the macrophages apoptosis via H3K9me3 mediated chromatin remodeling.


Assuntos
Mycobacterium tuberculosis , Apoptose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Montagem e Desmontagem da Cromatina , Endorribonucleases , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinases
9.
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
11.
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
12.
Nat Commun ; 12(1): 4375, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272366

RESUMO

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/química
13.
Dev Biol ; 478: 205-211, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34265355

RESUMO

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.


Assuntos
Olho Composto de Artrópodes/química , Olho Composto de Artrópodes/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Pigmentos Biológicos/análise , Alelos , Animais , Olho Composto de Artrópodes/ultraestrutura , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Cor de Olho , Mutação , Fenotiazinas/análise , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentação , Pteridinas/análise , Rodopsina/metabolismo
14.
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
15.
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
16.
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
17.
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
18.
Molecules ; 26(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200016

RESUMO

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.


Assuntos
Antibacterianos/farmacologia , Endorribonucleases/genética , Escherichia coli/enzimologia , Oligonucleotídeos/farmacologia , Sistema Livre de Células , Endorribonucleases/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Oligonucleotídeos/síntese química , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores
19.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206573

RESUMO

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Assuntos
RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , DNA Espaçador Ribossômico , Endorribonucleases , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
20.
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
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