RESUMO
The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein's structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies. IMPORTANCE: SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.
Assuntos
Proteínas do Nucleocapsídeo , Multimerização Proteica , Animais , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Cristalografia por Raios X , Suínos , Epitopos/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Ligação Proteica , Anticorpos Antivirais/imunologia , Humanos , Domínios Proteicos , Modelos MolecularesRESUMO
Pulmonary fibrosis (PF) is a chronic interstitial lung disease with no effective therapies. Galectin-3 (Gal-3), a marker of oxidative stress, plays a key role in the pathogenesis of PF. Fibroblast-myofibroblast differentiation (FMD) is an important source of fibrotic cells in PF. Previous studies showed that melatonin (MT) exerted anti-fibrotic effect in many diseases including PF through its antioxidant activity. In the present study we investigated the relationships among Gal-3, NRF2, ROS in FMD and their regulation by MT. We established an in vitro model of FMD in TGF-ß1-treated human fetal lung fibroblast1 (HFL1) cells and a PF mouse model via bleomycin (BLM) intratracheal instillation. We found that Gal-3 expression was significantly increased both in vitro and in vivo. Knockdown of Gal-3 in HFL1 cells markedly attenuated TGF-ß1-induced FMD process and ROS accumulation. In TGF-ß1-treated HFL1 cells, pretreatment with NRF2-specific inhibitor ML385 (5 µM) significantly increased the levels of Gal-3, α-SMA and ROS, suggesting that the expression of Gal-3 was regulated by NRF2. Treatment with NRF2-activator MT (250 µM) blocked α-SMA and ROS accumulation accompanied by reduced Gal-3 expression. In BLM-induced PF model, administration of MT (5 mg·kg-1·d-1, ip for 14 or 28 days) significantly attenuated the progression of lung fibrosis through up-regulating NRF2 and down-regulating Gal-3 expression in lung tissues. These results suggest that Gal-3 regulates TGF-ß1-induced pro-fibrogenic responses and ROS production in FMD, and MT activates NRF2 to block FMD process by down-regulating Gal-3 expression. This study provides a useful clue for a clinical strategy to prevent PF. Graphic abstract of the mechanisms. MT attenuated BLM-induced PF via activating NRF2 and inhibiting Gal-3 expression.
Assuntos
Melatonina , Fibrose Pulmonar , Animais , Humanos , Camundongos , Bleomicina/efeitos adversos , Fibroblastos , Galectina 3/efeitos dos fármacos , Galectina 3/metabolismo , Pulmão/patologia , Melatonina/farmacologia , Melatonina/uso terapêutico , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Fibroblast-myofibroblast differentiation (FMD) is a critical cellular phenotype during the occurrence and deterioration of pulmonary fibrosis (PF). FMD can increase with an elevated level of reactive oxygen species (ROS) on fibroblasts under oxidative stress. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that regulates the level of intracellular ROS. Nuclear factor erythroid 2-related factor 2 (Nrf2) can protect against FMD in PF. However, the relationship between Nrf2 and TXNIP in FMD remains elusive. Therefore, we established TGF-ß1-induced FMD in vitro and bleomycin (BLM)-induced mouse PF model in vivo to explore whether the activation of Nrf2 can inhibit TXNIP-mediated FMD in PF. Dimethyl itaconate (DMI) was selected to activate Nrf2. Our results showed that TXNIP was elevated and FMD was aggravated in mice lung tissues after BLM administration compared with the saline group. Inversely, Nrf2 decreased TXNIP expression and alleviated FMD in PF. In vitro, TXNIP overexpression enhanced FMD and increased the level of ROS. In contrast, TXNIP deficiency by small interfering RNA (siRNA) attenuated TGF-ß1-induced FMD and reduced ROS. An increase in ROS by H2 O2 can upregulate TXNIP expression. Moreover, Nrf2 also inhibited TGF-ß1-induced FMD and the increase of ROS, with reducing expression of TXNIP, and the inhibitory effect was better than TXNIP siRNA. These results suggest that activation of Nrf2 by DMI can protect against PF via inhibiting TXNIP expression. Our study may provide new therapeutic targets and treatment approaches for PF.
Assuntos
Antifibróticos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Succinatos/farmacologia , Tiorredoxinas/antagonistas & inibidores , Animais , Bleomicina , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Pulmonary fibrosis (PF) is a progressive and devastating lung disease of unknown etiology, excessive fibroblast proliferation serves as a key event to promote PF. Transcription factor forkhead box M1 (FOXM1) is not only a well-known proto-oncogene, but also an essential driver of cell proliferation. Recently, 5'-AMP-activated protein kinase (AMPK) is reported to reduce the incidence of PF. However, it remains elusive whether have an underlying relationship between AMPK and FOXM1 in fibroblast proliferation-mediated PF. Here, the progression of lung fibroblast proliferation and the expression levels of AMPK and FOXM1 were observed by intratracheally instilled of bleomycin (BLM) and intraperitoneal injection of metformin in C57BL/6 J mice. Meanwhile, human fetal lung fibroblast1 (HFL1) cells were respectively treated with AMPK activator metformin or AMPK inhibitor Compound C, or FOXM1 depletion by transfected small interfering RNA (siRNA) to unveil roles of AMPK, FOXM1 and the link between them on platelet-derived growth factor (PDGF)-induced fibroblast proliferation. Our results demonstrated that AMPK activated by metformin could down-regulate FOXM1 and alleviate BLM-induced mouse PF model. In vitro, activation of AMPK attenuated PDGF-induced fibroblast proliferation accompanied by the down-regulation of FOXM1. In contrast, inhibition of AMPK enhanced PDGF-induced fibroblast proliferation along with activating FOXM1. These findings suggest that AMPK can ameliorate the progression of fibroblast proliferation during PF via suppressing the expression of FOXM1 and provide new insight into seek PF treatment approaches.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Forkhead Box M1/metabolismo , Metformina/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologiaRESUMO
New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.
Assuntos
Técnicas Biossensoriais , Subtipo H7N9 do Vírus da Influenza A , Influenza Humana , Humanos , Hemaglutininas , Avaliação Pré-Clínica de Medicamentos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , AntiviraisRESUMO
Fibroblasts activation is a crucial process for development of fibrosis during idiopathic pulmonary fibrosis pathogenesis, and transforming growth factor (TGF)-ß1 plays a key regulatory role in fibroblast activation. It has been reported that metformin (MET) alleviated bleomycin (BLM)-induced pulmonary fibrosis (PF) by regulating TGF-ß1-induced fibroblasts activation, but the underlying mechanisms still deserve further investigations. In this study, MET blocked α-smooth muscle actin (α-SMA) accumulation in vivo accompanied with S100A4 expression and STAT3 phosphorylation inhibition, resulting in attenuating the progression of lung fibrosis after BLM administration. We determined that S100A4 plays critical roles in fibroblasts activation in vitro, evidenced by siRNA knockdown of S100A4 expression downregulated TGF-ß1 induced α-SMA production in Human fetal lung fibroblast (HFL1) cells. Importantly, we found for the first time that the expression of S100A4 in fibroblasts was regulated by STAT3. Stattic, an effective small molecule inhibitor of STAT3 phosphorylation, reduced S100A4 level in TGF-ß1- treated HFL1 cells accompanied with less α-SMA production. We further found that MET, which inhibits STAT3 phosphorylation by AMPK activation, also inhibits fibroblasts activation by targeting S100A4 in vitro. Together all these results, we conclude that S100A4 contributes to TGF-ß1- induced pro-fibrogenic function in fibroblasts activation, and MET was able to protect against TGF-ß1-induced fibroblasts activation and BLM-induced PF by down-regulating S100A4 expression through AMPK-STAT3 axis. These results provide a useful clue for a clinical strategy to prevent PF.
RESUMO
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death across the globe. Its repeated exacerbation will seriously worsen the quality of life, aggravate the patients' symptoms, and bring a heavy burden on the patients and the society. Understanding the current status of drug therapy and the role of pharmaceutical care is essential for the management of COPD. In addition to the drugs already on the market, recent clinical trials also show that emerging novel drugs for treating COPD are being developed to prevent the symptoms, reduce the frequency of acute exacerbation, and improve the quality of life. Recent progress in new drug research should lead to novel treatment options for COPD patients in future clinical practice. The pharmaceutical care has shown significantly favourable impacts on addressing drug-related problems, supporting its vital role in the management of COPD, especially when there are a wide range of therapeutic agents. This review not only provides an overview of current treatment strategies but also further underlines the importance of new drug development and pharmaceutical care for patients with COPD.