RESUMO
The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.
Assuntos
Crustáceos , Animais , Crustáceos/genética , Crustáceos/imunologia , Crustáceos/metabolismo , Crustáceos/microbiologia , Drosophila melanogaster , Lipopolissacarídeos , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Regulação para Cima , Vibrio , Transdução de Sinais , HumanosRESUMO
Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.
Assuntos
Autofagia , Ecdisterona , Helicoverpa armigera , Histona Acetiltransferases , Histonas , Processamento de Proteína Pós-Traducional , Acetilação , Autofagia/genética , Ecdisterona/metabolismo , Regiões Promotoras Genéticas , Helicoverpa armigera/genética , Helicoverpa armigera/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismoRESUMO
The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.
Assuntos
Proteínas de Ligação a DNA , Complexos Endossomais de Distribuição Requeridos para Transporte , Penaeidae , Replicação Viral , Vírus da Síndrome da Mancha Branca 1 , Animais , Vírus da Síndrome da Mancha Branca 1/fisiologia , Vírus da Síndrome da Mancha Branca 1/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Penaeidae/virologia , Penaeidae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Endossomos/metabolismo , Endossomos/virologia , Hemócitos/virologia , Hemócitos/metabolismo , Interações Hospedeiro-Patógeno , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Interferência de RNARESUMO
The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans; however, the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood. Using the holometabolous lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the steroid hormone 20-hydroxyecdysone (20E) upregulated the expression of transcription factor Krüppel-like factor (identified as Klf15) to promote macroautophagy/autophagy, apoptosis and gluconeogenesis during metamorphosis. 20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis. Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis. KLF15 promoted autophagic flux and transiting to apoptosis. KLF15 bound to the KLF binding site (KLF bs) in the promoter of Atg8 (autophagy-related gene 8/LC3) to upregulate Atg8 expression. Knockdown Atg8 reduced free fatty acids (FFAs), glycerol, free amino acids (FAAs) and glucose levels. However, knockdown of Klf15 accumulated FFAs, glycerol, and FAAs. Glycolysis was switched to gluconeogenesis, trehalose and glycogen synthesis were changed to degradation during metamorphosis, which were accompanied by the variation of the related genes expression. KLF15 upregulated phosphoenolpyruvate carboxykinase (Pepck) expression by binding to KLF bs in the Pepck promoter for gluconeogenesis, which utilised FFAs, glycerol, and FAAs directly or indirectly to increase glucose in the hemolymph. Taken together, 20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.
Assuntos
Ecdisterona , Mariposas , Animais , Autofagia/genética , Ecdisterona/metabolismo , Técnicas de Silenciamento de Genes , Gluconeogênese/genética , Glucose/metabolismo , Glicerol/metabolismo , Homeostase/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mariposas/genéticaRESUMO
BACKGROUND: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study. RESULTS: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling. CONCLUSIONS: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.
Assuntos
Encéfalo , Ácidos Graxos não Esterificados , Homeostase , Animais , Encéfalo/metabolismo , Encéfalo/crescimento & desenvolvimento , Ácidos Graxos não Esterificados/metabolismo , Lipase/metabolismo , Lipase/genética , Mariposas/crescimento & desenvolvimento , Mariposas/fisiologia , Mariposas/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Hormônios Juvenis/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Metamorfose Biológica/fisiologia , Ecdisterona/metabolismoRESUMO
Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with ß-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.
Assuntos
Ecdisterona/farmacologia , Proteínas de Insetos/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Clatrina/metabolismo , Ecdisterona/química , Ecdisterona/metabolismo , Endocitose , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.
Assuntos
Receptores de Imunoglobulina Polimérica , Vírus da Síndrome da Mancha Branca 1 , Antivirais/farmacologia , Calmodulina/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas/farmacologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/metabolismoRESUMO
The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control.
Assuntos
Proteínas de Artrópodes , Complexo do Signalossomo COP9 , Imunidade Inata , Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Penaeidae/genética , Penaeidae/imunologia , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/química , Imunidade Inata/genética , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterinária , Alinhamento de Sequência/veterinária , FilogeniaRESUMO
BACKGROUND: The regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis. RESULTS: Our findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation. CONCLUSIONS: The posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.
Assuntos
Ecdisterona , Insulina , Animais , Ecdisterona/farmacologia , Fosfoglicerato Quinase/genética , Fosforilação , Apoptose , LarvaRESUMO
Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Microbiota , Penaeidae/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Vibrio/fisiologia , Animais , Fatores de Transcrição Forkhead/genética , Hemócitos/imunologia , Homeostase , Imunidade Inata , Penaeidae/imunologia , Penaeidae/microbiologia , Fagocitose/imunologiaRESUMO
White spot syndrome virus (WSSV) is a threatening pathogenic virus in shrimp culture, and at present, no effective strategy can prevent and control the disease. Intestinal flora and its metabolites are important for the resistance of shrimp to lethal pathogenic viruses. However, the changes of metabolites in the shrimp intestines after WSSV infection remain unclear. We established an artificial oral infection method to infect shrimp with WSSV and analyzed the metabolites in intestinal content of shrimp by HPLC and tandem mass spectrometry. A total of 78 different metabolites and five different metabolic pathways were identified. Among them, we found that the content of linoleic acid, an unsaturated fatty acid, increased significantly after WSSV infection, indicating that linoleic acid might be involved in antiviral immunity in shrimp. Further study showed that, after oral administration of linoleic acid, WSSV proliferation decreased evidently in the shrimp, and survival rate of the shrimp increased significantly. Mechanical analysis showed that linoleic acid directly bound to WSSV virions and inhibited the viral replication. Linoleic acid also promoted the expression of antimicrobial peptides and IFN-like gene Vago5 by activating the ERK-NF-κB signaling pathway. Our results indicated that WSSV infection caused metabolomic transformation of intestinal microbiota and that the metabolite linoleic acid participated in the immune response against WSSV in shrimp.
Assuntos
Antivirais/farmacologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Ácido Linoleico/farmacologia , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Animais , Antivirais/metabolismo , Ácido Linoleico/metabolismo , Testes de Sensibilidade Microbiana , PenaeidaeRESUMO
Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.
Assuntos
Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/fisiologia , Receptores Dopaminérgicos/metabolismo , Animais , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Flupentixol/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/fisiologia , Muda/efeitos dos fármacos , Muda/fisiologia , Mariposas/efeitos dos fármacos , Interferência de RNA , Receptores Dopaminérgicos/genética , Células Sf9RESUMO
Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.
Assuntos
Penaeidae/imunologia , Receptores de Imunoglobulina Polimérica/imunologia , Vírus da Síndrome da Mancha Branca 1/metabolismo , Animais , Aquicultura/métodos , Vírus de DNA , Endocitose , Penaeidae/metabolismo , Penaeidae/patogenicidade , Ligação Proteica , Receptores de Imunoglobulina Polimérica/metabolismo , Proteínas do Envelope Viral , Internalização do Vírus , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/patogenicidadeRESUMO
The myeloid differentiation factor 2 (MD-2)-related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann-Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host-virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD-ML1-Dorsal-Vago signaling provided new insights into invertebrate antiviral immunity.
Assuntos
Colestadienos/imunologia , Interações Hospedeiro-Patógeno , Antígeno 96 de Linfócito/fisiologia , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , NF-kappa B/fisiologiaRESUMO
Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.
Assuntos
Cálcio/metabolismo , Ecdisterona/farmacologia , Agregados Proteicos/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Interação Estromal/deficiência , Molécula 1 de Interação Estromal/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Current chemotherapy regimens on acute myeloid leukemia (AML) still have some drawbacks, such as intolerance and drug resistance, which calls need for the development of targeted therapy. Signal transducer and activator of transcription 5 (STAT5) is often overexpressed or abnormally activated in leukemia and involved in cell self-renewal, proliferation, and stress adaptation. Overexpressed Aurora A (AURKA) is associated with poor prognosis in tumors, and inhibitors against AURKA are already in clinical trials. However, it has rarely been reported whether AURKA inhibitors restrain STAT5-activated leukemia cells. In this study, we constructed STAT5 constitutively activated (cS5) cells and found that STAT5 promoted cell proliferation and colony formation. Moreover, cS5 cells showed elevated reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, which indicated higher mitochondrial metabolism in cS5 cells. A novel AURKA inhibitor AKI604 was synthesized and showed significant inhibitory effects to the proliferation and colony formation in both STAT5 constitutively activated and nonactivated AML cells. AKI604 induced mitochondrial impairment, leading to the disruption of mitochondrial membrane potential and the elevation of ROS as well as cellular calcium (Ca2+ ) levels. AKI604 could also decline basal oxygen consumption rate and ATP biosynthesis, indicating the damage of oxidative phosphorylation. Furthermore, AKI604 exhibited significant antitumor effect in the HL-60 cS5 xenograft model of the BALB/c nude mice without an obvious influence on mice body weight and other healthy indicators. This study suggested that AKI604 was a potential strategy to overcome STAT5-induced leukemic proliferation in AML treatment by inducing mitochondrial impairment.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator de Transcrição STAT5/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
It has been identified that arginine vasopressin (AVP), vasopressin receptor 2(V2R), and the aquaporin 2 (AQP2) signaling pathway in the inner ear play important roles in hearing and balance functions through regulating the endolymph equilibrium; however, the contributions of this signaling pathway to the development of motion sickness are unclear. The present study was designed to investigate whether the activation of the AVP-V2R-AQP2 signaling pathway in the inner ear is involved in the induction of motion sickness and whether mozavaptan, a V2R antagonist, could reduce motion sickness. We found that both rotatory stimulus and intraperitoneal AVP injection induced conditioned taste aversion (a confirmed behavioral index for motion sickness) in rats and activated the AVP-V2R-AQP2 signaling pathway with a responsive V2R downregulation in the inner ears, and AVP perfusion in cultured epithelial cells from rat endolymphatic sacs induced similar changes in this pathway signaling. Vestibular training, V2R antagonist mozavaptan, or PKA inhibitor H89 blunted these changes in the V2R-AQP2 pathway signaling while reducing rotatory stimulus- or DDAVP (a V2R agonist)-induced motion sickness in rats and dogs. Therefore, our results suggest that activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness; thus, mozavaptan targeting AVP V2Rs in the inner ear may provide us with a new application option to reduce motion sickness. SIGNIFICANCE STATEMENT: Motion sickness affects many people traveling or working. In the present study our results showed that activation of the inner ear arginine vasopressin-vaspopressin receptor 2 (V2R)-aquaporin 2 signaling pathway was potentially involved in the development of motion sickness and that blocking V2R with mozavaptan, a V2R antagonist, was much more effective in reducing motion sickness in both rat and dog; therefore, we demonstrated a new mechanism to underlie motion sickness and a new candidate drug to reduce motion sickness.
Assuntos
Aquaporina 2/fisiologia , Arginina Vasopressina/fisiologia , Orelha Interna/fisiologia , Enjoo devido ao Movimento/etiologia , Receptores de Vasopressinas/fisiologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Arginina Vasopressina/sangue , Benzazepinas/uso terapêutico , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cães , Feminino , Masculino , Enjoo devido ao Movimento/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.
Assuntos
Proteínas de Artrópodes/metabolismo , Penaeidae/virologia , Proteínas/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Regulação da Expressão Gênica , Hemócitos/metabolismo , Mitocôndrias/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Penaeidae/classificação , Penaeidae/genética , Filogenia , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Superóxidos/metabolismo , Taxa de Sobrevida , Distribuição Tecidual , Replicação Viral/efeitos dos fármacos , Proteína Inibidora de ATPaseRESUMO
The nuclear receptor EcRB1, which is activated by the insect steroid hormone 20-hydroxyecdysone (20E), is reportedly phosphorylated by a protein kinase after 20E induction. However, the protein kinase has not been identified, and the significance of EcRB1 phosphorylation is unclear. In this study, we identified a protein kinase C δ (PKCδ) isoform (the E isoform) that phosphorylates EcRB1 in the lepidopteran Helicoverpa armigera, a serious agricultural pest worldwide, to promote apoptotic gene expression and apoptosis during metamorphosis. Through activation of the EcRB1/USP1 transcription complex by 20E, PKCδ expression was up-regulated in several tissues during the metamorphic stage. Knockdown of PKCδ caused failure to transition from larvae to pupae, prevented tissues from undergoing programmed cell death (PCD), and down-regulated the expression of the transcription factor Brz-7 and the apoptosis executors caspase-3 and caspase-6 The threonine residue at position 1343 of PKCδ was phosphorylated and was critical for its proapoptotic function. Overexpression of the PKCδ catalytic domain was localized to the nuclei in HaEpi cells, which increased caspase-3 activity and apoptosis. PKCδ directly phosphorylated a threonine residue at position 468 in the amino acid sequence of EcRB1. The phosphorylation of EcRB1 was critical for its heterodimeric interaction with the USP1 protein and for binding to the ecdysone response element. The data suggested that 20E up-regulates PKCδ expression to regulate EcRB1 phosphorylation for EcRB1/USP1 transcription complex formation, apoptotic gene transcription, and apoptosis.
Assuntos
Apoptose , Ecdisterona/farmacologia , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores de Esteroides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/genética , Receptores de Esteroides/genética , Elementos de Resposta , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.