RESUMO
Progestin resistance is the main obstacle for the conservative therapy to maintain fertility in women with endometrial cancer. Brusatol was identified as an inhibitor of the NRF2 pathway; however, its impact on progestin resistance and the underlying mechanism remains unclear. Here, we found that brusatol sensitized endometrial cancer to progestin by suppressing NRF2-TET1-AKR1C1-mediated progestin metabolism. Brusatol transcriptionally suppressed AKR1C1 via modifying the hydroxymethylation status in its promoter region through TET1 inhibition. Suppression of AKR1C1 by brusatol resulted in decreased progesterone catabolism and maintained potent progesterone to inhibit endometrial cancer growth. This inhibition pattern has also been found in the established xenograft mouse and organoid models. Aberrant overexpression of AKR1C1 was found in paired endometrial hyperplasia and cancer samples from the same individuals with progestin resistance, whereas attenuated or loss of AKR1C1 was observed in post-treatment samples with well progestin response as compared with paired pre-treatment tissues. Our findings suggest that AKR1C1 expression pattern may serve as an important biomarker of progestin resistance in endometrial cancer.
Assuntos
Hiperplasia Endometrial , Neoplasias do Endométrio , Humanos , Feminino , Camundongos , Animais , Hiperplasia Endometrial/tratamento farmacológico , Hiperplasia Endometrial/genética , Progestinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Progesterona , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a DNARESUMO
Macrocentrus cingulum is a principal endoparasite of Ostrinia furnacalis larvae. M. cingulum larvae repress host immune responses for survival and ingest host nutrients for development until emerging. However, most investigations focused on the mechanisms of how wasps repress the host immunity, the triggered immune responses and nutrient status altered by wasps in host are neglected. In this study, we found that parasitized O. furnacalis larvae activated fast recognition responses and produced some effectors such as lysozyme and antimicrobial peptides, along with more consumption of trehalose, glucose, and even lipid to defend against the invading M. cingulum. However, the expression of peroxidase 6 and superoxide dismutase 2 (SOD 2) was upregulated, and the messenger RNA (mRNA) levels of cellular immunity-related genes such as thioester-containing protein 2 (TEP 2) and hemocytin were also reduced, suggesting that some immune responses were selectively shut down by wasp parasitization. Taken together, all the results indicated that parasitized O. furnacalis larvae selectively activate the immune recognition response, and upregulate effector genes, but suppress ROS reaction and cellular immunity, and invest more energy to fuel certain immune responses to defend against the wasp invading. This study provides useful information for further identifying key components of the nutrition and innate immune repertoire which may shape host-parasitoid coevolutionary dynamics.
Assuntos
Transcriptoma , Vespas , Animais , Interações Hospedeiro-Parasita , Imunidade , LarvaRESUMO
Pathogen-induced host immune responses reduce the efficacy of pathogens used to control pests. However, compared to the well-deciphered immunity system of Drosophila melanogaster, the immunity system of agricultural pests is largely unconfirmed through functional analysis. Beginning to unveil mechanisms of transcription regulation of immune genes in the Asian corn borer, Ostrinia furnacalis, we cloned the complementary DNA (cDNA) of a transcription factor Relish by rapid amplification of cDNA ends. The 3164 bp cDNA, designated Of-Relish, encodes a 956-residue protein. Bioinformatic analysis showed that Of-Relish had a Rel homology domain, a predicted cleavage site between Q409 and L410 , six ankyrin repeats, and a death domain. The response of Of-Relish expression to the Gram-negative bacteria Pseudomonas aeruginosa was sooner and stronger than to the Gram-positive Micrococcus luteus. The antimicrobial peptide genes Attacin and Gloverin had similar expression patterns in response to the infections. Knockdown of Of-Relish led to a decrease in Attacin and Gloverin messenger RNA levels, suggesting that Attacin and Gloverin were regulated by Of-Relish. Together, the results suggested that Of-Relish is a key component of the IMD pathway in O. furnacalis, involved in defense against P. aeruginosa through activation of Attacin and Gloverin.
Assuntos
Regulação da Expressão Gênica , Mariposas/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila/genética , Genes de Insetos , Imunidade/genética , Proteínas de Insetos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mariposas/genéticaRESUMO
PURPOSE: Patients with Klinefelter syndrome (KS) who receive assisted reproductive technology (ART) treatment often experience poor pregnancy rates due to decreased fertilization, cleavage, and implantation rates and even an increased miscarriage rate. Mounting evidence from recent studies has shown that various technological advances and approaches could facilitate the success of ART treatment for KS patients. In this review, we summarize the methods for guiding KS patients during ART and for developing optimal strategies for preserving fertility, improving pregnancy rate and live birth rate, and avoiding the birth of KS infants. METHODS: We searched PubMed and Google Scholar publications related to KS patients on topics of controlled ovarian stimulation protocols, sperm extraction, fertility preservation, gamete artificial activation, round spermatid injection (ROSI), and non-invasive prenatal screening (PGD) methods. RESULTS: This review outlines the different ovulation-inducing treatments for female partners according to the individual sperm status in the KS patient. We further summarize the methods of retrieving sperm, storing, and freezing rare sperm. We reviewed different methods of gamete artificial activation and discussed the feasibility of ROSI for sterile KS patients who absolutely lack sperm. The activation of eggs in the process of intracytoplasmic sperm injection and non-invasive PGD are urgently needed to prevent the birth of KS infants. CONCLUSION: The integrated strategies will pave the way for the establishment of ART treatment approaches and improve the clinical outcome for KS patients.
Assuntos
Implantação do Embrião/genética , Síndrome de Klinefelter/terapia , Técnicas de Reprodução Assistida/tendências , Coeficiente de Natalidade , Feminino , Preservação da Fertilidade/tendências , Humanos , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/patologia , Masculino , Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/tendênciasRESUMO
TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.
Assuntos
Proteínas de Drosophila/genética , Lisossomos/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas/genética , Serina-Treonina Quinases TOR/genética , Animais , Proteínas de Ciclo Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epistasia Genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. Here we define opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Mio and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. To our knowledge, these studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. Our data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms.
Assuntos
Proteínas de Drosophila/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Oogênese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Meiose/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oogênese/efeitos dos fármacos , Sirolimo/farmacologia , Fatores de Transcrição/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a nearly lethal neoplasm. It is a remarkably stroma-rich, vascular-poor and hypo-perfused tumour, which prevents efficient drug delivery. Paradoxically, the neoplastic cells have robust glucose uptake, suggesting that the microvasculature has adopted an alternative method for nutrient uptake and cellular trafficking. Using adapted thick tumour section immunostaining and three-dimensional (3D) construction imaging in human tissue samples, we identified an undiscovered feature of the mature microvasculature in advanced PDAC tumours; long, hair-like projections on the basal surface of microvessels that we refer to as 'basal microvilli'. Functionally, these basal microvilli have an actin-rich cytoskeleton and endocytic and exocytic properties, and contain glucose transporter-1 (GLUT-1)-positive vesicles. Clinically, as demonstrated by PET-CT, the tumour microvasculature with the longest and most abundant basal microvilli correlated with high glucose uptake of the PDAC tumour itself. In addition, these basal microvilli were found in regions of the tumour with low GLUT-1 expression, suggesting that their presence could be dependent upon the glucose concentration in the tumour milieu. Similar microvasculature features were also observed in a K-Ras-driven model of murine PDAC. Altogether, these basal microvilli mark a novel pathological feature of PDAC microvasculature. Because basal microvilli are pathological features with endo- and exocytic properties, they may provide a non-conventional method for cellular trafficking in PDAC tumours.
Assuntos
Carcinoma Ductal Pancreático/irrigação sanguínea , Neoplasias Pancreáticas/irrigação sanguínea , Adaptação Fisiológica/fisiologia , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/patologia , Movimento Celular/fisiologia , Transportador de Glucose Tipo 1/metabolismo , Humanos , Microvasos/patologia , Neoplasias Pancreáticas/patologia , Neoplasias PancreáticasRESUMO
We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Testículo/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Testículo/crescimento & desenvolvimentoRESUMO
Centrosome linker tethers interphase centrosomes together allowing them to function as a single microtubule organization center. The centrosome linker is disrupted at the onset of mitosis to ensure timely centrosome disjunction and bipolar spindle formation and is reassembled at the end of mitosis. While the mechanism controlling centrosome linker disassembly at early mitosis has been well explored, little is known about how the linker is subsequently reassembled before mitotic exit. Here we report that ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are involved in centrosome linker reassembly. We showed that ASPP1/2 interacted with centrosome linker protein C-Nap1. Co-depletion of ASPP1 and ASPP2 inhibited re-association of C-Nap1 with centrosome at the end of mitosis. Moreover, ASPP1/2 facilitated the interaction between C-Nap1 and PP1α, and this interaction was significantly reduced by co-depletion of ASPP1/2. ASPP1/2 antagonized the NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner. Co-depletion of ASPP1 and ASPP2 inhibited dephosphorylation of C-Nap1 (Ser2417/2421) at the end of mitosis. Based on these findings, we propose that ASPP1/2 act as PP1-targeting subunits to facilitate C-Nap1 dephosphorylation and centrosome linker reassembly at the end of mitosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Células HeLa , Humanos , Mitose , Mapas de Interação de ProteínasRESUMO
Tssk4 belongs to the Testis Specific Serine/threonine protein Kinase (TSSK) family, members of which play an important role in spermatogenesis and/or spermiogenesis. Several Tssk family proteins have extensively been studied. However, the exact function of Tssk4 remains unclear. A Tssk4 knockout mouse model was generated and the males were subfertile due to seriously decreased sperm motility. The ultrastructure of the Tssk4(-/-)sperm tail is disorganized at the midpiece-principal piece junction, leading to a severe bend in the sperm flagellum. One or more axonemal microtubule doublets are absent and the midpiece is fused with the principal piece. Furthermore, we identified the association between Tssk4 and Odf2, a prominent cytoskeletal protein of the outer dense fiber (ODF) in sperm flagellum. Tssk4 can change the phosphorylation state of Odf2 and conversely Odf2 potentiates the autophosphorylation activity of Tssk4. These findings reveal that Tssk4 is required for maintaining the structural integrity of sperm flagellum and male fertility.
Assuntos
Fertilidade/fisiologia , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/fisiologia , Animais , Fertilidade/genética , Proteínas de Choque Térmico/genética , Masculino , Camundongos , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologiaRESUMO
Testis specific serine/threonine protein kinase 4 (TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed HeLa cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including HeLa, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.
Assuntos
Apoptose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Acute lung injury (ALI) is a severe respiratory disease with a high mortality rate. The integrity of the pulmonary endothelial barrier influences the development and prognosis of ALI. Therefore, it has become an important target for ALI treatment. Extracellular vesicles (EVs) are promising nanotherapeutic agents against ALI. Herein, endothelium-derived engineered extracellular vesicles (eEVs) that deliver microRNA-125b-5p (miRNA-125b) to lung tissues exerting a protective effect on endothelial barrier integrity are reported. eEVs that are modified with lung microvascular endothelial cell-targeting peptides (LET) exhibit a prolonged retention time in lung tissues and targeted lung microvascular endothelial cells in vivo and in vitro. To improve the efficacy of the EVs, miRNA-125b is loaded into EVs. Finally, LET-EVs-miRNA-125b is constructed. The results show that compared to the EVs, miRNA-125b, and EVs-miRNA-125b, LET-EVs-miRNA-125b exhibit the most significant treatment efficacy in ALI. Moreover, LET-EVs-miRNA-125b is found to have an important protective effect on endothelial barrier integrity by inhibiting cell apoptosis, promoting angiogenesis, and protecting intercellular junctions. Sequencing analysis reveals that LET-EVs-miRNA-125b downregulates early growth response-1 (EGR1) levels, which may be a potential mechanism of action. Taken together, these findings suggest that LET-EVs-miRNA-125b can treat ALI by protecting the endothelial barrier integrity.
Assuntos
Lesão Pulmonar Aguda , Vesículas Extracelulares , MicroRNAs , Humanos , Células Endoteliais , Pulmão , MicroRNAs/genética , Lesão Pulmonar Aguda/terapia , EndotélioRESUMO
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.
Assuntos
Dinaminas/fisiologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Endocitose , Concentração de Íons de Hidrogênio , Neuroblastoma/virologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clatrina/fisiologia , Primers do DNA , Neuroblastoma/patologia , RNA Interferente Pequeno , Ratos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The testis specific serine/threonine protein kinase family (Tssk) members play important roles in spermatogenesis and/or spermiogenesis. Similar to other Tssk family members, Tssk4 protein shows exclusive expression in testis, but its biochemical and biological functions are still largely unknown. In present work, we generate a polyclonal antibody which specifically recognizes Tssk4 but not the other three Tssk family members (Tssk1, Tssk2 and Tssk3). By using the qualified antibody, we show that Tssk4 protein is constantly expressed in testis from haploid round spermatids to morphological mature spermatozoa. Further experiments reveal that Tssk4 has autophosphorylation activity and self-association character in vitro. Importantly, we find that autophosphorylation of Tssk4 at Thr-197 in the T-loop region is essential to its kinase activity. Taken together, these findings suggest that autophosphorylation at Thr-197 plays a critical role in maintaining Tssk4 kinase activity, which might be involved in spermiogenesis.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Ativação Enzimática , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Espermatogênese/genética , Treonina/químicaRESUMO
The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Drosophila/genéticaRESUMO
The preservation of female fertility under unfavorable conditions is essential for animal reproduction. Inhibition of the target of rapamycin complex 1 (TORC1) is indispensable for Drosophila young egg chamber maintenance under nutrient starvation. Here, we show that knockdown of RagA results in young egg chamber death independent of TORC1 hyperactivity. RagA RNAi ovaries have autolysosomal acidification and degradation defects, which make the young egg chambers sensitive to autophagosome augmentation. Meanwhile, RagA RNAi ovaries have nuclear-localized Mitf, which promotes autophagic degradation and protects young egg chambers under stress. Interestingly, GDP-bound RagA rescues autolysosome defects, while GTP-bound RagA rescues Mitf nuclear localization in RagA RNAi young egg chambers. Moreover, Rag GTPase activity, rather than TORC1 activity, controls Mitf cellular localization in the Drosophila germ line. Our work suggests that RagA separately controls autolysosomal acidification and Mitf activity in the Drosophila young egg chambers.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Feminino , Drosophila/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Drosophila/metabolismo , Ovário/metabolismo , Células Germinativas/metabolismoRESUMO
Aging is a process accompanied by widespread degenerative changes which are a major cause of human disease and disability. One goal of aging research is to develop interventions or drugs that can extend organism lifespan and treat age-related diseases. Here, we report the identification of a broad spectrum anti-viral agent, ribavirin, as a potential pharmacological aging intervention. Ribavirin extended the lifespan and healthspan of Caenorhabditis elegans by inhibiting Target of Rapamycin (TOR) signaling and activating AMP-activated protein kinase (AMPK). Moreover, our data indicate that ribavirin activated AMPK by reducing the levels of adenosine triphosphate (ATP) and lysosomal v-ATPase-Ragulator-AXIN Complex. Thus, our studies successfully identify ribavirin as a potential anti-aging drug, and indicate that its anti-aging effect is mediated via AMPK-TOR signaling.
Assuntos
Caenorhabditis elegans , Longevidade , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Ribavirina/farmacologia , Transdução de SinaisRESUMO
FK506-binding protein 39kD (FKBP39) localizes in the nucleus and contains multiple functional domains. Structural analysis suggests that FKBP39 might function as a transcriptional factor and control juvenile hormone (JH) activity. Here, we show that FKBP39 expresses at a high level and localizes in the nucleolus of fat body cells during the first two larval stages and early third larval stage. The fkbp39 mutant displays delayed larval-pupal transition and an increased expression of Kr-h1, the main mediator of the JH pathway, at the early third larval stage. Moreover, the fkbp39 mutant has a fertility defect that is independent of JH activity. Interestingly, the expression of rp49, the most widely used reference gene for qRT-PCR in Drosophila, significantly decreased in the fkbp39 mutant, suggesting that FKBP39 might regulate ribosome assembly. Taken together, our data demonstrate the expression pattern and physiological roles of FKBP39 in Drosophila.
RESUMO
Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.
Assuntos
Calcineurina , Óxido Nítrico , Monofosfato de Adenosina/metabolismo , Animais , Peptídeos Antimicrobianos , Calcineurina/metabolismo , Drosophila , Escherichia coli/metabolismo , Larva/microbiologia , Óxido Nítrico/metabolismoRESUMO
The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3'-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly.