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1.
Nat Commun ; 11(1): 858, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051406

RESUMO

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons.


Assuntos
Proteínas Argonauta/classificação , Proteínas de Drosophila/química , Drosophila/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Bombyx/metabolismo , Linhagem Celular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , RNA não Traduzido
2.
Artigo em Inglês | MEDLINE | ID: mdl-32058017

RESUMO

The vitellogenin receptor (VgR) plays a critical role in egg development by mediating endocytosis of the major yolk protein precursor vitellogenin (Vg). Therefore, identifying the VgR of beneficial insects and its characterization could lead to the development of novel egg production strategies to enhance their commercial values. Here, we present the cloning, expression, and functional characterization of the VgR from an economically important eri silkworm, Samia ricini. The complete mRNA sequence was 6002 bp with an ORF of 5484 bp, encoding a protein of 1827 amino acids. Sequence analyses revealed that the SrVgR contained all of the conservative structural motifs characteristics of LDLR family members. The SrVgR was specifically expressed in the ovary, and the mRNA level increased steadily in pupal stages, reached its peak on day 9, and then declined to a bare minimum in adults. RNA interference (RNAi) clearly reduced the VgR transcript levels, disrupted the ovarian development resulting in malformed ovarioles and abnormal development of eggs. Taken together, these data provide conclusive evidence for the essential roles of VgR in insect reproduction.


Assuntos
Bombyx/metabolismo , Proteínas do Ovo/metabolismo , Proteínas de Insetos/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Bombyx/genética , Clonagem Molecular , Proteínas do Ovo/genética , Feminino , Proteínas de Insetos/genética , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos
3.
Artigo em Inglês | MEDLINE | ID: mdl-31931329

RESUMO

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.


Assuntos
Bombyx/química , Cromatografia de Afinidade/métodos , Hemolinfa/química , Larva/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Bombyx/metabolismo , Eletroforese em Gel de Poliacrilamida , Larva/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
4.
Insect Mol Biol ; 29(1): 66-76, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31301266

RESUMO

Storage proteins are haemolymph-specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune-responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real-time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co-immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP-6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two-hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Animais , Bombyx/genética , Bombyx/imunologia , Linhagem Celular , Corpo Adiposo/metabolismo , Proteínas de Fluorescência Verde , Hemolinfa/imunologia , Imunidade Inata , Proteínas de Insetos/genética , Larva/genética , Larva/imunologia , Larva/metabolismo , Proteínas Recombinantes
5.
Insect Mol Biol ; 29(1): 48-55, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31294881

RESUMO

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.


Assuntos
Bombyx/enzimologia , Monoéster Fosfórico Hidrolases/química , Serina/biossíntese , Sequência de Aminoácidos , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Seda
6.
Insect Biochem Mol Biol ; 117: 103293, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31809784

RESUMO

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.


Assuntos
Proteínas de Transporte/genética , Proteínas de Insetos/genética , Mariposas/genética , Animais , Transporte Biológico , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Insetos/metabolismo , Ligantes , Metabolismo dos Lipídeos , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo
7.
Insect Biochem Mol Biol ; 117: 103279, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31756435

RESUMO

In the present study, we demonstrated that bombyxin, an insect insulin-like peptide, modulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) through redox signaling. Our results showed that bombyxin treatment resulted in a transient increase in intracellular reactive oxygen species (ROS) concentration, as measured using 2',7'-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant N-acetylcysteine (NAC) abolished the bombyxin-induced increase in fluorescence in Bombyx PGs. Furthermore, bombyxin-induced ROS production was inhibited by mitochondrial oxidative phosphorylation inhibitors (rotenone and antimycin A), indicating mitochondria-mediated ROS production. The stimulation of ROS production in response to bombyxin appears to undergo development-specific changes. We further investigated the action mechanism of bombyxin-stimulated ROS signaling. Results showed that in the presence of either NAC, rotenone, or antimycin A, bombyxin-stimulated phosphorylation of insulin receptor, Akt, and 4E-binding protein (4E-BP) was blocked and bombyxin-stimulated ecdysteroidogenesis in PGs was greatly inhibited. From these results, we conclude that ROS signaling appears to be involved in bombyxin-stimulated ecdysteroidogenesis of PGs in B. mori by modulating the phosphorylation of insulin receptor, Akt, and 4E-BP. To our knowledge, this is the first demonstration of redox regulation in insulin signaling in an insect system.


Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Bombyx/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/metabolismo
8.
Insect Biochem Mol Biol ; 116: 103258, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678582

RESUMO

The protease inhibitors found in silkworm cocoons can be divided into several families, a majority of which contain serpin, TIL, or Kunitz domains. Previously, it has been reported that TIL-type protease inhibitors have antimicrobial activity. To date, however, it has not been determined whether the Kunitz-type protease inhibitor BmSPI51, the most abundant of cocoon protease inhibitors, plays an antimicrobial role. Thus, in this study, we sought to determine the biological role of BmSPI51 in silkworm cocoons. Our results obtained from real-time quantitative reverse transcription PCR and immunofluorescence analyses indicate that BmSPI51 is expressed exclusively in the silk glands during the larval fifth instar stage and is subsequently secreted into cocoon silk. Moreover, at a molar ratio of 1:1, BmSPI51 produced via prokaryotic expression exhibited inhibitory activity against trypsin and also proved to be highly stable over wide ranges of temperature and pH values. The expression of BmSPI51 was also found to be significantly upregulated in the larval fat body after infection with three species of fungi, namely, Candida albicans, Beauveria bassiana, and Saccharomyces cerevisiae. In vitro inhibition tests revealed that BmSPI51 significantly inhibited the sporular growth of all three of these fungal species. Further, results obtained from a binding assay showed that BmSPI51 binds to ß-d-glucan and mannan on the surface of fungal cells. In this study, we, thus, revealed the antimicrobial activity of BmSPI51 and its underlying mechanism in silkworm, thereby contributing to our present understanding of defense mechanisms in silkworm cocoons.


Assuntos
Bombyx/metabolismo , Bombyx/microbiologia , Proteínas de Insetos/genética , Inibidores de Serino Proteinase/genética , Animais , Antifúngicos/metabolismo , Beauveria/fisiologia , Bombyx/crescimento & desenvolvimento , Candida albicans/fisiologia , Corpo Adiposo/química , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/microbiologia , Saccharomyces cerevisiae/fisiologia , Inibidores de Serino Proteinase/metabolismo
9.
Arch Insect Biochem Physiol ; 103(2): e21627, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31701579

RESUMO

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , MicroRNAs/genética , Sericinas/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , MicroRNAs/metabolismo , Sericinas/metabolismo
10.
Chemosphere ; 245: 125660, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31869670

RESUMO

A comparative transcriptome analysis was conducted to investigate the gene expression changes in the fat body of silkworm after treatment with different concentrations (50 µM and 200 µM) of selenium (Se). 912 differential expression genes (DEGs) (371 up-regulated and 541 down-regulated) and 1420 DEGs (1078 up-regulated and 342 down-regulated) were identified in silkworm fat body treated with 50 µM and 200 µM of Se, respectively. In case of 50 µM group, DEGs were mainly enriched in the peroxisome pathway and fatty acid metabolism pathway, and later were associated with antioxidant defense and nutrition regulation. After 200 µM Se-treatment, DEGs were mainly located in the glycerolipid metabolism and arachidonic acid metabolism pathways, which further encoded detoxification related genes. Furthermore, 32 candidate DEGs from these pathways had been selected to confirm the RNA-seq data. Among these DEGs, 14 genes were up-regulated in the 50 µM Se-treated group (only three genes in the 200 µM Se-treated group) which were involved in lipid metabolism and antioxidant defense, and 13 up-regulated genes (only two genes were up-regulated in the 50 µM Se-treated group) were involved in detoxification of the 200 µM Se-treated group. These changes showed that lower concentration of Se could regulate the nutrition and promote antioxidation pathways; whereas, high levels of Se promoted the detoxification of silkworm. These findings can be helpful to understand the possible mechanisms of Se action and detoxification in silkworm and other insects.


Assuntos
Bombyx/fisiologia , Selênio/metabolismo , Tecido Adiposo/metabolismo , Animais , Bombyx/genética , Bombyx/metabolismo , Regulação para Baixo , Corpo Adiposo/metabolismo , Corpo Adiposo/fisiologia , Perfilação da Expressão Gênica , Inativação Metabólica , Metabolismo dos Lipídeos , Transcriptoma
11.
Carbohydr Polym ; 228: 115382, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31635752

RESUMO

Chitins of different purity grades (45%, 89.7% and 93.3%) were efficiently extracted from Bombyx eri larva and fully physico-chemically characterized. Compared to commercially available and extracted α-chitin from shrimp shell, the collected data showed that insect chitins had similar characteristics in terms of crystallographic structures (α-chitin), thermal stability and degree of acetylation (>87%). The major differences lay in the crystallinity indexes (66% vs 75% for shrimp chitin) and in the morphological structures. Furthermore, low ash contents were determined for the insect chitins (1.90% vs 21.73% for shrimp chitin), making this chitin extraction and purification easier, which is highly valuable for an industrial application. Indeed, after only one step (deproteinization), the obtained chitin from Bombyx eri showed higher purity grade than the one extracted from shrimp shells under the same conditions. Insect chitins were then subjected to room temperature ionic liquid (RTIL) pretreatment prior to enzymatic degradation and presented a higher enzymatic digestibility compared to commercial one whatever their purity grade and would be thus a more relevant source for the selective production of N-acetyl-D-glucosamine (899.2 mg/g of chitin-2 stepsvs 760 mg/g of chitin com). Moreover, for the first time, the fermentescibility of chitin hydrolysates was demonstrated with Scheffersomyces stipitis used as ethanologenic microorganism.


Assuntos
Bombyx/metabolismo , Quitina , Crustáceos/metabolismo , Animais , Quitina/química , Quitina/isolamento & purificação , Larva/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 107: 110197, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761195

RESUMO

Silks, in particular silkworm silks, have been studied for decades as possible candidate materials for biomedical applications. Recently, great attentions have been paid to spider silks, mainly due to their unique and remarkable mechanical properties. Both materials express singular interactions with cells through specific biorecognition moieties on the core proteins making up the two silks. In this work, the silk from a Colombian spider, Linothele megatheloides (LM), which produces a single type of silk in a relatively large amount, was studied in comparison with silk from Bombyx mori silkworm, before and after degumming, with the evaluation of their chemical, mechanical and biological properties. Unexpected biological features in cell culture tests were found for the LM silk already at very early stage, so suggesting further investigation to explore its use for tailored biomedical applications.


Assuntos
Bombyx/metabolismo , Seda/química , Aranhas/metabolismo , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Módulo de Elasticidade , Camundongos , Células NIH 3T3 , Seda/farmacologia , Resistência à Tração
13.
J Insect Sci ; 19(6)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31765475

RESUMO

The present study was carried out to determine the influence of 2% aqueous honey (Apis dorsata Fabricius, 1793 [Hymenoptera: Apidae]) on larval growth and silk cocoon yield of fifth-instar larvae of the silkworm, Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae). The larvae of silkworms (Chinese HUAKAND2) were divided into a control and an experimental groups (n = 20 in each group). Control group was fed with plain mulberry leaves throughout the fifth instar, whereas the experimental group was offered mulberry leaves dipped in 2% aqueous solution of honey every other day for 4 d (days 1, 3, 5, and 7). On the other days (days 2, 4, 6, and 8), plain mulberry leaves were offered to larvae. Results showed that the average weight gain in larvae of the experimental group was 348.23 and 204.54% in case of the control group. Uneaten mulberry leaves were weighed; the control group left 34.05% of their leaves and the treated group 28.54%. The cocoon formation in the honey-treated larvae was more uniform in shape than the control group. Furthermore, honey-treated larvae began to form cocoons 7.8 ± 0.23 h earlier than the control group. We also recorded an increase of 15.34% in average weight of cocoons of the experimental group when compared with the control. Average shell percentage of fresh silk cocoons of the control and experimental groups was 20.5 and 23.5%, respectively. It is concluded from the study that 2% aqueous honey has positive impact on the larval growth and cocoon yield of B. mori.


Assuntos
Bombyx/crescimento & desenvolvimento , Mel , Larva/crescimento & desenvolvimento , Seda/biossíntese , Animais , Abelhas , Bombyx/metabolismo , Larva/metabolismo
14.
Int J Mol Sci ; 20(19)2019 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-31569473

RESUMO

Sterols, especially cholesterol (Chl), are fundamental for animal survival. Insects lacking the ability to synthesize Chl are sterol auxotrophic animals and utilize dietary Chl and phytosterols to survive. The sterols obtained from a diet are distributed to the tissues; however, sterol homeostasis in insect tissues remains to be elucidated. This study sought to understand the sterol characteristics of insect tissues through detailed sterol quantification and statistics. The combination of sterol quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS) and principal component analysis (PCA) revealed tissue-specific sterol characteristics in the silkworm, Bombyx mori, a phytophagous insect. We found that insect tissues have tissue-intrinsic sterol profiles. The brain has a unique sterol composition as compared to other tissues-high concentration of Chl and less accumulation of phytosterols. Other tissues also have intrinsic sterol characteristics, which when defined by dietary sterols or Chl metabolites, indicate preference for a sterol and consistently manage their own sterol homeostasis. Though most tissues never change sterol profiles during development, the brain drastically changes its sterol profile at the wandering stage, indicating that it could alter sterol composition in preparation for metamorphosis. These results suggest the existence of tissue- and sterol-specific systems for sterol homeostasis in insects.


Assuntos
Bombyx/metabolismo , Encéfalo/metabolismo , Cromatografia Líquida , Esteróis/metabolismo , Espectrometria de Massas em Tandem , Animais , Colesterol/metabolismo , Hemolinfa/metabolismo , Especificidade de Órgãos
15.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470538

RESUMO

Protein conjugations at post-translational levels are known to be essential to protein stability and function. Recently, it has been proven that the split protein CnaB2 (SpyTag/SpyCatcher, ST/SC) from Streptococcus pyogenes can induce covalent conjugation rapidly and efficiently under various conditions. The protein of interest fused with the split protein SC/ST could be assembled spontaneously. In light of this finding, we introduced the ST/SC protein coupling concept into the silkworm-bacmid protein expression system (SpyBEVS). As a proof of concept, we first examined and confirmed that a competent ligation occurred between ST/SC-fused protein partners in vitro in cultured silkworm cells and in vivo in silkworm larvae by co-infection of several recombinant baculoviruses. The protein conjugation could be also achieved sufficiently by a simple one-step mixture of purified ST/SC-tagged peptide-protein pairs in vitro. Given the flexibility and robustness of silkworm-BEVS, our results on SpyBEVS show an alternative method for enabling the production of protein decorations in vitro and inside of silkworms.


Assuntos
Bombyx/genética , Vetores Genéticos/genética , Proteínas de Insetos/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bombyx/citologia , Bombyx/metabolismo , Células Cultivadas , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Engenharia de Proteínas/métodos , Estabilidade Proteica , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
16.
Chemosphere ; 236: 124379, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31545189

RESUMO

In order to study the role of mulberry (Moms alba L) as an economic crop for remediation of cadmium (Cd) contaminated soil, the transport of Cd from mulberry to silkworm were investigated. Three varieties of mulberry (Yuesang-11, Nongsang-14, and Qiangsang-1) with three planting densities were cultivated in two heavy metal-contaminated fields named Dongkou in Shaoyang city and Linxiang in Yueyang city in Hunan province respectively. The both field soils were contaminated by heavy metals, especially by Cd. The potential risks of heavy metals in Linxiang's soil were higher than those in Dongkou's because of higher concentrations of Cd. Since the promotion of Cd concentrations in aerial parts (stem, branch and leaf) resulted from the increase of planting density, the method of high planting density is beneficial to improve the efficiency of the remediation of Cd contaminated soil. The percentages of average Cd contents of mulberry in Dongkou accounted for 44%, 20%, 18% and 16% in roots, stems, branches and leaves respectively, while the Cd contents were 38%, 27%, 19% and 16% distributed in roots, stems, branches and leaves respectively. Mulberry leaves from contaminated soils was applied in food source of silkworms in this study. Although there is Cd uptake occurred in silkworm growth and its products (cocoons and chrysalis), Cd contents in cocoons are lower than the national standard (100 µg*kg-1) for textile industry of China. Therefore, mulberry can be regarded as an economical crop to control soil contamination with Cd.


Assuntos
Cádmio/metabolismo , Recuperação e Remediação Ambiental/métodos , Morus/metabolismo , Poluentes do Solo/metabolismo , Animais , Biodegradação Ambiental , Bombyx/química , Bombyx/metabolismo , Cádmio/análise , China , Recuperação e Remediação Ambiental/economia , Frutas/química , Frutas/metabolismo , Morus/química , Morus/classificação , Folhas de Planta/química , Folhas de Planta/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Poluentes do Solo/análise
17.
Mol Biotechnol ; 61(11): 852-859, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473916

RESUMO

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Vírus da Dengue/metabolismo , Corpo Adiposo/metabolismo , Expressão Gênica , Vetores Genéticos , Hemolinfa/metabolismo , Heparina/metabolismo , Larva/metabolismo , Sinais Direcionadores de Proteínas/genética , Sorogrupo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Vírion/ultraestrutura
18.
Nucleic Acids Res ; 47(16): 8708-8719, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392993

RESUMO

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented.


Assuntos
Enzimas de Restrição do DNA/genética , DNA Cruciforme/genética , Elementos Nucleotídeos Longos e Dispersos , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , Transcrição Reversa , Animais , Bombyx/genética , Bombyx/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA , Primers do DNA/genética , Primers do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo
19.
Pestic Biochem Physiol ; 159: 41-50, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400783

RESUMO

Emerging fungal phytodiseases are a food security threat and novel fungicides are in an urgent need. Herein, a series of isobutyrophenone derivatives were designed and synthesized. The derivatives exhibited excellent fungicidal activities against seven fungi. The structure-activity relationship (SAR) study indicated that the introduction of a bromo group at the position 3 or 5 of the phenyl ring, as well as esterification of the 4-hydroxy with a chloroacetyl group, could substantially increase the antifungal activity and spectrum of the compounds. Among all 23 compounds, 2-bromo-3-hydroxy-4-isobutyryl-6-methylphenyl 2-chloroacetate (12b) showed the highest fungicidal activity against all seven tested fungal pathogens with EC50 values ranging from 1.22 to 39.94 µg/mL and exhibited the most potent inhibition against class II fructose-1,6-bisphosphate aldolase with an IC50 of 3.63 µM. The lead compounds were proven to be safe to NIH3T3/293 T cells and silkworm larvae, and relatively stable under different harsh conditions. Detached fruit tests showed the practical potential of lead compounds for fruit (or plant) protection. Taken together, our results indicated that the isobutyrophenone derivatives could be further optimized and developed as advanced leads for new fungicides.


Assuntos
Antifúngicos/química , Antifúngicos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Animais , Bombyx/metabolismo , Linhagem Celular , Frutose-Bifosfato Aldolase/genética , Humanos , Larva/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Células NIH 3T3 , Relação Estrutura-Atividade
20.
Insect Biochem Mol Biol ; 114: 103225, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31446032

RESUMO

Negative regulation of the immune signaling pathway involves diverse negative regulators that target different signaling molecules. One of the signaling molecules, DREDD, which activates the NF-κB transcription factor Relish in the IMD pathway, is a homolog of mammalian caspase-8. Some structural related proteins have been identified to regulate the activity of caspase-8 in signaling complex assembly. However, it is unknown in insects whether the IMD pathway undergoes such a down-regulation. In this study, we explored the regulatory role of a newly identified protein BmCaspase-8 like (BmCasp8L) in silkworm, which displays high sequence similarity with the N-terminus of BmDREDD to the IMD pathway, and investigated its mechanism. Domain prediction, phylogenic analysis and gene architecture suggests BmCasp8L acts as a potential inhibitor to BmDREDD. We then found it is highly expressed in the fat body and hemocytes, and suppresses the cleavage of BmRelish and BmIMD mediated by BmDREDD upon PGN stimulation, resulting in deficiency in antimicrobial peptides production. Besides the inhibitory role in the IMD pathway, it also suppresses the BmDREDD-induced apoptosis. By investigating the amyloidal activity of BmCasp8L and its interaction with BmDREDD and BmFADD, we demonstrated that BmCasp8L forms amyloid-like aggregates in vitro as well as in vivo, and it inactivates BmDREDD by blending into the amyloidal speck-like structure formed by BmDREDD and BmFADD that is required for BmDREDD activity. Taken together, our results demonstrate BmCasp8L inhibits the IMD signaling pathway via forming amyloidal aggregates with BmDREDD, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.


Assuntos
Amiloide/metabolismo , Bombyx/metabolismo , Caspases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas de Insetos/metabolismo , Animais , Bombyx/imunologia , Linhagem Celular , Imunidade Inata , Transdução de Sinais
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