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1.
Hepatology ; 71(2): 463-476, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31278760

RESUMO

Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3' exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2-NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2-NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus-strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV-DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.

2.
Biol Open ; 8(5)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30992325

RESUMO

Impaired osteoblast differentiation may result in bone metabolic diseases such as osteoporosis. It was reported recently that hedgehog (Hh) signaling and autophagy are two important regulators of bone differentiation. In order to further dissect their relationship in bone development, we used a zebrafish larvae model to investigate how disruption of one of these signals affects the function of the other and impacts osteoblast differentiation. Our results showed that activation of Hh signaling negatively regulated autophagy. However, suppression of autophagy by knocking down atg5 expression did not alter Hh signaling, but dramatically upregulated the expression of osteoblast-related genes and increased bone mineralization, especially in the den region. On the contrary, inhibition of the Hh signaling pathway by cyclopamine treatment suppressed the expression of osteoblast-related genes and decreased bone mineralization. In agreement with these findings, blocking Hh signaling through knockdown SHH and Gli2 genes led to defective osteoblast differentiation, while promoting Hh signaling by knockdown Ptch1 was beneficial to osteoblast differentiation. Our results thus support that activation of the Hh signaling pathway negatively regulates autophagy and consequentially promotes osteoblast differentiation. On the contrary, induction of autophagy inhibits osteoblast differentiation. Our work reveals the mechanism underlying Hh signaling pathway regulation of bone development.

3.
ACS Infect Dis ; 5(5): 659-674, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29893548

RESUMO

Hepatitis B virus (HBV) has infected one-third of world population, and 240 million people are chronic carriers, to whom a curative therapy is still not available. Similar to other viruses, persistent HBV infection relies on the virus to exploit host cell functions to support its replication and efficiently evade host innate and adaptive antiviral immunity. Understanding HBV replication and concomitant host cell interactions is thus instrumental for development of therapeutics to disrupt the virus-host interactions critical for its persistence and cure chronic hepatitis B. Although the currently available cell culture systems of HBV infection are refractory to genome-wide high throughput screening of key host cellular factors essential for and/or regulating HBV replication, classic one-gene (or pathway)-at-a-time studies in the last several decades have already revealed many aspects of HBV-host interactions. An overview of the landscape of HBV-hepatocyte interaction indicates that, in addition to more tightly suppressing viral replication by directly targeting viral proteins, disruption of key viral-host cell interactions to eliminate or inactivate the covalently closed circular (ccc) DNA, the most stable HBV replication intermediate that exists as an episomal minichromosome in the nucleus of infected hepatocyte, is essential to achieve a functional cure of chronic hepatitis B. Moreover, therapeutic targeting of integrated HBV DNA and their transcripts may also be required to induce hepatitis B virus surface antigen (HBsAg) seroclearance and prevent liver carcinogenesis.

4.
ACS Infect Dis ; 5(5): 759-768, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30525438

RESUMO

Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.

5.
Antiviral Res ; 159: 1-12, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30201396

RESUMO

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.


Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , RNA/metabolismo , Proteínas do Core Viral/genética , Montagem de Vírus , Regulação Alostérica , Proteínas do Capsídeo/metabolismo , Eletroforese , Ensaio de Desvio de Mobilidade Eletroforética , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , RNA Viral/metabolismo , Replicação Viral
6.
J Virol ; 92(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669831

RESUMO

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.


Assuntos
Genoma Viral , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Precursores de RNA/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Regulação Alostérica , Capsídeo/metabolismo , Células Cultivadas , Hepatite B/genética , Hepatite B/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fosforilação , Precursores de RNA/genética , RNA Viral/genética , Proteínas do Core Viral/genética , Replicação Viral
7.
J Biol Chem ; 292(44): 18062-18074, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28928221

RESUMO

Parkinson's disease (PD) is one of the most epidemic neurodegenerative diseases and is characterized by movement disorders arising from loss of midbrain dopaminergic (DA) neurons. Recently, the relationship between PD and autophagy has received considerable attention, but information about the mechanisms involved is lacking. Here, we report that autophagy-related gene 5 (ATG5) is potentially important in protecting dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in zebrafish. Using analyses of zebrafish swimming behavior, in situ hybridization, immunofluorescence, and expressions of genes and proteins related to PD and autophagy, we found that the ATG5 expression level was decreased and autophagy flux was blocked in this model. The ATG5 down-regulation led to the upgrade of PD-associated proteins, such as ß-synuclein, Parkin, and PINK1, aggravation of MPTP-induced PD-mimicking pathological locomotor behavior, DA neuron loss labeled by tyrosine hydroxylase (TH) or dopamine transporter (DAT), and blocked autophagy flux in the zebrafish model. ATG5 overexpression alleviated or reversed these PD pathological features, rescued DA neuron cells as indicated by elevated TH/DAT levels, and restored autophagy flux. The role of ATG5 in protecting DA neurons was confirmed by expression of the human atg5 gene in the zebrafish model. Our findings reveal that ATG5 has a role in neuroprotection, and up-regulation of ATG5 may serve as a goal in the development of drugs for PD prevention and management.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica , Terapia Genética , Transtornos Parkinsonianos/prevenção & controle , Proteínas de Peixe-Zebra/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , DNA Recombinante/uso terapêutico , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/patologia , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Larva , Microinjeções , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/uso terapêutico , Neuroproteção/efeitos dos fármacos , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
8.
Sci Rep ; 7(1): 11250, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28900156

RESUMO

Autophagy and immune response are two defense systems that human-body uses against viral infection. Previous studies documented that some viral mechanisms circumvented host immunity mechanisms and hijacked autophagy for its replication and survival. Here, we focus on interactions between autophagy mechanism and innate-immune-response in HCV-subgenomic replicon cells to find a mechanism linking the two pathways. We report distinct effects of two autophagy-related protein ATG10s on HCV-subgenomic replication. ATG10, a canonical long isoform in autophagy process, can facilitate HCV-subgenomic replicon amplification by promoting autophagosome formation and by combining with and detaining autophagosomes in cellular periphery, causing impaired autophagy flux. ATG10S, a non-canonical short isoform of ATG10 proteins, can activate expression of IL28A/B and immunity genes related to viral ds-RNA including ddx-58, tlr-3, tlr-7, irf-3 and irf-7, and promote autophagolysosome formation by directly combining and driving autophagosomes to perinuclear region where lysosomes gather, leading to lysosomal degradation of HCV-subgenomic replicon in HepG2 cells. ATG10S also can suppress infectious HCV virion replication in Huh7.5 cells. Another finding is that IL28A protein directly conjugates ATG10S and helps autophagosome docking to lysosomes. ATG10S might be a new host factor against HCV replication, and as a target for screening chemicals with new anti-virus mechanisms.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Hepacivirus/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Isoformas de Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral , Linhagem Celular , Hepacivirus/fisiologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos
9.
Yao Xue Xue Bao ; 49(6): 843-8, 2014 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-25212030

RESUMO

To investigate vincristine-induced dopaminergic neurons toxicity and mechanism, and explore the molecular target to reduce the toxicity, zebrafish was chosen as a model animal, based on RT-PCR, Western blotting, whole mount in situ immunofluorescence and other technical means. The results showed that the transcription levels of tyrosine hydroxylase gene and dopamine transporter protein gene were inhibited. Furthermore, the number of dopaminergic neurons was decreased by vincristine. Autophagy was suppressed and beclin1 gene expression was inhibited in a dose-dependent manner by vincristine in larval zebrafish. Up-regulated beclin1 partly reduced vincristine-induced neurotoxicity, and down-regulated beclin1 increased toxicity. Beclin1 plays an important role in vincristine-induced dopaminergic neurons toxicity.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Vincristina/efeitos adversos , Proteínas de Peixe-Zebra/metabolismo , Animais , Autofagia , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Larva/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra
10.
PLoS One ; 8(3): e56985, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469178

RESUMO

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-ß, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , RNA Mensageiro/antagonistas & inibidores , Proteínas do Core Viral/genética , Peixe-Zebra/virologia , Animais , Antivirais/farmacologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Genes Reporter , Proteínas de Fluorescência Verde , Hepacivirus/crescimento & desenvolvimento , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Larva/efeitos dos fármacos , Larva/virologia , Fígado/efeitos dos fármacos , Fígado/virologia , Proteínas Mutantes Quiméricas/antagonistas & inibidores , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Ribavirina/farmacologia , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/metabolismo , Vitamina B 12/farmacologia
11.
Yi Chuan ; 34(9): 1165-73, 2012 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-23017458

RESUMO

To further understand the neural toxicity and teratogenicity of antiepileptic drugs in clinic, we established a zebrafish model for antiepileptic toxicity using trimethadione as a probe drug. The results indicated that embryonic malformation occurred under trimethadione treatment in a concentration-dependent manner. The defects included growth retardation, small head, eyes and acoustic capsule, deficient semicircular canals and otolith, and abnormal cardiovascular system. The number of hair cells in neuromast ML2 was obviously reduced in the treated larvae. Whole mount in situ hybridization indicated that the gene expression patterns of brain marker genes, such as zic1 and xb51, and autophagic gene atg5 was changed significantly. The result of RT-PCR showed that the expressions of hearing genes val and hmx2 were also changed in the trimethadione-treated embryos. All these findings suggest that brain tissue and the neural sensors for body balance and hearing are the main targets of trimethadione toxicity, and that zebrafish is able to mimic mammal responses to the teratogenicity and the neural toxicity of trimethadione in the embryonic and larva development.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Teratogênios/toxicidade , Trimetadiona/toxicidade , Peixe-Zebra/embriologia , Anormalidades Múltiplas/induzido quimicamente , Animais , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos
12.
Autophagy ; 7(12): 1514-27, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22082871

RESUMO

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, ß-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12­ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.


Assuntos
Autofagia/genética , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Organogênese/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Sirolimo/farmacologia , Fatores de Tempo , Transcrição Genética/efeitos dos fármacos , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
13.
BMC Mol Biol ; 10: 50, 2009 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-19470186

RESUMO

BACKGROUND: Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated. RESULTS: Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae. CONCLUSION: Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.


Assuntos
Autofagia , Bombyx/citologia , Bombyx/genética , Clonagem Molecular , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Bombyx/química , Bombyx/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Transdução de Sinais
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