RESUMO
Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases. In this study, we isolated the caspase gene (Cscaspase-1) of Chilo suppressalis (one of the leading pests responsible for destruction of rice crops). It possesses the open reading frame (ORF) of 295 amino acids including prodomain, large subunit and small subunits, and two cleavage sites (Asp23 and Asp194) were found to be located among them. In addition to these profiles, Cscaspase-1 contains two active sites (His134 and Cys176). Genomic analysis demonstrated there was no intron in the genome of Cscaspase-1. The Cscaspase-1 transcripts were found in all tissues of the fifth instar larvae, and higher levels were found in the midgut, hindgut and Malpighian tubules. Examination of Cscaspase-1 expression in different developmental stages indicated low constitutive levels in the eggs and early larvae stages, and higher abundances were exhibited in the last larvae and pupae stages. The relative mRNA levels of Cscaspase-1 were induced by heat and cold temperatures. For example, the highest increase of Cscaspase-1 transcription was at -3 °C and 36 °C respectively. In a word, Cscaspase-1 plays a role of effector in the apoptosis of C. suppressalis. It also correlates with development, metamorphosis and thermotolerance of C. suppreassalis.
Assuntos
Caspase 1/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Proteínas de Insetos/genética , Lepidópteros/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Caspase 1/química , Caspase 1/classificação , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/classificação , Lepidópteros/enzimologia , Lepidópteros/crescimento & desenvolvimento , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , TemperaturaRESUMO
BACKGROUND: The cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species. RESULTS: In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their Drosophila counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species. CONCLUSIONS: The basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.
Assuntos
Caspases/genética , Lepidópteros/enzimologia , Sequência de Aminoácidos , Animais , Caspase 1/análise , Caspase 1/classificação , Caspase 1/genética , Caspase 3/análise , Caspase 3/classificação , Caspase 3/genética , Caspase 6/análise , Caspase 6/classificação , Caspase 6/genética , Caspases/análise , Caspases/classificação , Drosophila/enzimologia , Drosophila/genética , Etiquetas de Sequências Expressas , Lepidópteros/genética , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Members of the caspase family play a central and evolutionary role in programmed cell death (PCD), which removes unwanted, damaged and dangerous cells during development to maintain homeostasis. In this paper, we describe the cloning and characterization of a caspase from the cotton bollworm, Helicoverpa armigera, named Hearm caspase-1. The 1,350 bp full-length cDNA contains an 885 bp open reading frame (ORF) that encodes a Hearm caspase-1 proenzyme of 294 amino acids. The deduced protein is highly homologous to Spodoptera frugiperda Sf caspase-1 and Drosophila melanogaster ICE and has the highly conserved pentapeptide QACQG, the recognized catalytic site of caspases, suggesting that it is an effector caspase of the cotton bollworm. Northern blot and RT-PCR analyses demonstrate that Hearm caspase-1 is expressed in embryos and the fat body, midgut and haemocytes of feeding and wandering larvae. Expression of Hearm caspase-1 in the haemocytes appears to be correlated with the pulse of ecdysone, and it is up-regulated by ecdysone agonist RH-2485, implying that Hearm caspase-1 activation is regulated by ecdysone.
