title: Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori.
abstract: We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Ikue
authors.initials: I
authors.lastname: Suganuma
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Ushiyama
authors.forename: Hayato
authors.initials: H
authors.lastname: Yamada
authors.forename: Asako
authors.initials: A
authors.lastname: Iwamoto
authors.forename: Michihiro
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Motoko
authors.initials: M
authors.lastname: Ikeda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D012831:Bombyx / Q000201:enzymology*; D020169:Caspases / Q000737:chemistry*; D002460:Cell Line; D003609:Dactinomycin; D029721:Drosophila Proteins / Q000737:chemistry*; D004926:Escherichia coli; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D029721:Drosophila Proteins; D003609:Dactinomycin; D020169:Caspases; C118731:dronc protein, Drosophila