Silkworm 30K protein inhibits ecdysone-induced apoptosis by blocking the binding of ultraspiracle to ecdysone receptor-B1 in cultured Bm5 cells.
This study investigates the mechanism through which increased 30K protein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) after transfection with the pIZT/V5-His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti-30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5-His-transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5-His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR-B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR-B1-containing complexes from Bm5 cells transfected with control pIZT/V5-His vector and treated with 20E showed that EcR-B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR-B1 through formation of a 30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cell apoptosis.
School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
Mi Young
MY
Kim
Hwa Young
HY
Song
Jin Hee
JH
Kim
Bo Yong
BY
Kim
Sun Whan
SW
Park
Dong Kyung
DK
Sung
Hun Hee
HH
Park
Soung Hoo
SH
Jeon
In Sik
IS
Chung
Bong Hee
BH
Lee
D000818:Animals; D017209:Apoptosis; D012831:Bombyx / Q000378:metabolism*; D002460:Cell Line; D004440:Ecdysone / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D019476:Insect Proteins; D011987:Receptors, Steroid; D011994:Recombinant Proteins; C055469:ecdysone receptor; D004440:Ecdysone