SILKWORM 30K PROTEIN INHIBITSECDYSONE-INDUCED APOPTOSISBY BLOCKING THE BINDING OFULTRASPIRACLE TO ECDYSONERECEPTOR-B1 IN CULTURED Bm5CELLS

摘要

This study investigates the mechanism through which increased 30Kprotein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovariancell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) aftertransfection with the pIZT/V5-His control vector triggered apoptosis, but20E treatment did not trigger apoptosis in Bm5 cells transfected with thepIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis,30K protein was first purified from Escherichia coli transformed with a30K expression vector and used to generate specific antibodies in mice.Anti-30K antiserum was used to confirm synthesis of the 30K protein inpIZT/30K/V5-His-transfected Bm5 cells and to detect 30K proteinbinding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum wasused to immunoprecipitate protein complexes containing 30K from Bm5cells transfected with pIZT/30K/V5-His vector and treated with 20E. Weobserved that 30K proteins bound primarily to the EcR-B1 and not toultraspiracle (USP). Reciprocal immunoprecipitation ofEcR-B1-containing complexes from Bm5 cells transfected with controlpIZT/V5-His vector and treated with 20E showed that EcR-B1 bound toUSP in the absence of 30K but did not bind to USP inpIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that30K proteins block USP binding to EcR-B1 through formation of a30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cellapoptosis.