SILKWORM 30K PROTEIN INHIBITS ECDYSONE‐INDUCED APOPTOSIS BY BLOCKING THE BINDING OF ULTRASPIRACLE TO ECDYSONE RECEPTOR‐B1 IN CULTURED Bm5 CELLS

Abstract

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.

The stability and antiapoptotic activity of Bm30K‐3 can be improved by lysine acetylation in the silkworm, Bombyx mori

“…This result was an update from a previous analysis based on the newly identified 30K proteins in the UniProt database, as in previous analysis, we only identified 11 acetylated 30K proteins containing 23 Kac sites (Nie et al, ). As members of the lipoprotein family, 30K proteins mainly perform the lipid loading (J. P. Yang et al, ) and antiapoptosis functions (E. J. Kim et al, ; E. J. Kim, Rhee, & Park, ; M. Y. Kim et al, ; Pakkianathan et al, ; Yu et al, ). The widespread acetylation of B. mori 30K proteins might suggest important roles of acetylation with respect to the function of 30K proteins in silkworms, which requires additional studies.…”

“…J. Kim, Rhee, & Park, 2001; M. Y. Kim et al, 2012;Pakkianathan et al, 2015;Yu et al, 2013). The widespread acetylation of B. mori 30K proteins might suggest important roles of acetylation with respect to the function of 30K proteins in silkworms, which requires additional studies.…”

“…Because these proteins have molecular weights of approximately 30 kDa, they are named 30K proteins (Tojo, Betchaku, Ziccardi, & Wyatt, ). Previous studies have shown that the 30K proteins not only have the function of lipid loading (J. P. Yang et al, ) but also have a significant suppressive effect on mammalian and insect cell apoptosis (E. J. Kim, Park, & Park, ; M. Y. Kim et al, ; Pakkianathan, Singh, Konig, & Krishnan, ; Yu, Li, Yao, Quan, & Zhang, ). In addition, 30K proteins also perform other important functions, such as anti‐fungus (Li et al, ; Ujita et al, ), protein delivery (Park et al, ), and production and sialylation of recombinant proteins (Koo, Park, Park, & Park, ; Z. Wang, Park, Park, Tan, & Park, ).…”

Functional analysis of 30K proteins during silk gland degeneration by a caspase‐dependent pathway in Bombyx

“…In previous studies, 30K proteins have been shown to play a role in antiapoptosis (Kim et al ., , , ; Kim & Park, ; Choi et al ., ; Song et al ., ). At the PP stage and on PP1, genomic DNA is gradually concentrated in SG, and it is rapidly and completely degraded on PP1 according to the DNA ladder.…”

“…30Kc6 protein inhibits apoptosis in human and insect cells (Kim et al ., ; Kim & Park, ; Choi et al ., ). It is gradually synthesized in the Bombyx motorneurones and results in inhibition of programmed cell death in vitro by blocking ultraspiracle binding to ecdysone receptor‐B1 (EcR‐B1) through the formation of a 30K/EcR‐B1 complex (Kim et al ., ; Song et al ., ). 30Kc19 is not only an apoptosis‐inhibiting factor but also perhaps a proteinaceous factor involved in the uptake of the chymotrypsin inhibitor (CI‐8) into the midgut through the membrane (Kim et al ., ; Ueno et al ., ).…”