Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells.
MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment.
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Ming-Ming
MM
Ji
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Jae Man
JM
Lee
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Hiroaki
H
Mon
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Jian
J
Xu
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Tsuneyuki
T
Tatsuke
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: kusakabe@agr.kyushu-u.ac.jp.
Takahiro
T
Kusakabe
D000595:Amino Acid Sequence; D000818:Animals; D000071182:Autophagosomes / Q000187:drug effects* / Q000502:physiology; D001343:Autophagy / Q000187:drug effects*; D000071190:Autophagy-Related Protein 8 Family / Q000737:chemistry / Q000378:metabolism; D015415:Biomarkers; D012831:Bombyx / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology*; D008247:Lysosomes / Q000187:drug effects; D018942:Macrolides / Q000494:pharmacology; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D059748:Proteolysis / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology
D000071190:Autophagy-Related Protein 8 Family; D015415:Biomarkers; D019476:Insect Proteins; D007976:Leupeptins; D018942:Macrolides; D061988:Proteasome Inhibitors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C040929:bafilomycin A1; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D020123:Sirolimus
Atg8; Autophagy; Bombyx mori; Insect; MG132; Silkworm