Ji Ming-Ming, Lee Jae Man, Mon Hiroaki, Xu Jian, Tatsuke Tsuneyuki, Kusakabe Takahiro
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan.
Biochem Biophys Res Commun. 2016 Oct 28;479(4):690-696. doi: 10.1016/j.bbrc.2016.09.151. Epub 2016 Sep 28.
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.
MG132已被用作家蚕细胞中的蛋白酶体抑制剂，但其对自噬的生理作用仍未阐明。在本研究中，我们发现脂化的BmAtg8，即作为自噬体标记蛋白的BmAtg8-PE，仅定位于膜上。然后我们建立了监测家蚕细胞自噬通量的系统：自噬的诱导减少了外源性BmAtg8和外源性BmAtg8-PE，促进了自噬体的形成，雷帕霉素和巴弗洛霉素A1共同处理后绿色EGFP-BmAtg8斑点表明了这一点，并导致自溶酶体中EGFP-BmAtg8裂解产生的游离EGFP积累。使用这些建立的系统，我们发现当泛素化蛋白在家蚕细胞中明显积累时，MG132的暴露会抑制基础自噬和雷帕霉素诱导的自噬。有趣的是，我们发现这些细胞中自噬的减弱归因于MG132处理后自噬体形成的明显抑制。
