Tumor Susceptibility Gene 101 facilitates rapamycin-induced autophagic flux in neuron cells
Tumor Susceptibility Gene 101 facilitates rapamycin-induced autophagic flux in neuron cells
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DOI：
10.1016/j.biopha.2020.111106
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发表时间：
2021-02-01
影响因子：
7.5
通讯作者：
Shu, Chih-Wen
中科院分区：
文献类型：
--
作者：
Cheng, Jiin-Tsuey;Liu, Pei-Feng;Shu, Chih-Wen
肿瘤易感基因101（TSG 101）是内体分选复合物的成员，负责与自噬过程相关的内吞途径。然而，TSG 101在自噬中的作用仍不清楚。为了研究TSG 101对神经元细胞中膜结合的MAP 1 LC 3-II、p62和泛素化蛋白水平的影响，使用免疫印迹来评估用针对TSG 101的siRNA沉默并用自噬诱导剂雷帕霉素处理的细胞中的影响。使用GFP-MAP 1 LC 3和串联荧光标记的LC 3（mTagRFP-mWasabi-MAP 1 LC 3）报告载体，使用共聚焦显微镜监测细胞中的自噬。自噬泡的形态进一步用透射电镜证实。我们的研究结果表明，TSG 101的表达略有增加，在神经元细胞暴露于雷帕霉素。用siRNA耗尽TSG 101导致MAP 1 LC 3-II、GFP-MAP 1 LC 3斑点和自噬空泡在细胞中积累。在TSG 101沉默的细胞中，雷帕霉素升高的MAP 1 LC 3-II周转率和RFP+Wasabi puncta被抑制，表明TSG 101参与了雷帕霉素诱导的细胞自噬通量。此外，沉默TSG 101减少Rab 7，MAP 1 LC 3和细胞活力的共定位，增加神经元细胞中的p62，泛素化蛋白。综上所述，我们的结果表明，TSG 101可能需要两性体形成，以促进自噬通量在神经元细胞暴露于雷帕霉素。
Tumor Susceptibility Gene 101 (TSG101) is a member of endosomal sorting complexes responsible for endocytic pathway, which is associated with autophagic process. However, the role of TSG101 in autophagy remains unclear. To investigate the effect of TSG101 on the membrane-bound MAP1LC3-II, p62 and ubiquitinated protein levels in neuron cells, immunoblotting was used to evaluate the effects in cells silenced with siRNA against TSG101 and treated with autophagy inducer rapamycin. GFP-MAP1LC3 and tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-MAP1LC3) reporter vectors were used to monitor autophagy in cells using confocal microcopy. The autophagic vacuoles were further validated with transmission electron microscopy. Our results showed that TSG101 expression was slightly increased in neuron cells when exposed to rapamycin. Depletion of TSG101 with siRNA lead to accumulation of MAP1LC3-II, GFP-MAP1LC3 puncta and autophagic vacuoles in the cells. Rapamycin-elevated MAP1LC3-II turnover and RFP+Wasabi puncta were repressed in TSG101 silenced cells, indicating that TSG101 is involved in rapamycin-induced autophagic flux in cells. Moreover, silencing TSG101 reduced colocalization of Rab7, MAP1LC3 and cell viability, increased p62, ubiquitinated proteins in the neuron cells. Taken together, our results suggested that TSG101 might be required for amphisome formation to promote autophagic flux in neuron cells when exposed to rapamycin.