Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters.
Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters.
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DOI：
10.4161/auto.5.4.7761
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发表时间：
2009-05
期刊：
影响因子：
13.3
通讯作者：
Weihl CC
中科院分区：
文献类型：
--
作者：
Ju JS;Miller SE;Jackson E;Cadwell K;Piwnica-Worms D;Weihl CC
细胞和组织中自噬的分析主要通过定性测量进行。这些测定鉴定自噬体或测量LC 3 I向LC 3 II的转化。然而，定性分析不能定量自噬底物的降解，因此只能间接测量完整的自噬系统。“自噬通量”可以使用通过自噬降解的长寿命蛋白质来测量。我们开发了一种可定量的荧光素酶报告基因测定法，该测定法测量长寿命的聚谷氨酰胺蛋白聚集体polyQ 80-荧光素酶的降解。使用该报告基因，在处理4小时后，与非聚集的polyQ 19-荧光素酶相比，细胞中通过饥饿或雷帕霉素诱导自噬优先降低polyQ 80-荧光素酶。这种反应是时间和浓度依赖性的，被自噬抑制剂阻止，并且在ATG 5敲除细胞中不存在。我们通过将polyQ 19-荧光素酶和polyQ 80-荧光素酶表达构建体分别电穿孔到小鼠的右胫骨前肌（TA）和左胫骨前肌（TA）中来使该测定适应于活体动物。通过体内生物发光成像定量自噬刺激或抑制之前和之后polyQ 80-荧光素酶与polyQ 19-荧光素酶信号的比率的变化。在饥饿或腹膜内雷帕霉素处理两天后，polyQ 80：polyQ 19-荧光素酶活性的比率降低约35%，与polyQ 80蛋白的选择性自噬降解一致。在体内骨骼肌中的这种自噬反应通过与氯喹的共治疗和在ATG 16 L1亚型小鼠中被废除。我们的研究证明了一种方法来量化的自噬通量的扩大聚谷氨酰胺通过荧光素酶报告在体外和体内。
The analysis of autophagy in cells and tissue has principally been performed via qualitative measures. These assays identify autophagosomes or measure the conversion of LC3I to LC3II. However, qualitative assays fail to quantitate the degradation of an autophagic substrate and therefore only indirectly measure an intact autophagic system. “Autophagic flux” can be measured using long-lived proteins that are degraded via autophagy. We developed a quantifiable luciferase reporter assay that measures the degradation of a long-lived polyglutamine protein aggregate, polyQ80-luciferase. Using this reporter, the induction of autophagy via starvation or rapamycin in cells preferentially decreases polyQ80-luciferase when compared with a non-aggregating polyQ19-luciferase after four hours of treatment. This response was both time- and concentration-dependent, prevented by autophagy inhibitors and absent in ATG5 knockout cells. We adapted this assay to living animals by electroporating polyQ19-luciferase and polyQ80-luciferase expression constructs into the right and left tibialis anterior (TA) muscles of mice, respectively. The change in the ratio of polyQ80-luciferase to polyQ19-luciferase signal before and after autophagic stimulation or inhibition was quantified via in vivo bioluminescent imaging. Following two days of starvation or treatment with intraperitoneal rapamycin, there was a ~35% reduction in the ratio of polyQ80:polyQ19-luciferase activity, consistent with the selective autophagic degradation of polyQ80 protein. This autophagic response in skeletal muscle in vivo was abrogated by co-treatment with chloroquine and in ATG16L1 hypomorphic mice. Our study demonstrates a method to quantify the autophagic flux of an expanded polyglutamine via luciferase reporters in vitro and in vivo.
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影响因子：
46.9
作者：
Lindsten, K;Menéndez-Benito, V;Dantuma, NP
通讯作者：
Dantuma, NP
影响因子：
3.3
作者：
Mizushima, N;Yamamoto, A;Ohsumi, Y
通讯作者：
Ohsumi, Y
影响因子：
3.5
作者：
Ravikumar, B;Duden, R;Rubinsztein, DC
通讯作者：
Rubinsztein, DC
影响因子：
37.8
作者：
Tannous, Paul;Zhu, Hongxin;Hill, Joseph A.
通讯作者：
Hill, Joseph A.
影响因子：
46.9
作者：
Dantuma, NP;Lindsten, K;Masucci, MG
通讯作者：
Masucci, MG