A HaloTag-based reporter processing assay to monitor autophagic flux
A HaloTag-based reporter processing assay to monitor autophagic flux
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基于 HaloTag 的报告处理分析，用于监测自噬通量
DOI：
10.1080/15548627.2022.2123638
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发表时间：
2022
期刊：
影响因子：
13.3
通讯作者：
Mizushima Noboru
中科院分区：
文献类型：
--
作者：
Wen-You Yim Willa;Yamamoto Hayashi;Mizushima Noboru
在大多数自噬研究中，监测哺乳动物巨噬/自噬通量是必要的，但通常很难做到。在这里，我们讨论了我们最近报道的一种基于halotag的处理方法，该方法可以直接读出自噬通量。我们发现自标记蛋白HaloTag在被其配体标记时变得抵抗蛋白水解。将HaloTag与自噬蛋白（如LC3）融合产生报告蛋白，该报告蛋白在进入溶酶体时被完全降解，但当用HaloTag配体脉冲标记时，在被溶酶体酶处理时释放出游离的HaloTag配体。通过免疫印迹或凝胶内荧光检测，可定量观察游离盐配体的量，反映自噬通量。除了与荧光显微镜和流式细胞术应用兼容外，该定量分析还可以很容易地用于监测大多数自噬途径或感兴趣的蛋白质的自噬降解。
Monitoring mammalian macroautophagic/autophagic flux is necessary in most autophagy studies but has generally been difficult to do. Here, we discuss our recent report of a HaloTag-based processing method that offers a straightforward readout for autophagic flux. We found that the self-labeling protein HaloTag becomes resistant to proteolysis when labeled with its ligand. Fusing HaloTag to an autophagy protein such as LC3 results in a reporter that is completely degraded when delivered into lysosomes but, when pulse-labeled with HaloTag ligand, releases free HaloTagligandwhen processed by lysosomal enzymes. The quantifiable amount of free HaloTagligand, observed by immunoblotting or in-gel fluorescence detection, reflects autophagic flux. Besides being compatible with fluorescence microscopy and flow cytometry applications, this quantitative assay can be readily adapted to monitor most autophagy pathways or the autophagic degradation of a protein of interest.