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Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells

机译:基于荧光的伴侣细胞介导的自噬和微自噬活性的评估

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The autophagy-lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70-dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA-mediated knockdown of CMA- and mA-related proteins. Knockdown of LAMP2A, a CMA-related protein, and TSG101, an mA-related protein, significantly but only partially decreased the punctate accumulation of GAPDH-HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH-HT puncta in TSG101-knockdown cells, but not in LAMP2A-knockdown cells, suggesting that punctate accumulation of GAPDH-HT under LAMP2A- and TSG101-knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.
机译:自噬溶酶体蛋白降解进一步分类为大自噬(MA),微自噬(mA)和伴侣蛋白介导的自噬(CMA)。尽管MA参与了各种功能和疾病的发病机制,但对CMA和mA的了解却很少,因为缺乏简便的方法来评估其活性。我们最近建立了一种使用3-磷酸甘油醛脱氢酶(GAPDH),CMA底物和HaloTag(HT)系统评估CMA活性的方法。另一小组最近确定了哺乳动物mA途径,其中底物以热休克同源蛋白(Hsc)70依赖性方式递送至晚期内体。由于Hsc70也参与CMA,因此我们的方法将同时检测CMA和mA活性。在这项研究中,我们试图通过siRNA介导的CMA和mA相关蛋白的敲低分别评估CMA和mA的活性。击倒CMA相关蛋白LAMP2A和mA相关蛋白TSG101,可显着但仅部分减少GA293在HT293细胞和原代培养的大鼠皮质神经元中的点状积累。激活CMA的化合物在TSG101敲除细胞中显着增加了GAPDH-HT的点数,但在LAMP2A敲除细胞中则没有,这表明GAPDH-HT在LAMP2A和TSG101敲除下的点状积累分别代表mA和CMA活性。我们成功建立了通过荧光观察分别评估CMA和mA活性的方法。

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