AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells
AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells
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AU4S：一种新型合成肽，用于测量活细胞中 ATG4 的活性
DOI：
10.1080/15548627.2015.1009773
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发表时间：
2015-02-01
期刊：
影响因子：
13.3
通讯作者：
He, Fengtian
中科院分区：
文献类型：
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作者：
Ni, Zhenhong;Gong, Yi;He, Fengtian
ATG 4在自噬诱导中起着关键作用，但在活细胞中监测ATG 4活性的方法有限。在这里，我们设计了一种新的荧光肽命名为AU 4S的非侵入性检测的活性在活细胞中，它由细胞穿透肽（CPP），ATG 4识别序列“GTFG”，和荧光团FITC。另外，使用抗ATG 4肽AG 4 R作为对照。CPP可以帮助AU 4S或AG 4 R有效地穿透细胞膜。ATG 4能识别AU 4S而不能识别AG 4 R，并能将其裂解，导致荧光强度发生变化。因此，在相同样品中AU 4S-和AG 4 R-测量的荧光值之间的差异，定义为“F-D值”，可以反映ATG 4活性。通过检测F-D值，我们发现ATG 4活性与雷帕霉素处理的细胞中的LC 3B-II水平相关，但既不与饥饿细胞中的LC 3B-II水平相关，也不与健康供体或白血病患者的WBC中的LC 3B-II积累相关。然而，当DTT加入到系统中时，ATG 4活性不仅在存在或不存在自噬抑制剂的情况下降低饥饿细胞中的LC 3B-II水平，而且与白血病患者WBC中的LC 3B-II积累呈正相关（R2 = 0.5288）。总之，本研究提供了一种方便、快速、定量的方法来监测活细胞中ATG 4的活性，这可能有利于自噬的基础和临床研究。
ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R2 = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.