AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

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 (<i>R²</i> = 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.

ATG4在自噬诱导过程中发挥关键作用，但当前用于活细胞内监测ATG4活性的方法较为有限。本研究设计了一种名为AU4S的新型荧光肽，用于活细胞内ATG4活性的无创检测，该肽由细胞穿透肽（cell-penetrating peptide, CPP）、ATG4识别序列“GTFG”以及荧光基团异硫氰酸荧光素（FITC）组成。此外，本研究使用了抗ATG4肽AG4R作为对照。CPP可帮助AU4S或AG4R高效穿透细胞膜。AU4S可被ATG4识别并切割，而AG4R则无法被ATG4识别切割，这一过程会导致荧光强度发生变化。因此，将同一样本中AU4S与AG4R检测所得的荧光值之差定义为“F-D值”，该指标可反映ATG4的活性水平。通过检测F-D值，本研究发现：雷帕霉素处理的细胞中，ATG4活性与LC3B-II水平呈正相关；但在饥饿处理的细胞中，二者并未呈现此种关联；在健康供者或白血病患者的外周血白细胞（white blood cells, WBCs）中，ATG4活性也与LC3B-II积累无相关性。然而，当向反应体系中加入二硫苏糖醇（dithiothreitol, DTT）后，无论是否添加自噬抑制剂，饥饿处理细胞中的ATG4活性均与LC3B-II水平呈正相关；同时，白血病患者外周血白细胞中的ATG4活性也与LC3B-II积累呈显著正相关（决定系数R²=0.5288）。综上，本研究提供了一种便捷、快速且可定量的活细胞ATG4活性监测方法，有望为自噬相关的基础与临床研究提供助力。