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 (R(2) = 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.
Keywords: 3-MA, 3-methyladenine; AG4R, ATG4-resistant peptide; ATG4; ATG4, autophagy-related 4, cysteine peptidase; AU4S; AU4S, autophagy-related 4 substrate; Ac, acetyl; CFP, cyan fluorescent protein; CPP, cell-penetrating peptide; CQ, chloroquine; DTT, dithiothreitol; EBSS, Earle's balanced salt solution; FITC, fluorescein isothiocyanate; HIV, human immunodeficiency virus; LC3; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; NAC, N-acetyl-L-cysteine; NRK, normal rat kidney cell line; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PLA2, phospholipase A2; PMSF, phenylmethanesulfonyl fluoride; PtdIns3K, phosphatidylinositol 3-kinase; ROS; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; WBCs, white blood cells; YFP, yellow fluorescent protein; autophagy.