title: AU4S: a novel synthetic peptide to measure the activity of ATG4 in living cells.
abstract: 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.
authors.affiliation: a Department of Biochemistry and Molecular Biology; College of Basic Medical Sciences ; Third Military Medical University ; Chongqing , China.
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Ni
authors.forename: Yi
authors.initials: Y
authors.lastname: Gong
authors.forename: Xufang
authors.initials: X
authors.lastname: Dai
authors.forename: Wen
authors.initials: W
authors.lastname: Ding
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Gong
authors.forename: Liyan
authors.initials: L
authors.lastname: Qin
authors.forename: Panke
authors.initials: P
authors.lastname: Cheng
authors.forename: Song
authors.initials: S
authors.lastname: Li
authors.forename: Jiqin
authors.initials: J
authors.lastname: Lian
authors.forename: Fengtian
authors.initials: F
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005453:Fluorescence; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D051381:Rats
chemical_list: D000071183:Autophagy-Related Proteins; D008840:Microfilament Proteins; D010455:Peptides; C474025:ATG4A protein, human; D003546:Cysteine Endopeptidases
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
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