A high-throughput FRET-based assay for determination of Atg4 activity

Abstract

Atg4 is required for cleaving Atg8, allowing it to be conjugated to phosphatidylethanolamine on phagophore membranes, a key step in autophagosome biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a regulatory step in controlling autophagy. Therefore, the activity of Atg4 is important for autophagy and could be a target for therapeutic intervention. In this study, a sensitive and specific method to measure the activity of two Atg4 homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two substrates that could be differentially cleaved by Atg4A and Atg4B, were fused with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. The FRET signals decreased in proportion to the Atg4-mediated cleavage, which separated the two fluorescent proteins. This method is highly efficient for measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro conditions. Applications of the assay indicated that the activity of Atg4B was dependent on its catalytic cysteine and expression level, but showed little changes under several common autophagy conditions. In addition, the assays displayed excellent performance in high throughput format and are suitable for screening and analysis of potential modulators. In summary, the FRET-based assay is simple and easy to use, is sensitive and specific, and is suitable for both routine measurement of Atg4 activity and high-throughput screening.

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

“…Therefore, the BRET-based assay could be used to monitor the rate of autophagy in leaf tissue during a specific biological process. Recently, fluorescence resonance energy transfer (FRET)-based human Atg8 substrates have been described for quantitative measurement of hAtg4 activity in vitro and in mammalian cell lysates (22). Although the FRET-based assay is highly sensitive, the direct excitation of the acceptor fluorophore by the external light for activation of the donor fluorophore influences successful FRET (23).…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…120 It is also possible to use assays with an artificial fluorogenic substrate, or a fusion of LC3B to phospholipase A(2) that allows the release of the active phospholipase for a subsequent fluorogenic assay, 121 and there is a FRETbased assay utilizing CFP and YFP tagged versions of LC3B and GABARAPL2 that can be used for high-throughput screening. 122 Another method to monitor ATG4 activity in vivo uses the release of Gaussia luciferase from the C terminus of LC3 that is tethered to actin. 123 Note that there are four Atg4 homologs in mammals, and they have different activities with regard to the Atg8 subfamilies of proteins.…”

Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B

“…A very recent publication has given strong direct evidence for the potential of inhibitors of ATG4B in treating cancer. Atkin et al 27 identified a small molecule ATG4B inhibitor with moderate potency (IC 50 51 lM as tested in an assay developed by Li et al) 28 from in silico docking of small molecules from the NCI library into the ATG4B active site. In spite of its rather low potency, the authors showed that the compound, NSC185058, suppressed formation of autophagic vesicles in starved cells, suppressed activation and lipidation of LC3 and, most significantly, suppressed autophagic markers in livers of fasted mice, and suppressed autophagy and growth of osteosarcoma tumors both in vitro and in vivo in immunodeficient mice.…”

“…During the past several years we, 29 and others, 28,[30][31][32] have developed a number of protocols for assaying small molecule ATG4B inhibitors. We previously developed an assay based on the cleavage of pro-LC3 followed by mass spectrometry (LC-MS assay).…”

“…Unfortunately none of these hits have yet yielded leads we consider useful for optimization. Other researchers have reported on HTS assays for ATG4B including another FRET assay based on a fluorescent LC3 construct 28 and a coupled reporter assay based on a fusion protein between LC3 and phospholipase A 2 (PLA 2 ) where PLA 2 activity is monitored and hits are counter-screened against PLA 2 to identify false positives. 31,32 Thus far there are no published reports of the use of the Li FRET assay for HTS but the LC3-PLA 2 assay developed by Shu 31 was used to initially screen small test libraries where it performed well with a low rate of false positives due to PLA 2 inhibition.…”