Quantitation of autophagy by luciferase release assay

Ketteler, Robin; Seed, Brian

doi:10.4161/auto.6401

Cited by 47 publications

(72 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A similar procedure has been reported before. 15 Basically, HEK-293T cells (human embryonic kidney cells, ATCC, CRL-11268) were seeded in 6-well plate with a density of 70% confluence. One microgram of enzyme expression plasmid and 2 μg of substrate expression plasmid were cotransfected …”

Section: ■ Experimental Methodsmentioning

confidence: 99%

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

Qiu

Kuhn

Aebi

et al. 2016

ACS Med. Chem. Lett.

View full text Add to dashboard Cite

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure−activity relationship (SAR) and protease selectivity are also discussed.KEYWORDS: ATG4B, autophagy, covalent inhibitor, fluoromethylketone, peptidomimetics A utophagy is an evolutionarily conserved process essential for cell homeostasis and housekeeping by catabolizing aggregated proteins and damaged cellular components. 1 The hallmark of autophagy is the formation of autophagosomes and subsequent fusion with lysosomes to achieve degradation of their contents. Dysregulation of autophagy has been recently described in the pathogenesis of a variety of diseases such as cancer, neurodegenerative and metabolic disorders, and viral infections. 2,3 Modulation of autophagy has become a very active area of preclinical and clinical research, and particularly, there is high interest to identify potent and specific autophagy inhibitors. 4 ATG4 or autophagins are a class of cytosolic cysteine proteases that cleave ATG8 family proteins, such as light chain 3 (LC3). ATG4 plays essential roles in the formation and maturation of autophagosomes. 5,6 Among all four human ATG4 orthologues, ATG4B is functionally dominant, and it is the sole enzyme reported to efficiently cleave LC3 precursors and to deconjugate lipid from membrane bound LC3-phosphatidylethanolamine (LC3-PE). 7 ATG4B has been considered as a potential therapeutic target in the development of chemosensitizer for the treatment of certain cancer types. Recently, a number of small molecule ATG4B inhibitors such as Z-L-Phe-chloromethylketone and NSC185058 were reported to have modulatory effects on the autophagy process. 8−10 However, because of its high chemical reactivity, the chloromethylketone moiety is usually associated with significant cytotoxicity. NSC185058, however, is only a weak ATG4B inhibitor with an IC 50 of 51 μM in an assay detecting the cleavage of LC3-GST. 10 Previously, we reported the identification of Z-FA-FMK (1, Figure 1) as a covalent active-site directed ATG4B inhibitor from a TR-FRET based focused library screening. 11 The hit expansion of 1 led to the discovery of Z-FG-FMK (2), which was 10-times more potent than 1 with an IC 50 of 1.2 μM in the biochemical assay. 11 Herein we disclose the structure-guided optimization of 2 toward highly potent fluoromethylketone (FMK)-based ATG4B inhibitors.As an early step in the autophagosome formation, ATG4B cleaves proLC3 to expose the C-terminal Gly120 for subsequent PE-conjugation and autophagosome membrane insertion. 5 The cocrystal structures of catalytically inert C74S or

show abstract

Section: ■ Experimental Methodsmentioning

confidence: 99%

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

Qiu

Kuhn

Aebi

et al. 2016

ACS Med. Chem. Lett.

View full text Add to dashboard Cite

show abstract

“…All three proteins have previously been found to be valuable markers of autophagy also when fused to fusion partners such as green fluorescent protein (GFP) and luciferase. 13,17,19,[21][22][23][24] To estimate the autophagic flux of these proteins we developed an inducible expression system to monitor their degradation following promoter shut-off (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

A reporter cell system to monitor autophagy based on p62/SQSTM1

Larsen¹,

Lamark²,

Øvervatn³

et al. 2010

Autophagy

139

105

View full text Add to dashboard Cite

M acroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy, cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut-off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFPp62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform the best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knockdown of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that A reporter cell system to monitor autophagy based on p62/SQSTM1

show abstract

“…5), suggesting a role for ATG4B phosphorylation in promoting the binding of ATG4B with LC3-PE and hence aiding in LC3 delipidation. Using the Actin-LC3B-dNGLUC assay, which serves as a cell-based assay to detect the cleavage of both pro-LC3 and LC3-PE (31,32,34), we showed that activity of the ATG4B S383A/S392A mutant toward LC3 cleavage was greatly decreased. It is possible that phosphorylation of the flexible C-terminal region of ATG4B induces a conformation where accessibility of ATG4B to its membraneassociated substrate, LC3-PE is more favorable.…”

Section: Discussionmentioning

confidence: 99%

“…The evidence for an important role of phosphorylation in controlling ATG4B's LC3-directed protease activity included experiments in which ATG4B recovered from cells was dephosphoryated in vitro using phosphatases and studies of phosphorylation site mutants (particularly the S383A, S392A double mutant) recovered from cells. For these experiments, the protease activity of ATG4B was measured in vitro using an engineered substrate LC3-PLA 2 and in intact cells using the substrate actin-LC3B-dNGLUC assay (27,29,34). Using these assays, we found that phosphorylation-site mutants of ATG4B showed reduced cellular protease activity.…”

Section: Discussionmentioning

confidence: 99%

“…We then employed a cellular ATG4B protease assay for quantifying the intracellular proteolysis of LC3 based on non-conventional secretion of Gaussia luciferase (dNGLUC) (29,34). In this system, the LC3B-dNGLUC substrate is anchored inside the cell by fusion to ␤-actin and the cleaved product is secreted after release from the cytoskeleton.…”

Section: Atg4b Is Phosphorylated In Cells-mentioning

confidence: 99%

See 1 more Smart Citation

ATG4B (Autophagin-1) Phosphorylation Modulates Autophagy

Yang

Wilkie-Grantham

Yanagi

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: ATG4B mediates the cleavage of pro-LC3 and removes lipid conjugates from LC3 during autophagy. Results: We determined that defects in phosphorylation of ATG4B reduced its hydrolyase activity and impaired autophagic flux. Conclusion: Phosphorylation of ATG4B plays an important role in modulating its hydrolyase activity. Significance: This is the first report showing a role for phosphorylation of an ATG4-family protease in control of autophagy.

show abstract

Quantitation of autophagy by luciferase release assay

Cited by 47 publications

References 73 publications

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

A reporter cell system to monitor autophagy based on p62/SQSTM1

ATG4B (Autophagin-1) Phosphorylation Modulates Autophagy

Contact Info

Product

Resources

About