2018
DOI: 10.3389/fcell.2018.00148
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Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells

Abstract: Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order … Show more

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Cited by 20 publications
(21 citation statements)
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“…This inhibitory loop is displaced upon binding to LC3/GABARAP and, in addition, is most likely subject to conformational change induced by post-translational modification. For instance, Ser34, which is located at the hinge of the N-terminal domain, has been shown to be phosphorylated by AKT kinase [14], and a phospho-mimetic S34D mutant displays enhanced ATG4B activity [72], suggesting that phosphorylation results in a conformational opening of the auto-inhibitory loop.…”
Section: Atg4b As Drug Targetmentioning
confidence: 99%
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“…This inhibitory loop is displaced upon binding to LC3/GABARAP and, in addition, is most likely subject to conformational change induced by post-translational modification. For instance, Ser34, which is located at the hinge of the N-terminal domain, has been shown to be phosphorylated by AKT kinase [14], and a phospho-mimetic S34D mutant displays enhanced ATG4B activity [72], suggesting that phosphorylation results in a conformational opening of the auto-inhibitory loop.…”
Section: Atg4b As Drug Targetmentioning
confidence: 99%
“…The mechanism of GLUC release in this system is not well understood, but it does not require autophagy as it is not affected in ATG5 knockout cells [111], making this a specific ATG4B activity assay. This assay has been employed to identify regulators of ATG4B activity and has become one of the main methods to monitor cell-based ATG4 activity [12,59,72]. The assay has also been adapted to monitor LC3A and LC3B2 [9], thus broadening its application to other ATG4 family members, although a similar approach to monitor GABARAP cleavage instead of LC3 resulted in very high background release of GLUC, and is thus impractical for use as a phenotypic reporter for screening (R.K., unpublished observation).…”
Section: Cell-based Assaysmentioning
confidence: 99%
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“…Large-scale screening was performed on 31,954 chemical compounds from the ChemiBank(UCL) collection (http://www.ucl.ac.uk/chemibank/) [66]. The flasks were coated with 0.1 mg/ml Matrigel (BD bioscience) diluted in DMEM (Gibco, Life Technology).…”
Section: High-throughput Compound Screeningmentioning
confidence: 99%
“…The growth medium consists of 20% fetal bovine serum (FBS, Gibco), 2% L-glutamine (Sigma), and 1% penicillin/streptomycin (Gibco), plus 2% chick embryo extract (Sera lab) and c-IFN (2 ll/ml, Merck). Large-scale screening was performed on 31,954 chemical compounds from the ChemiBank(UCL) collection (http://www.ucl.ac.uk/chemibank/) [66]. Molecular properties ranges were as follows: molecular weight between 126 and 600, AlogP between À3.5 and 6, hydrogen bond donors between 0 and 6, hydrogen bond acceptors between 0 and 12, rotatable bonds between 0 and 15, and number of rings between 1 and 8.…”
Section: High-throughput Compound Screeningmentioning
confidence: 99%