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Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in

Niccolo Pengo1, Krisna Prak1, Joana R Costa1

  • 1MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.

Frontiers in Cell and Developmental Biology
|November 17, 2018
PubMed

Insights

Researchers identified regulators of autophagy protease ATG4B, crucial in cancer. A new assay revealed compounds activating ATG4B and identified AKT2 kinase as a key enhancer of its cellular activity.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Autophagy protease ATG4B regulates the LC3/GABARAP system, essential for autophagosome formation.
  • Dysregulation of ATG4B and related proteins is linked to diseases, notably cancer, making ATG4B a therapeutic target.
  • Post-translational modifications, such as phosphorylation and dephosphorylation, are known regulators of ATG4B activity.

Purpose of the Study:

  • To identify novel regulators of cellular ATG4B activity.
  • To develop and validate a cell-based assay for screening ATG4B modulators.
  • To discover small molecules and protein kinases/phosphatases that impact ATG4B function.

Main Methods:

  • Optimization of a cell-based luciferase assay measuring ATG4B-dependent Gaussia luciferase release.
  • Proof-of-concept small molecule compound screen to identify ATG4B activators.
  • High-throughput screening using siRNA knockdown and cDNA overexpression to identify regulatory kinases and phosphatases.

Main Results:

  • Successful optimization of a cell-based assay for ATG4B activity.
  • Identification of small molecule compounds that enhance cellular ATG4B activity.
  • Preliminary evidence suggests the kinase AKT2 positively regulates ATG4B activity in cells.

Conclusions:

  • Novel insights into the post-translational regulation of ATG4B activity have been uncovered.
  • The developed assay serves as a valuable tool for future drug discovery targeting ATG4B.
  • Understanding ATG4B regulation is critical for developing targeted cancer therapies.
Keywords:
AKT2ATG4BcDNAkinasephosphatasescreensiRNAsmall molecule

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