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Updated: Jul 23, 2025
Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells
Published on: June 30, 2023
Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase
Denise R B Pilger1, Christin Luft1, Robin Ketteler2
1Laboratory for Molecular Cell Biology, University College London.
Abstract:
Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.
Insights
High autophagic flux fuels tumor growth and resistance to cancer therapies. Researchers developed a new assay to measure ATG4B protease activity, a potential cancer drug target, for high-throughput screening.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- High autophagic flux correlates with tumor progression and resistance to cancer therapies.
- ATG4B protease is a potential therapeutic target for cancer, as its inhibition improves survival.
- Monitoring autophagy proteins is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To adapt a selective luciferase-based assay for monitoring ATG4B activity.
- To enable automated high-throughput screening of ATG4B activity in cells.
- To optimize the workflow for analyzing cellular ATG4B activity.
Main Methods:
- Developed a reporter assay using Gaussia princeps luciferase (GLUC) tagged to LC3B, ATG4B's substrate.
- Linked the GLUC-LC3B reporter to the actin cytoskeleton for cytoplasmic retention.
- Measured secreted GLUC in cell culture supernatants as a correlate of ATG4B activity.
Main Results:
- Successfully adapted the ATG4B activity assay for automated high-throughput screening.
- Established a workflow for efficient analysis of cellular ATG4B activity.
- Demonstrated the assay's utility for monitoring a key autophagy protease relevant to cancer.
Conclusions:
- The developed assay provides a robust method for high-throughput screening of ATG4B activity.
- This assay facilitates the discovery of novel cancer therapeutics targeting the autophagy pathway.
- Measuring ATG4B activity is a viable strategy for advancing cancer therapy research.
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