Analysis of ATG4C function in vivo

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Autophagy is a crucial cellular process for degrading and recycling damaged components.
• ATG8 family proteins (e.g., MAP1LC3/LC3, GABARAP) are key players in autophagosome formation.
• ATG4 proteases are essential for processing ATG8 proteins, but their specific substrate preferences and functions remain incompletely understood.

Purpose of the Study:

• To elucidate the distinct roles of the four mammalian ATG4 proteases (ATG4A, ATG4B, ATG4C, ATG4D).
• To determine the specific ATG8 family proteins (including MAP1LC3A/LC3A, MAP1LC3B/LC3B, GABARAP, GABARAPL1, GABARAPL2) cleaved by each ATG4 paralog.
• To investigate the impact of ATG4 activity on lipidation of ATG8 proteins with phosphatidylethanolamine (PE).

Main Methods:

• In vitro protease assays using recombinant ATG4 proteins and ATG8 substrates.
• Cell-based assays to assess ATG8 processing and lipidation in cells with depleted or overexpressed ATG4s.
• Mass spectrometry to identify cleavage sites and modified ATG8 proteins.

Main Results:

• Differential substrate specificity was observed among ATG4 paralogs for ATG8 proteins.
• ATG4B and ATG4D showed significant activity towards MAP1LC3B/LC3B and GABARAPL1.
• ATG4A and ATG4C exhibited distinct cleavage patterns and influenced PE conjugation of specific ATG8 proteins.

Conclusions:

• Mammalian ATG4 proteases possess specialized functions in cleaving ATG8 proteins, contributing to the fine-tuning of autophagy.
• The specific ATG4-ATG8 interactions regulate key steps in autophagosome biogenesis and cargo recognition.
• Understanding these specificities is critical for deciphering autophagy regulation in health and disease.