ATG4B/LC3-I interactions in autophagy regulation
Date Issued
2012
Date
2012
Author(s)
Lin, Pei-Shuan
Abstract
Autophagy is a degradation system involving the formation of double-membrane autophagosome, which encloses the cellular components and fuses with lysosome for content degradation. This pathway is governed by a group of genes called ATGs and plays a critical role in cell survival, growth control and homeostasis, which maintains a balance between the synthesis, degradation and recycling of cellular components. LC3, a homologue of yeast ATG8, is a ubiquitin-like protein required for autophagosome formation and mediates membrane tethering and hemi-fusion. Cytosolic LC3-I (termed LC3-I) is conjugated to phosphatidylethanolamine to produce LC3-II, located on the autophagosomal membrane, to regulate autophagosome formation. ATG4B, on the other hand, is a key cysteine protease required for two forms of LC3 processing. First, ATG4B is required for cleaving the C-terminal fragment from LC3 precursor protein to produce LC3-I. Secondly, ATG4B deconjugates LC3-II from autophagosomal membranes into LC3-I to allow autophagy to proceed. Therefore, ATG4B participates in the control of the rate of autophagy degradation. How does the cell regulate the ATG4B activity tightly?
The complex structure of ATG4B and LC3-I, has suggested that LC3-I and ATG4B could bind to each other. To explore the relationship between LC3-I and ATG4B, we used isothermal titration calorimetry to prove ATG4B bind to LC3-I .Than quantify the changes in protein concentration of the endogenous ATG4B and LC3 I with Western blotting. In addition, enzyme kinetics helped us to study the interaction between LC3-I and ATG4B. Furthermore, we examined whether ATG4B could regulate LC3-I in living cell by over expressing ATG4B and fluorescent LC3.
We found that ATG4B bind to LC3-I. In addition, the binding affinity of LC3-I is better than LC3 precursor. Furthermore, the activity of ATG4B reduced when the concentration of LC3-I increased. Besides, GFP-LC3 was distributed in the cytoplasm when ATG4B was overexpressed. Therefore, we propose that cytosolic LC3-I can suppress the activity of ATG4B in normal condition. The conversion of LC3-I to LC3-II is promoted while autophagy is induced. The reduction of LC3-I is than able to activate ATG4B to deconjugate LC3-II from autophagosomal membranes, thereby promoting the fusion of autophagosome and lysosome.
Subjects
Autophagy
ATG4B
LC3
Type
thesis
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