Identification of autophagy-specific cargo and characterization of b-catenin as LC3/GABARAP interacting protein

Abstract

Autophagy is an evolutionary conserved eukaryotic degradation system induced under cellular stress conditions in which cellular components, such as damaged organelles, and aggregated proteins sequestrated through lysomal fusion. Alterations in the process of autophagy can trigger several pathological conditions, such as neurodegeneration, infectious diseases, and cancer. Autophagy research is hampered mainly due to limited research tools and need for better understanding of its molecular mechanisms. Therefore, dissecting autophagy from a proteomics perspective has become a genuine interest of many studies for the characterization of the autophagy process. Nevertheless, whole-cell proteomics studies were not effective enough to selectively identify proteins targeted by autophagic degradation or or regulate autophagy. Thus, the composition of autophagosomes is more selectively subjected to proteomic analysis using biochemical fractionation, affinity purification, and close-proximity labelling methods. These kinds of approaches can identify autophagy-specific substrates/regulators and provide significant insights on cellular functions that autophagy affects. Here, this study aims to create the basis for understanding the functions of autophagy by studying the composition of autophagosomes isolated from tumour cells. Consistent with this aim, a robust autophasome purification technique combining fractionation and immune isolation methods was developed. This technique enabled purification of pure and intact autophagomomes from cancer cells at a sufficient amount to analyse their content by proteomics. Subsequent analysis of the autophagosome content revealed many novel potential candidates might be involved in the autophagy process. Biochemical analyses confirmed some of the candidates as autophagy substrates. EEAT1 protein was shown to be degraded by autophagy for the first time. Maintaining cellular homeostasis requires a careful balance between many proliferative systems under physiological conditions and during cellular stress. Wnt/b-catenin signalling is an evolutionary conserved regulatory pathway that governs numerous normal cellular and developmental processes, such as cell fate specification, stem cell maintenance, cell proliferation, and migration. However, aberrant Wnt/b-catenin signalling has also been identified as a critical mechanism in several types of cancer, with b-catenin accumulation and dysregulated b-catenin/TCF/LEF target gene expression as a major cancer driver. Recent evidence points toward autophagy as an alternative b-catenin degradation pathway. Given the importance of Wnt/b-catenin signalling and autophagy, this study aims to investigate the role of autophagy in regulating Wnt/b-catenin signalling by identifying a link between these pathways. For that purpose, a physical connection between LC3/GABARAP and b-catenin proteins was identified. An atypical LIR motif in the structure of b-catenin was shown to facilitate its binding to LC3/GABARAP proteins with a stronger binding to the GABARAP family. However, this interaction does not lead b-catenin to autophagic degradation. Instead, it was shown that LC3/GABARAP proteins enhance the transcriptional activity of b-catenin. In addition, b-catenin was found to regulate autophagy in a cell type-dependent manner.