Exploration of the autophagy degradome and MAP1LC3B interactome using novel tools in combination with quantitative proteomics

Abstract

This thesis describes our efforts to study autophagy’s role in cancer by developing new assays that allow extensive profiling of the autophagy degradome and MAP1LC3B interactome. Autophagy plays a central role in many cellular processes but has also been extensively implicated in the pathology of many diseases, including in cancer. However, the role of autophagy in cancer appears both complex, paradoxical and cancer specific. The exact nature of its role appears to be context dependent and is yet to be elucidated. This project aimed to study autophagy’s role in cancer by developing new assays that have enabled elucidation of the autophagy degradome and MAP1LC3B interactome. We have utilised novel cell permeating peptides, derived from Phytophthora infestans virulence factors, which allow temporal and selective inhibition of autophagy, in combination with quantitative mass spectrometry based proteomics to optimise multiple whole-cell proteomics, proximity dependent and IP-MS workflows. Using these optimised workflows, we have profiled the MAP1LC3B interactome and autophagy degradome, whilst also delineating LIR dependent interactors of MAP1LC3B. These optimised protocols have been expanded in a pan-cancer fashion to establish high confidence, cancer-specific autophagy cargo and interactors, which have the potential to explain the differential role of autophagy between cancers and identify cancer-specific therapeutic vulnerabilities.