Characterization of molecular mechanisms governing selective autophagy

Abstract

This thesis delves into the molecular underpinnings of autophagy, a critical cellular mechanism for maintaining cellular homeostasis and defence against pathogens, through the lens of novel protein-protein interactions and structural insights. We pioneer the use of the AF2-multimer protein modelling approach to accurately identify both canonical and non-canonical AIM/LIR motifs, crucial for autophagy receptor and adaptor function. This innovative methodology not only sheds light on the interaction between ATG8/LC3 proteins and various autophagy components but also highlights its potential in uncovering ATG8-LIR/AIM associations across kingdoms, thus offering new perspectives on the cross-talk between autophagy pathways and pathogen virulence factors. Exploration into the ribosomal protein RPLP2 unveils its unexpected role within autophagic dynamics, characterized by a novel non-canonical binding motif facilitating its interaction with ATG8. This interaction points to RPLP2 as a potential selective receptor in ribophagy, expanding our understanding of autophagy's selectivity and complexity. Moreover, the application of the AF2-multimer prediction pipeline to viral proteomes reveals diverse strategies by which viral effectors can manipulate host autophagy, providing insights into the intricate dance between host defence mechanisms and viral evasion strategies. Lastly, the thesis presents a comprehensive study on Joka2, a selective cargo receptor in plants, elucidating its structure-function relationships through AlphaFold2 predictions, circular dichroism spectroscopy, and negative stain electron microscopy. The challenges encountered in structural analysis underscore the dynamic nature of autophagy-related proteins and the complexity of achieving high-resolution structural data. Together, our results contribute significantly to the understanding of autophagy's molecular basis, offering novel insights into the structural and functional nuances of autophagy components and their interactions with pathogens. This work not only broadens our comprehension of autophagy but also opens avenues for future research in cellular biology, biochemistry and structural biology.